ORIGINAL PAPER

Brain angioarchitecture and intussusceptive microvasculargrowth in a murine model of Krabbe disease

Arianna Giacomini1 • Maximilian Ackermann2 • Mirella Belleri1 • Daniela Coltrini3 •

Beatrice Nico4 • Domenico Ribatti4,5 • Moritz A. Konerding2 • Marco Presta1,3 •

Marco Righi6,7,8

Received: 19 May 2015 / Accepted: 13 August 2015 / Published online: 27 August 2015

� Springer Science+Business Media Dordrecht 2015

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 neuro-

logical dysfunctions and accumulation of neurotoxic psy-

chosine 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 reduc-

tion 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 micro-

scopy 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, con-

firmed by histological analysis, was paralleled by alter-

ations 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.

Keywords Angioarchitecture � Brain � Computational

analysis � Corrosion casting � Neurodegenerative Krabbe

disease � Intussusceptive angiogenesis

Introduction

Globoid cell leukodystrophy (GLD), or Krabbe disease, is

an autosomal recessive sphingolipidosis caused by the

genetic deficiency of the lysosomal hydrolase

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10456-015-9481-6) contains supplementarymaterial, which is available to authorized users.

& Marco Presta

marco.presta@unibs.it

& Marco Righi

m.righi@in.cnr.it

1 Unit of Experimental Oncology and Immunology, University

of Brescia, Brescia, Italy

2 Institute of Functional and Clinical Anatomy, University

Medical Center of the Johannes Gutenberg-University Mainz,

Mainz, Germany

3 Unit of Histology, Department of Molecular and

Translational Medicine, University of Brescia, viale Europa

11, 25123 Brescia, Italy

4 Unit of Human Anatomy and Histology, Department of Basic

Biomedical Sciences, University of Bari, Bari, Italy

5 National Cancer Institute ‘‘Giovanni Paolo II’’, Bari, Italy

6 CNR – Institute of Neuroscience, Milan, Italy

7 Department of Medical Biotechnology and Translational

Medicine, University of Milano, Milan, Italy

8 Consiglio Nazionale delle Ricerche, Institute of

Neuroscience, Via Vanvitelli 32, 20129 Milan, Italy

123

Angiogenesis (2015) 18:499–510

DOI 10.1007/s10456-015-9481-6

b-galactosylceramidase (GALC) [1]. The disease is char-

acterized by degeneration of oligodendroglia and progres-

sive demyelination of the peripheral and central nervous

system (CNS). Clinically, GLD manifests in early infancy

and results in a severe neurological dysfunction that often

leads to death by 2 years of age [2–4]. GALC degrades

galactosylceramide (a major component of myelin) and

other terminal b-galactose-containing sphingolipids,

including b-galactosylsphingosine (psychosine). Psy-

chosine is a bioactive water-soluble sphingolipid with

cytotoxic properties: normally undetectable; this sphingoid

base accumulates at micromolar concentrations in the CNS

of GLD patients and it is strongly implicated in the

molecular pathogenesis of the disease [5]. At present, the

only clinical treatment for GLD is bone marrow or

umbilical cord blood cell transplantation for late-onset and

presymptomatic patients [3, 6]. Thus, understanding the

molecular pathogenesis of GLD remains a high priority

from a clinical standpoint.

Brain is a highly vascularized organ, total length of

blood capillaries in adult human brain being equal to

approximately 6.5 9 105 m [7]. Accordingly, angiogenesis

plays an important role in the development of CNS and in

neurological disorders [8, 9]. The tight cross-talk among

glial, neuronal and endothelial cells in CNS [9] is under-

lined by the capacity of angiogenic factors, including

vascular endothelial growth factor-A (VEGF) and fibrob-

last growth factor-2 (FGF2), to modulate neurogenesis and

neuroprotection. In turn, neurotrophic factors may regulate

angiogenesis. Thus, vascular alterations appear to be

implicated in the pathogenesis of stroke and neurodegen-

erative disorders, including Alzheimer’s disease, Parkin-

son’s disease, multiple sclerosis and amyotrophic lateral

sclerosis [7–10].

Recently, the effects of GALC deficiency on CNS

microvascularization and angiogenesis have been investi-

gated in twitcher mice, an authentic murine model of GLD

harboring a naturally occurring premature stop codon in the

murineGALC gene [11]. GALC deficiency, with consequent

psychosine accumulation, induces significant defects in the

endothelium of the postnatal brain of twitcher mice. More-

over, twitcher endothelium shows a progressively reduced

capacity to respond to pro-angiogenic factors. In addition,

RNA interference-mediated GALC gene silencing hampers

the pro-angiogenic response of human endothelial cells to

VEGF. Accordingly, microvascular alterations occur in a

cortical brain biopsy from a GLD patient [11]. These

observations suggest that GALC deficiency affects not only

the glial/neuronal compartment of the neurovascular brain

unit but also its vascular moiety. Thus, given the interactions

between neurogenesis and angiogenesis and the pivotal role

played by neovascularization in postnatal neuroprotection,

alterations in blood vessel development are likely to

contribute to the injury occurring in the nervous system of

GLD patients during early infancy.

