1

Supplementary Information for Nogo-A Targeted Therapy Promotes Vascular Repair and Functional Recovery Following Stroke

Authors

Ruslan Rust1,2,*, Lisa Grönnert1, Christina Gantner3, Alinda Enzler2, Geertje

Mulders1, Rebecca Z. Weber1, Arthur Siewert4, Yanuar D.P. Limasale5, Andrea

Meinhardt5, Michael A. Maurer1, Andrea M. Sartori1,2, Anna-Sophie Hofer1,2,

Carsten Werner5, Martin E. Schwab1,2,*

Affiliations 1 Institute for Regenerative Medicine, University of Zurich, 8952 Schlieren,

Switzerland, 2 Dept. of Health Sciences and Technology, ETH Zurich, 8092 Zurich,

Switzerland, 3 Dept. of Biology, ETH Zurich, 8093 Zurich, Switzerland, 4

Department of Technology, Bielefeld University, 33615 Bielefeld, Germany, 5 Leibniz Institute for Polymer Research, 01069 Dresden, Germany

Correspondence

Ruslan Rust

Rust@irem.uzh.ch

This PDF file includes:

SI Materials and Methods

Figs. S1 to S9

Tables S1-S2

www.pnas.org/cgi/doi/10.1073/pnas.1905309116

2

SI Materials and Methods

Study design

The goal of the present study was to test if blockage of the neurite outgrowth

inhibitor Nogo-A improves vascular repair in mice with cerebral ischemia. We

hypothesized that high Nogo-A levels restrict the formation of new blood vessels

in the ischemic border zone, similar to its previously reported role in negative

regulation of developmental CNS vasculature. Its inhibition may facilitate the

generation of a more permissive environment for neuroprotective and

regenerative processes that ultimately might result in improved function.

To test this, we first examined genetic mouse models deficient for Nogo-A (N=9)

or its corresponding receptor S1PR2 (N=5), or neutralized Nogo-A with a

function-blocking anti-Nogo-A antibody (N=12). Control animals in all

experiments were wildtype (WT) litter mates (N=13) or animals receiving an IgG

isotype control antibody (N=13). We assessed motor skills of the stroked animals

at baseline (BL) and at 3, 7, 14, and 21 days after injury, respectively. Ischemic

cortical tissue was histologically analyzed after three weeks for vascular function,

cell survival, synaptic and neurotransmitter activity and regenerative processes.

A subset of Nogo-A–/– and WT animals underwent functional blood perfusion

analysis with Laser Doppler Flowmetry (N=4 per group). To link improved

vascular repair with functional improvement after Nogo-A blockage, we blocked

post-stroke angiogenesis of Nogo-A–/– mice by systemic application of anti-VEGF

antibodies for 10 days after stroke (N=6-7, per group). Moreover, in an in vitro

3D hydrogel model we tested the effects of Nogo-A components together with

VEGF on the vascular network (N=5-7, per group).

All animals are presented in the study; no statistical outliers were excluded. Data

was acquired blinded. The surgery, behavioral testing and analysis of the data

was performed by different investigators that were blinded in all steps of analysis.

3

Animals

We used wildtype, Nogo-A−/− and S1PR2−/− C57BL/6 mice. Animals were

maintained at the Brain Research Institute Zurich on a 12 light/dark cycle with

food and water provided ad libitum. Mice were housed in standard Type II/III

cages at least in pairs. Animals used were 10-14-week-old females. Validation

experiments were also performed with age-matched males (SI Appendix, Fig.

S7). All experiments were conducted in accordance with the applicable national

regulations and approved by the Cantonal Veterinary Department of Zurich.

Photothrombotic stroke and antibody treatment

Animals were anaesthetized with 2% isoflurane followed by an intraperitoneal

injection of fentanyl (20ug/kg), midazolam (1mg/kg), medetomidine (200ug/kg). A

photo-thrombotic stroke to unilaterally lesion the sensorimotor cortex was

introduced on the right hemisphere, as previously described.