In this report, in order to get further insights about blood

vessel alterations consequent to GALC deficiency, we

performed a quantitative assessment of the 3D microvas-

cular architecture of the postnatal brain cortex of twitcher

mice by computational analysis of CD31-immunostained

histological sections [12] and by microvascular corrosion

casting followed by scanning electron microscopy (SEM)

morphometry [13]. The results highlight significant alter-

ations in the angioarchitecture of the frontal cortex of

twitcher mice with a uniform, generalized reduction in

microvascular density. In parallel, vascular remodeling and

intussusceptive angiogenesis were observed in twitcher

brain as a possible mechanism of vascularization alterna-

tive to impaired sprouting angiogenesis.

Methods

Mice

Breeder twitcher heterozygous mice (C57BL/6 J; Jackson

Laboratories, ME, USA) were maintained under standard

housing conditions. Animal handling protocols were in

accordance with Italian institutional guidelines for animal

care and use. Twitcher mutation was determined by poly-

merase chain reaction (PCR) on DNA extracted from

clipped tails [14]. In all the experiments, littermate wild-

type (wt) and homozygous (twi/twi) animals were used at

postnatal days 34–36 (P34–P36).

Immunostaining of brain microvasculature

Brains from P34 to P36 wt and twi/twi mice were either

fixed overnight at 4 �C in 4 % paraformaldehyde (PFA) or

frozen. For histological analysis of endothelial intraluminal

tissue folds, sections (8-lm-thick) of PFA-fixed paraffin-

embedded samples were stained with an aqueous solution

of 0.1 % toluidine blue. For 3D analysis, PFA-fixed 6 %

agarose-embedded samples were cut at a thickness of

100 lm using a vibratome. Free-floating sections were first

blocked in 2 % BSA (bovine serum albumine) and 0.2 %

Triton X-100 for 45 min at RT and then incubated over-

night at 4 �C with an anti-CD31 antibody (Dianova GmbH,

Hamburg, Germany, EU) diluted 1:50 in PBS containing

0.2 % BSA and 0.1 % Triton X-100. After several rinses in

PBS, sections were incubated overnight at 4 �C with sec-

ondary AlexaFluor594-conjugated antibody (Invitrogen,

Carlsbad, CA, USA) diluted 1:200 in PBS containing

0.1 % Triton X-100. For double-immunofluorescent anal-

ysis, sections (4-lm-thick) of frozen brains were fixed with

500 Angiogenesis (2015) 18:499–510

123

acetone (for 5 min at 4 �C), rinsed with PBS, and blocked

with 2 % BSA in PBS. Sections were first incubated for 1 h

at RT with the anti-CD31 antibody and for 30 min at RT

with the secondary AlexaFluor594-conjugated antibody.

After several rinses in PBS, terminal deoxynucleotidyl

transferase dUTP nick-end labeling (TUNEL) staining

(Roche, Milano, Italy, EU) was performed according to the

manufacturer’s instructions to highlight apoptotic cells that

were detected in the green channel. In order to analyze

pericyte coverage, CD31-stained sections were incubated

for 1 h at RT with an anti-desmin antibody (Dako, Milano,

Italy, EU). After washing with PBS, sections were incu-

bated for 30 min at RT with the appropriate secondary

AlexaFluor488-conjugated antibody (Invitrogen). After

further rinsing in PBS, fluorescent-stained sections were

mounted in a drop of anti-bleaching mounting medium

(Vectashield, Vector Laboratories, Burlingame, CA, USA)

and examined under an Axiovert 200 M microscope

equipped with ApoTome optical sectioning device (Carl

Zeiss, Oberkochen, Germany, EU).

Quantification of endothelial intraluminal tissue

folds

Thirty transversally sectioned vessel profiles with a single

layer of endothelial cells, either without or with a thin

basement membrane, were randomly chosen for wt and

twi/twi animals. Measurements were performed at 409

magnification using a computer-assisted image analysis

system (AnalySIS, Olympus Italia). For each vessel, the

number of connections of intraluminal tissue folds with the

opposite vascular wall, expression of intussusceptive

microvascular growth, was counted. They were recognized

by moving the focus through the thickness of the histo-

logical slide and counting only the strands completely

crossing the lumen. Those appearing incomplete and cross

sections of pillars within the vascular lumen were not

considered. Within each sample, the number of lumen

crossing endothelial folds per vessel was averaged to pro-

vide a representative value of each parameter for that

sample.

Image sampling, acquisition and pre-processing

of CD31-immunostained samples

For each treatment, images were acquired from samples

obtained from two independent experiments with two or

more animals per group. At least six Z-series of vascular

fields from different sections per animal group were

acquired from 100-lm-thick samples using an Axiovert

200 M microscope equipped with ApoTome optical sec-

tioning device (Carl Zeiss) with a 209 objective at

3.125 pixels/lm nominal resolution. In the vertical axis,

optical slices were 1 lm apart. Acquired images were

scaled to 1.56 pixels/lm, cropped to 512 9 512 pixels and

organized as 8-bit image stacks which were made isotropic

using the ImageJ plug-in developed by Cooper (http://

rsbweb.nih.gov/ij/plugins/make-isotropic/index.html). The

stacks were then converted to binary using a custom rou-

tine devised to recover faint signals while discarding

excessive random noise. Briefly, after a median filter, the

variance of each single slice was used to calculate an

integrated density value. If variance was higher than a fixed

arbitrary threshold, all the signals from the slice were

discarded as spurious. The resulting stack was then nor-

malized, and background was subtracted (‘‘rolling ball’’

method) to obtain a ‘‘clean’’ stack. At this point, data in the

stack were processed with the Renyi entropy algorithm to

build a binary mask which was dilated by two cycles using

standard mathematical morphology [15, 16]. This proce-

dure gave a 3D binary map roughly reporting vessel

positions as found in the original stack. Finally, the

‘‘clean’’ stack was filtered using the spatial map as a mask,

and the recovered grayscale signal was converted to binary

using the default algorithm present in ImageJ. As the very

last step, each slice was filtered by size, keeping particles

sized 1.6 lm2 (4 pixels) or more. This procedure and the

arbitrary thresholds used were validated on a set of at least

three image stacks for each experimental condition by

visual inspection of a trained operator.