Briefly the animal was fixed in a stereotactic frame, the skull exposed through a

midline incision, cleared of connective tissue and dried. A cold light source

(Olympus KL 1500LCS, 150W, 3000K) was positioned over an opaque template

with an opening for the light source (5 x 3 mm) 2.5 mm to -2.5 mm anterior and 0

mm to 3 mm lateral to Bregma. Rose Bengal (13mg/kg body weight, 10mg/ml

Rose Bengal in 0.9% NaCl solution) was intraperitoneally injected and after 5

min, the brain was illuminated through the intact skull for 10.5 min.

For constant delivery of the function blocking Ig G1 mouse monoclonal anti-

Nogo-A antibody 11C7 (7 mg/ml), antibodies were stereotaxically administered

24 hours after injury through subcutaneously implanted mini-osmotic pumps

(Alzet, 1002) with a flow rate of 0.25 µl/h to a cohort of 15 12-week-old female

C57BL/6 mice for 14 days. The control cohort received Ig G1 isotype control

(FG12). After photothrombotic stroke induction a cannula was inserted into the

left ventricle (anterior/posterior -0.57 mm, medial/lateral -1.5 mm, dorsal/ventral -

2.1 mm to Bregma), thereby sparing the motor area. The osmotic pump was

placed in the midscapula region to permit free head and neck movement.

Although the osmotic pump begins operating immediately, we adapted the set-up

4

to allow a 24h delay of the application. This was achieved by filling the tip of the

catheter with a control solution (0.9% saline) according to the manufacturer's

recommendations. Thereafter, antibodies were released over 14 days with a flow

rate of 0.25 µl/h. To antagonize the anaesthesia, Antisedan (0.15 mg/kg body

weight) and Flumazenil (40 µg/kg body weight) were s.c. injected. For

postoperative care, all animals received analgesics (Dafalgan Sirup, Braun, per

os in the drinking water) and antibiotics (Baytril, 5 mg/kg body weight, Bayer, s.c.)

once a day for at least 3 days after surgery. Sham operated animals underwent

the entire surgical process but did not received Rose Bengal.

Tissue processing for high throughput qPCR

Brains from WT animals were rapidly dissected in RNA Later solution (Ambion)

under a stereotaxic microscope. Stroked cortical tissue and the corresponding

contralesional site were placed in Eppendorf tubes, flash frozen in liquid nitrogen,

and stored at -80 °C until RNA extraction. Total RNA was prepared using the

RNeasy RNA isolation kit (Qiagen) including a DNase treatment to digest

residual genomic DNA. For reverse transcription, equal amounts of total RNA

were transformed by oligo(dT) and M-MLV reverse transcriptase (Promega).

Primer pairs were designed to span intronic sequences or to cover exon-intron

boundaries. Real-time quantitative PCR was performed in a Fluidigm system

consisting of a BioMark HD instrument, IFC HX controller and 96 × 96 dynamic

array, as described in the manufacturer’s user guide. Expression of selected

genes was confirmed by qPCR. Therefore, ten nanograms of cDNA were

amplified in the Light Cycler 480 thermocycler (Roche Diagnostics AG) with the

polymerase ready mix (SYBR Green I Master; Roche Diagnostics AG).

Tissue processing for histological analysis

Animals were deeply anaesthetized by intraperitoneal injection of pentobarbital

(150 mg/kg bodyweight) and subsequently transcardially perfused with isotonic

Ringer solution (5 ml/l Heparin, B. Braun) and 4% Paraformaldehyde (PFA, in 0.2

M phosphate buffer, pH 7.4). Brains were removed from the skulls and post-fixed

5

for 4 h at 4 °C. For cryoprotection, brains were transferred to 30% sucrose and

kept overnight at 4 °C. Coronal sections with a thickness of 40 µm were cut using

a sliding microtome (Microm HM430, Leica). Sections were collected and stored

as free-floating sections in cryoprotectant solution at -20 °C until further

processing. For immunohistochemical staining brain sections were washed with

0.1 M phosphate buffer (PB) and then incubated with a blocking and

permeabilization solution (TNB, 0.1% TBST, 3% normal goat serum) for 30 min

at RT shaking. Sections were incubated with primary antibodies (SI Appendix,

Table 1) overnight at 4 °C. The next day sections were washed and incubated

with secondary antibodies (SI Appendix Table 2) for 2 h at RT. All antibodies

were diluted in blocking and permeabilization solution (TNB, 0.1% TBST, 3%

normal goat serum). Nuclei were counterstained with DAPI (1:2000 in 0.1 M PB).