Voxel classification according to vessel caliber

Binary voxels were assigned to vascular components of

different caliber using cross-sectional areas projected on

Cartesian planes according to a previously described

workflow [12]. After fill-up of hollow vessels, we classified

the resulting voxels according to the area of the smallest

cross section of the particle they belonged to. To this

respect, we considered only cross sections defined by the

three Cartesian planes passing through the voxel. This step

allowed to built maps of caliber-filtered vessels in the

analyzed volumes. Then, these maps were intersected with

the input binary volume to obtain the final classification of

input voxels.

Analyses of CD311 percent volume and spatial

dispersion

The endothelial percent volume of a binary sample was

calculated as the ratio of all CD31? voxels to the total

voxels of the volume. Then, to quantitate the spatial dis-

persion, we considered the 3D fill-up of each volume using

a mathematical morphology procedure we have previously

validated [12]. Accordingly, we counted the number of

dilation cycles needed to fill each volume up to 90 %

Angiogenesis (2015) 18:499–510 501

123

starting from the original distribution of CD31? voxels and

following a rhombicuboctahedral expansion scheme. For

each analysis, we compared only equal volumes and used

the greatest amount of signal observed among all volumes

to normalize the amount of input voxels. Thus, for each

sample, the normalized result was obtained subtracting the

number of dilation cycles needed to reach the normaliza-

tion value from the overall number of cycles needed to fill

90 % of the volume.

Automatic quantification of vascular lengths

To obtain these data, we reduced filled vessels to their basic 3D

skeletons using the ImageJ plug-in Skeletonize (2D/3D) (http://

imagejdocu.tudor.lu/doku.php?id=plugin:morphology:skeleto

nize3d:start) developed by Arganda-Carreras and based on

the decision tree algorithm created by Lee et al. [17]. The

plug-in was applied recursively to each input image until no

further skeletonization was observed. Then, taking advan-

tage of the analytical ImageJ plugin Analyze Skeleton (2D/

3D) (http://imagejdocu.tudor.lu/doku.php?id=plugin:analy

sis:analyzeskeleton:start) developed by Arganda-Carreras

et al. [18], we used a custom ImageJ S1 script to approximate

vessel lengths (the script and its instructions/rationale are

enclosed as Online Resource 1 and Online Resource 2,

respectively). The sum of all contributes was then multiplied

for the size of the voxel, to yield the total vascular length of

the analyzed volume.

Automatic classification of skeletonized voxels

and assessment of free ends

Quantification of skeletonized ends was performed using

the ImageJ S1 script mentioned above applied to the

ImageJ plug-in Analyze Skeleton (2D/3D) that provides

also basic voxel classification (node, stalk or end). These

preliminary results were integrated with a custom auto-

matic test to state whether tentative ‘‘ends’’ and ‘‘nodes’’

should be really considered as such on the basis of their

degree of connection with surrounding noncontiguous

voxels. This step was performed considering a 3x3x3 voxel

cube centered around each signal (black) voxel from

skeletonized samples. For each cube, we counted the iso-

lated groups of external black voxels connected through

faces (6-based connectivity). Given that all external voxels

connect with the central one, the center voxel was classi-

fied as an ‘‘end’’ when we identified only one group of

connected voxels (see the script rationale in Online

Resource 2); the voxel was considered a ‘‘stalk’’ when

connected to two groups and a ‘‘node’’ when connected

with more than two noncontiguous voxels or groups of

voxels. This approach eased the classification of end voxels

involved in skeleton kinks. Script functions were integrated

with a basic 3D filter to ignore end-to-end vessels (and

their ends) shorter than an arbitrary length, as calculated by

the Analyze Skeleton (2D/3D) plug-in in voxel units. A

folder with image files suitable for testing the S1 script can

be provided by the authors on request.

Corrosion casting

P36 wt and twi/twi mice were thoracotomized under deep

pentobarbital anesthesia. The left ventriclewas cannulated, and

the ascending aorta was entered with an olive-tipped cannula.

The entire vasculature of the animal was thoroughly rinsed by

perfusing with lukewarm saline (10–20 ml) and then fixed by

perfusing with 2.5 % buffered glutaraldehyde (10–20 ml,

860 mosmol, pH7.4). Finally, up to 15 ml of the polyurethane-

based casting resin PU4ii (vasQtec, Zurich, Switzerland, EU)

was gently perfused as a casting medium [19]. After complete

polymerization in a lukewarm water bath, the brains were

excised. The specimens were macerated in 5 % potassium

hydroxide, rinsed, dried and mounted on stubs for scanning

electron microscopy. The microvascular corrosion casts were

viewed on after coating with gold in argon atmosphere with a

Philips ESEM XL 30 scanning electron microscope (FEI,

Eindhoven, Netherlands, EU). From all specimens, areas were

recorded as stereo images using a tilt angle of 6�.

Morphometry

Pairs of stereo images with a tilt angle difference of 6�obtained using an eucentric specimen holder were used

after digitization and 3D reconstruction with an image

analysis program (Kontron KS 300, Kontron, Eching,

Germany, EU) to calculate parameters describing the

microvascular network architecture. For details of the

reconstruction and calculation, see [20].

Quantitative RT-PCR (RT-qPCR) analysis

Brains from P35 wt and twi/twi mice were analyzed for the

expression of the indicated genes by RT-qPCR, and data

were normalized for Gapdh expression as described [21].

To this purpose, total RNA was extracted from frozen

samples and contaminating DNA was digested using

DNAse, following indications reported in RNeasy Micro

Handbook (Qiagen, Valencia, CA, USA). Two micrograms

of total RNA was retrotranscribed with MMLV reverse

transcriptase (Invitrogen, Carlsbad, CA, USA) using ran-

dom hexaprimers in a final 20 ll volume. Quantitative

PCR was performed with a ViiATM 7 Real-Time PCR

Detection System (Applied Biosystems) using a iQTM

SYBR Green Supermix (Bio-Rad) according to the man-

ufacturer’s instructions. The primers are listed in Supple-

mentary Table S1 in Online Resource 3.

502 Angiogenesis (2015) 18:499–510

123

Software and statistical analyses

All the images from CD31?-stained samples were analyzed

using ImageJ software v. 1.48. Vascular tree renderings

were obtained with the ImageJ 3D viewer rendering tool.

The ImageJ scripts already published and the ImageJ plug-

in for 3D spatial dispersion can be found as supplementary

information in [12] together with instructions for use

(Sa_ImageJ_routines.pdf). Statistical analyses and tests

were performed using the Prism software environment

(GraphPad, La Jolla, CA, USA) and its basic statistical

package. Student’s t test for unpaired data (two-tailed) was

used to test the probability of significant differences

between two groups of samples. Differences were consid-

ered significant when P\ 0.05.

Results

Computational analysis of the 3D microvascular

architecture of the brain cortex of twitcher mice

Immunohistochemical analysis performed using the vas-

cular markers FVIII/vWF and CD31 had shown a signifi-

cant decrease in the density of capillary blood vessels in the

brain cortex of twi/twi mice when compared to wt animals

[11]. On this basis, in order to get further insights about

blood vessel alterations consequent to GALC deficiency,

we performed a quantitative assessment of the microvas-

cular architecture of the postnatal brain cortex of twitcher

mice by computational analysis of 3D reconstructions of

CD31-immunostained histological sections.

To this aim, the vascularization of the frontal brain

cortex of wt and twi/twi mice was quantified in a first set of

experiments by analysis of Z-stacks images after CD31

immunostaining of free-floating, 100-lm-thick tissue sec-

tions. We identified vessel walls by applying a custom

threshold algorithm to isotropic grayscale stacks repre-

senting nominal volumes of 327 9 327 9 70 lm3. Direct

inspection (Fig. 1a, b) and analysis of the volumes

(Fig. 1c) pointed out that the frontal cortex of twi/twi mice

presented reduced CD31? vessels (expressed as CD31?

percent volume) than wt controls. Conversely, we did not

observe inhomogeneities upon analysis of vessel spatial

dispersion in each volume (Fig. 1d). The reduction in

CD31? vessels in twitcher samples was paralleled by a

significant decrease in total microvascular length per unit

of tissue volume, as assessed after vessel skeletonization

(Fig. 1e). Also, direct inspection of twi/twi samples

(Fig. 1a, b) pointed out marked changes in the organization

of CD31? voxels that appeared to represent a pool of

fragmented vessels more than a well-organized vascular

tree. In order to quantify these changes, we calculated the

number of free CD31? segment ends in each sample that

were normalized for the total vascular length measured in

the same sample after removal of isolated fragments

shorter than 2.4–3.0 lm. The results, shown in Fig. 1f,

confirmed that the normalized amounts of free vascular

ends were significantly increased in the cerebral tissue of

twi/twi animals with respect to wt controls in both exper-

iments. Finally, normalized CD31 stainings, defined as the

amounts of CD31 signal per unit of vascular length,

demonstrated the absence of significant differences in

specific staining between twi/twi and wt samples (Fig. 1g).

This indicates that the reduction in CD31? signal in twi/twi

samples reflects a real paucity of blood vessels and not a

reduction in their CD31 immunoreactivity. Similar results

were obtained in a second independent experiment, con-

firming the reproducibility of these observations (Supple-

mentary Fig. S1a–e in Online Resource 3). Thus, this

preliminary analysis suggested a homogeneous reduction in

the vascularization of the frontal brain cortex of twitcher

mice, extending previous indications obtained by standard

histological techniques [11].

In order to deepen these observations, we continued

sorting out CD31? voxels according to the approximate

caliber of the microvessel to which they belonged [12]. To

this aim, we classified image voxels considering cross-

sectional surface classes ranging from 1.6 to about 95 lm2

(theoretical diameters between 1.4 and 11 lm assuming a

circular cross section). In this process, we took advantage

of the vascular maps of reconstructed filled microvessels to

assign poorly connected voxels to their correct class

(Fig. 2a). Then, we quantified the differences in brain

cortex vascularization by analyzing samples from twi/twi

and wt mice to obtain the percent fraction of CD31? voxels

in each volume for each class of vascular calibers.

According to our classification, the majority of signal

belonged to vessels with a diameter ranging between 4.0

and 5.5 lm for both healthy and diseased brains (Fig. 2b).

However, twi/twi mice showed a marked drop in the

amount of positive voxels for most class of calibers, with

up to 45–50 % signal reduction for vessels with a diameter

ranging between 2.8 and 5.5 lm (Table 1). The statistical

significance of these differences varied from class to class

of calibers but was markedly significant for well-populated

classes. Similar results were obtained in a second inde-

pendent experiment (Supplementary Fig. S1f in Online

Resource 3 and Table 1).

To rule out the possibility that the observed differences

in vascularization were the mere consequence of a different

diffusion of the anti-CD31 antibody through the whole

100-lm-thick tissue sections, the computational analysis

was repeated on the topmost 23 lm of all samples. Even

under these experimental conditions, we obtained a similar

distribution of signal losses for twi/twi samples in the two

Angiogenesis (2015) 18:499–510 503

123

independent experiments (Supplementary Table S2 in

Online Resource 3).

Cytotoxic psychosine, present at high micromolar con-

centrations in the CNS of patients with GLD and twitcher

mice, is detectable at very low levels in GALC-deficient

non-nervous tissues/organs (see [11] and references

therein). On this basis, to investigate whether vascular

alterations in twitcher mice were specific for cerebral tis-

sue, we repeated these vascular analyses on kidney

explants from wt and twi/twi animals. At variance with

data from brain samples, no significant difference in the

amount of CD31? vascular signals, total vascular length

and normalized amounts of free vascular ends was

observed in the kidneys of control and diseased animals

(Supplementary Fig. S2 in Online Resource 3).

Ultrastructural analysis of the frontal cortex vasculature

of twi/twi mice had shown significant alterations of

microvascular endothelial cells that were characterized by

swollen glial endfeet, altered tight junctions, decrease in

the expression of the tight junction-associated protein ZO-1

and increased permeability. Also, the microvasculature of

brain biopsies obtained from a 2.5-year-old GLD patient

showed an irregularly shaped Factor VIII? endothelium

with discontinuous wrapping by smooth muscle actin cells

[11]. Accordingly, the endothelium of the brain cortex of

twitcher mice is characterized by the disorganization of

Fig. 1 Vascular alterations in the brain of twitcher mice by

computational analysis. Z-series of vascular fields from 100-lm-

thick CD31-immunostained sections of the frontal cortex from the

brains of P35 wt and twitcher mice were acquired using an Axiovert

200 M microscope equipped with ApoTome optical sectioning device

(Carl Zeiss) with a 920 objective at 3.125 pixels/lm nominal

resolution. a Acquired images were scaled to 1.56 pixels/lm, cropped

to 512 9 512 pixels and organized as 8-bit grayscale image stacks

which were made isotropic using the ImageJ plug-in developed by

Cooper (http://rsbweb.nih.gov/ij/plugins/make-isotropic/index.html).

b The 8-bit grayscale stacks were then converted to binary using a

custom routine devised to recover faint signals while discarding

excessive random noise (for more details see ‘‘Materials and meth-

ods’’ section). c Quantification of vascular density expressed as per-

cent ratio of CD31? voxels/total voxels (V %). d Quantification of

spatial dispersion expressed considering the number of expansion

cycles needed to fill 90 % of the volume following a rhombicuboc-

tahedral expansion scheme [12]. Sample normalization as described

in ‘‘Materials and methods’’ section. Data are shown as classical box

and whiskers graphs: the boxes extend from the 25 to the 75th per-

centiles, the lines indicate the median values, and the whiskers indi-

cate the range of values. e Quantification of oriented vascular lengths

calculated in whole samples after vessel skeletonization (for more

details see ‘‘Materials and methods’’ section). f Number of vascular

ends normalized by the sum of the total oriented vascular lengths

determined after vessel skeletonization. g Amounts of CD31? signal

normalized by the sum of the total oriented vascular lengths deter-

mined after vessel skeletonization. In each graph, data are the

mean ± SD, n = 8; ***P\ 0.001; **P\ 0.01; n.s., not significant

504 Angiogenesis (2015) 18:499–510

123

perivascular desmin? pericytes, leading to a significant

reduction in endothelial coverage (Fig. 3), thus confirming

that GALC deficiency results in profound alteration of the

blood–brain barrier functionality.

SEM morphometry of the 3D microvascular

architecture of the brain cortex of twitcher mice

Microvascular corrosion casting followed by SEM mor-

phometry allows a fine qualitative and quantitative char-

acterization of the vascular networks restricted to patent

vessels connected to the bloodstream [22]. On this basis, in

order to validate the information provided by the compu-

tational analysis of immunofluorescence data, microvas-

cular corrosion casts of the brain cortex of P36 twi/twi

mice were studied by SEM morphometry.

Morphometric analysis of corrosion casts (Fig. 4) indi-

cates that, when compared to wt mice, the patent

microvasculature of the brain cortex of twi/twi animals

shows a significant increase in the intervascular distance, a

morphometric measure of vascular density, thus confirming

the data obtained by computational analysis of CD31?

microvasculature (see above). In addition, direct inspection

of SEM images of corrosion casts indicated that twitcher

microvasculature is characterized by dilated vessels with

alterations in vessel diameter, vascular leakage and

endothelial dehiscence (Fig. 5a). Remarkably, vascular

branch remodeling and intussusceptive angiogenesis,

characterized by the presence of intussusceptive pillars,

were observed in twitcher mice but not in control animals

(Fig. 5b). In keeping with these observations, microscopic

analysis of coronal sections of the brain cortex of P36 twi/

twi mice stained with toluidine blue demonstrated the

presence of microvessels with morphological aspects sug-

gestive of intussusceptive microvascular growth in form of

intraluminal tissue folds connecting the opposite vascular

walls. These aspects were not recognizable in control

specimens (Fig. 5c). Accordingly, the average number of

endothelial intraluminal tissue folds was equal to 3 ± 1

lumen-crossing pillars per vessel in twitcher mice and

equal to 0 ± 0 in control animals (n = 30) (Fig. 5d).

Fig. 2 Quantification of vascular CD31? signals classified by vessel

calibers. a Color-coded renderings of wt and twi/twi microvasculature

after vessel classification by approximated cross section. Classes are

identified by their theoretical diameters (in lm) as it follows: magenta

1.4–2.0, red 2.0–2.8, yellow 2.8–4.0, green 4.0–5.5, cyan 5.5–8, blue

8.0–11.0. b Quantification of CD31? signals for each classes of vessel

calibers from wt (closed color bars) and twi/twi samples (color-

bordered open bars). Data are the mean ± SD, n = 8. Color codings

as in panel a. ***P\ 0.001; **P\ 0.01; *P\ 0.05; n.s., not

significant. (Color figure online)

Table 1 Percent vascular volume differences between wt and twi/twi brain cortex samples

Vessel diameter (lm) Experiment 1 Experiment 2

Loss in vol (%) Statistical significancea Loss in vol (%) Statistical significancea

1.4–2.0 8.2 n.s. -16.4 n.s.

2.0–2.8 33.4 P\ 0.05 29.5 P\ 0.05

2.8–4.0 44.5 P\ 0.001 51.2 P\ 0.01

4.0–5.5 48.7 P\ 0.001 68.5 P\ 0.001

5.5–8.0 33.5 n.s. 73.4 P\ 0.01

8.0–11.0 15.3 n.s. 64.2 n.s.

n = 8 n = 6

Values are expressed as percent reduction among the different classes of vessels grouped according to their theoretical diameter in twi/twi with

respect to wt samples as observed in two independent experimentsa Statistical significance was measured by Student’s t test; n.s., not significant

Angiogenesis (2015) 18:499–510 505

123

Expression of intussusceptive angiogenesis-related

genes in the brain of twitcher mice

Intussusceptive angiogenesis is a morphogenetic, non-

sprouting angiogenic process in which a single vessel is

split into two lumens. A distinguishing anatomic feature of

intussusceptive angiogenesis is the intussusceptive pillar, a

transluminal tissue bridge that spans the vessel lumen.

Physical expansion or growth of the pillar along the vessel

axis divides the lumen resulting in vascular duplication

[23]. Even though the molecular mechanisms leading to

intussusceptive angiogenesis are not fully elucidated,

recent observations have shown that inhibition of Notch

signaling in already existing vascular beds induces exten-

sive intussusceptive angiogenesis [24]. The process, char-

acterized by pericyte detachment, increased vessel

permeability and extravasation of mononuclear cells,

occurs in parallel with downregulation of Notch-dependent

Hes5 and Tie-2 gene expression and Fgf2 and Cxcr4

upregulation.

On this basis, we evaluated the expression levels of

intussusception-related genes in the total brain extracts of

twitcher mice at P35. RT-qPCR analysis demonstrates a

significant downregulation of the Notch target gene Hes5.

Also, we observed a significant upregulation of Fgf2 and

Cxcr4 expression as well as of various pro-inflammatory

cytokines and chemotactic chemokines, including Tumor

necrosis factor-a, Interleukin-1a and Cxcl-1, paralleled by

a sustained inflammatory infiltrate as indicated by the

significant increase in CD45 mRNA levels (Fig. 6). This

occurs in the absence of significant changes in the

expression levels of Notch1 and of its ligands Dll4 and

Jag1 (data not shown).

Discussion

Previous observations from our laboratory had shown that

GALC deficiency induces significant defects in the

endothelium of the postnatal brain of twitcher mice, an

authentic murine model of GLD [11]. Here, we extend these

observations by showing that quantitative computational

analysis of the three-dimensional CD31? microvascular

architecture of the postnatal frontal cortex coupled with

microvascular corrosion casting/SEM morphometry [22]

highlights significant alterations in the angioarchitecture of

the brain of twitcher mice with a uniform, generalized

reduction in microvascular density. In parallel, vascular

branch remodeling and intussusceptive angiogenesis were

observed in twitcher brain as a tentative mechanism of vas-

cularization alternative to impaired sprouting angiogenesis.

CD31? vessel quantification was initially approached by

customized ImageJ scripts developed in order to speed up

Fig. 3 Immunofluorescent analysis of endothelial coverage. a Frontalcortex microvessels of P36 wt and twi/twi mice were double-stained

with anti-CD31 and anti-desmin antibodies. Scale bar: 30 lm.

b Quantification of the percentage of pericyte coverage calculated

as desmin? area/CD31? area ratio. Data are the mean ± SEM.

***P\ 0.001. (Color figure online)

Fig. 4 Morphometric analysis of microvascular corrosion casts. The

analysis was performed on corrosion casts of the frontal cortex of P36

wt and twi/twi mice. Vessel diameters are expressed as box plots (left)

and cumulative percentual frequency graphs (right). The boxes extend

from the 25th to the 75th percentiles, the lines indicate the median

values, and the whiskers indicate the range of values; the mean values

are indicated with a dashed line. ***P\ 0.001

506 Angiogenesis (2015) 18:499–510

123

the analysis of significant numbers of isotropic 3D samples.

The analysis provided cumulative information about

functional and non-functional vessels (including frag-

mented vessels apparently not connected with the blood-

stream) and was integrated by SEM observations of the

corrosion casts of patent vessels. The results demonstrate a

remarkable reduction in CD31? vascularity in twitcher

brains analyzed at an advanced stage of disease, possibly

due to a decrease in the angiogenic process that drives

postnatal brain vascularization [11] and/or to cytotoxic

effects due to the accumulation of high levels of psy-

chosine in the CNS [5] with consequent degenerative

neuroinflammation [6, 25]. Taken together, our current and

previous observations [11] indicate that GALC deficiency

may affect not only the glial/neuronal compartment of the

neurovascular brain unit but also its vascular moiety. A

tight cross-talk exists between angiogenesis and

neurogenesis, and neovascularization plays an important

role in postnatal neuroprotection [8–10, 26]. Consequently,

alterations in blood vessel development may contribute

greatly to the CNS damage occurring in GLD. It remains

challenging to establish whether vascular alterations in

GLD represent the outcome of the astrocytic/neuronal

injury, with a consequent deficiency in the production of

trophic angioneurins [27], or are due to a direct damage of

endothelial cell functions by the loss of GALC function

and psychosine accumulation. Relevant to this point,

TUNEL staining of postnatal frontal cortex sections

showed a significant overall increase in apoptotic events in

twitcher mice when compared to control animals. How-

ever, they were limited to non-endothelial CNS compo-

nents, as shown by double TUNEL/CD31 staining that

failed to indicate the presence of apoptotic events in brain

endothelium (Supplementary Fig. S3 in Online Resource

Fig. 5 Scanning electron micrographs of the frontal cortex microvas-

culature of twitcher mice. Microvascular corrosion casting/SEM

analysis of the frontal cortex of P36 twi/twi mice. a The vasculature

was characterized by dilated vessels and high frequency of diameter

changes (black asterisks). The dehiscences in endothelium result in a

leakage of vasculature (white arrows). Scale bar: 10 lm. b Example

of intussusceptive site in twitcher mice characterized by vessel

bifurcation reflecting structural intussusceptive branch remodeling

(see magnified 1, scale bar: 10 lm) and intussusceptive pillar

indicating vessel duplication (see magnified 2, scale bar: 10 lm).

Scale bar: 20 lm. c Frontal cortex microvessels of P36 wt and twi/twi

mice stained with toluidine blue showing the presence of intraluminal

tissue fold connecting the opposite vascular walls (black arrows) in

twi/twi mice but not in wt mice. Scale bar: 100 lm. d Quantification

of endothelial intraluminal tissue folds. Transversally sectioned vessel

profiles with a single layer of endothelial cells were randomly chosen

for wt and twi/twi animals. For each vessel, the number of

connections of intraluminal tissue folds with the opposite vascular

wall, expression of intussusceptive microvascular growth, was

counted. Data are the mean ± SD, n = 30. *P\ 0.05

Angiogenesis (2015) 18:499–510 507

123

3). This is in keeping with the capacity of psychosine to

induce oligodendrocyte apoptosis [28] with no effect on

endothelial cell survival [11].

Additional suggestions come from the analysis of the

spatial distribution of CD31? vessels. In the search for

focal sites where vascular pruning and remodeling might

be taking place, we did not observe marked differences

between control and twitcher animals. Previous observa-

tions had shown that brain neovascularization is reduced in

twi/twi mice also at postnatal stages that precede the

occurrence of significant signs of disease [11]. Thus, a

progressive slowdown of vascular development, and mat-

uration, might explain why twitcher brains showed a larger

amount of fragmented vessels with no apparent continuity

with the vascular tree. Further experiments will be required

to depict the exact spatiotemporal contribution of reduced

neovascularization and psychosine toxicity to the observed

decrease in functional microvascular density in the brain of

twitcher mice. Unfortunately, it must be pointed out that, to

this respect, the increased vascular leakage of the GALC-

deficient endothelium precludes the possibility to use

bloodstream-injected molecular tracers for a direct visual-

ization and quantification of patent, functional vessels [11].

Search for signs of compensatory neovascularization in

twitcher brains did not reveal evident hot spots of sprouting

vessels or a marked increase in microvessels with small

cross sections. Indeed, previous observation had shown a

progressively reduced capacity of twitcher endothelium to

respond to pro-angiogenic factors [11]. In contrast, SEM

and histological analyses showed increased signs of

intussusceptive angiogenesis. This occurs in parallel with

the modulation of genes associated with this process (e.g.,

Hes5, Cxcr4, Fgf2) [24] and in the presence of a sustained

inflammatory response. Intussusceptive (non-sprouting)

angiogenesis is a well-characterized morphogenetic pro-

cess of blood capillaries that produces two lumens from a

single vessel by intravascular septation, the intussusceptive

pillar being a 1- to 5-lm transluminal tissue bridge that

spans the vessel lumen [29]. Intussusceptive angiogenesis

is distinct from sprouting angiogenesis because it does not

require cell proliferation. It might represent a general fast

recovery adaption of vascularization to growth necessities

with a rapid expansion of the existing microvascular net-

work [23]. In inflammatory tissue, a shift is observed from

sprouting angiogenesis toward intussusceptive angiogene-

sis [23]. Thus, the dynamic process of intussusceptive

angiogenesis by rapidly increasing capillary exchange

surface area appears to be a relevant adaptive mechanism

in inflammatory and ischemic tissues [23, 30, 31] and is

characterized by pericyte detachment, increased vessel

permeability and extravasation of mononuclear cells [24].

Our data suggest that structural and architectural angioad-

aptations may occur in twi/twi mice in response to GALC

deficiency [6, 25], intussusceptive angiogenesis represent-

ing a possible mechanism of escape in the attempt to

overcome defective sprouting angiogenic responses. On

note, no difference in mRNA and protein levels of hypoxia-

inducible factor-1a was observed in the total brain extracts

of wt and twi/twi animals (A.G., unpublished observa-

tions). Further experiments will be required to elucidate the

role of the neuroinflammatory process and the molecular

mechanism leading to intussusceptive microvascular

growth following GALC deficiency.

The pathogenesis of GLD has been proposed to arise

from the accumulation of psychosine, the neurotoxic

lysolipid metabolite detected at high levels in the CNS of

GLD patients and twitcher mice [5, 32, 33]. Accumulation

of psychosine leads to cytotoxic effects on oligodendroglial

cells, triggering apoptotic cell death in vitro and in vivo

[28, 34, 35]. Given the important role played by oligo-

dendrocytes in brain vascularization [36], it is conceivable

that the vascular alterations observed in the brain of

twitcher mice may represent the outcome of the astrocytic/

neuronal injury, with a consequent deficiency in the pro-

duction of trophic angioneurins [27] and activation of

inflammatory responses [6, 25]. Accordingly, at variance

with the results of the computational analysis of brain

cortex microvasculature, no significant alterations of the

three-dimensional CD31? microvascular architecture were

observed in the kidneys of twitcher mice in which psy-

chosine is detectable at very low levels when compared to

CNS [37]. On the other hand, endothelial cells of different

origins express GALC in vitro and in vivo, and GALC

deficiency in twitcher mice affects the capacity of periph-

eral endothelium to respond to pro-angiogenic factors [11].

Thus, GALC loss-of-function may affect the postnatal

microvasculature of different organs besides the CNS.

Fig. 6 RT-qPCR analysis of intussusceptive angiogenesis-related

genes in the brain of twitcher mice. Total brain extracts from P35 wt

and twi/twi mice (n = 3–7 per group) were analyzed for the

expression of the indicated genes by RT-qPCR, and data were

normalized for Gapdh expression. Data are the mean ± SD;

**P\ 0.01

508 Angiogenesis (2015) 18:499–510

123

Indeed, significant vascular permeability defects occur in

different visceral organs of twi/twi mice, including kidney,

lung and liver [11]. Taken together, these data suggest that

GALC deficiency may cause a generalized endothelial cell

dysfunction in peripheral tissues, leading to increased

vessel permeability, with no significant consequence on

their angioarchitecture.

Lysosomal storage disorders represent one of the most

frequent classes of human genetic diseases. They are

characterized by the accumulation of disease-specific

metabolic intermediates within lysosomes, leading to sev-

ere organ damages and premature death [32]. Besides

GLD, brain alterations of the microvascular angioarchi-

tecture may occur also in adrenoleukostrophy, Canavan’s

disease and Alexander’s disease patients [38, 39]. Of note,

b-glucosylpsychosine (1-b-D-glucosylsphingosine) puri-

fied from human Gaucher spleen exerts an inhibitory effect

on endothelial cell proliferation and motility similar to

psychosine [11], and ultrastructural abnormalities occur in

capillary endothelial cells in skin biopsies from patients

with acute neuronopathic infantile Gaucher disease [40].

Together, these observations point to endothelial dysfunc-

tion as a common factor in severe lysosomal diseases. At

present, the only clinical treatment for GLD is bone mar-

row or umbilical cord blood cell transplantation for late-

onset and presymptomatic patients [3, 6]. A better under-

standing of the alterations of CNS and peripheral

microvascular angioarchitecture and of endothelial cell

dysfunctions may provide further insights into the patho-

genesis of lysosomal storage disorders and pave the way

toward more efficacious therapeutic approaches. To this

respect, our observations show that quantitative vascular

analysis by a simple, automatic computational approach

may provide relevant information when applied to twitcher

mice, an authentic model of Krabbe disease. Given the

increasing availability of image analysis tools and the

intimate link between vascular and neurological systems,

we consider that time is ripe for a systematic analysis of

vascular trees in animal models of neurodegenerative

pathologies.

Acknowledgments This work is dedicated in the memory of late

Prof. Moritz A. Konerding. The work was supported in part by the

CNR Research Project on Aging (Regione Lombardia MbMM-con-

venzione No. 18099/RCC) to the Institute of Neuroscience (Milan,

Italy) and by Grants from Ministero dell’Istruzione, Universita e

Ricerca (FIRB Project RBAP11H2R9 2011), Associazione Italiana

per la Ricerca sul Cancro (AIRC Grant No. 14395) to M.P., A.G. was

supported by a Fondazione Italiana per la Ricerca sul Cancro

Fellowship.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict

of interest.

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