Sections were mounted in 0.1 M PB on Superfrost PlusTM microscope slides

(Thermo Fisher) and coverslipped using Mowiol.

Laser Doppler Flowmetry

Animals were anaesthetized with 2% isoflurane followed by an intraperitoneal

injection of fentanyl (20ug/kg), midazolam (1mg/kg), medetomidine (200ug/kg).

Animals were fixed in a stereotactic frame, the skull exposed through a midline

incision, cleared of connective tissue and dried. 3x3 coordinates were placed on

an opaque template to record the relative blood perfusion in the ischemic border

regions. A laser Doppler fiber (Oxford Optronix) was successively placed at the

region of interest and recorded the relative blood perfusion for 20 seconds.

Perfusion was measured at baseline, 4 h, and 4, 7, and 21 days post injury.

Vascular tracing

After deep anesthesia and shortly before perfusion, 50 µl of 1mg/ml Lycopersicon

Esculentum lectin conjugated to DyLight594 (DL-1177, VactorLabs) were

transcardially injected. After 1 min animals were transcardially perfused with

isotonic Ringer solution (5 ml/l Heparin, B. Braun) and 4% Paraformaldehyde

(PFA, in 0.2 M phosphate buffer, pH 7.4).

6

Behavioral testing and analysis

In order to evaluate recovery of motor function following stroke behavioral testing

of all animals was performed 3, 7, 14 and 21 days after stroke, respectively, and

compared to baseline performances recorded 3 days prior to surgery.

Horizontal ladder: To assess locomotor function after stroke, animals were

placed on an elevated horizontal ladder and trained to cross the device. To

prevent habituation to a specific bar distance, bars were irregularly spaced (1–4

cm) and each animal had to cross the ladder in both directions. For behavioral

testing, a total of three runs per animal were recorded (Panasonic HDC-SD800

High Definition Camcorder) and paw misplacements were analyzed frame-by-

frame (Fiji). The error rate was calculated by dividing the amount of incorrect

steps by the number of total steps taken with the respective paw in the 3 test

runs.

Cylinder test: To evaluate locomotor asymmetry mice were placed in an open-

top, clear plastic cylinder for about 10 min to record their forelimb activity while

rearing against the wall of the arena. Forelimb use is defined by the placement of

the whole palm on the wall of the arena, which indicates its use for body support.

Forelimb contacts while rearing were scored with a total of 20 contacts recorded

for each animal. Three parameters were analysed which include paw preference,

symmetry and paw dragging. Paw preference was assessed by the number of

impaired forelimb contacts to the total forelimb contacts. Symmetry was

calculated by the ratio of asymmetrical paw contacts to total paw contacts. Paw

dragging was assessed by the ratio of the number of dragged impaired forelimb

contacts to total impaired forelimb contacts.

EdU administration

To label proliferating vascular endothelial cells mice received three consecutive

i.p. injections of 5-ethynyl-2’-deoxyuridine (EdU, 50 mg/kg body weight,

ThermoFischer) on day 6, 7 and 8 after stroke. EdU incorporation was detected

7

21 days after stroke using the Click-it EdU Alexa Fluor 647 Imaging Kit

(ThermoFischer) on 40 µm free floating coronal sections.

Fluorescence microscopy and histological analysis

Imaging of brain sections three weeks after stroke was performed with an Axio

Scan.Z1 slide scanner (Zeiss) and an Olympus FV1000 laser scanning confocal

microscope equipped with 10x, 20x and 40x objectives. Images were processed

using Zen 2 software, Fiji (ImageJ) and Adobe Illustrator CS6. First, stroke sizes

were compared between animals in 40 µm coronal sections stained with DAPI.

The dorso-ventral, medio-lateral, and anterior-posterior stroke extent was used to

modulate a precise ellipsoid with the coordinates relative to Bregma.

All other analysis steps were performed in the same region of interest (ROI), a

peri-infarct region distal to the stroke core with a width of 300 µm. All histological

samples received a code and were analyzed blinded. To assess post-stroke

angiogenesis an ImageJ (Fiji) script was established to automatically calculate 1)

area fraction of blood vessels, 2) number of blood vessels, 3) number of

branches and junctions, 4) distance between blood vessels and 5) vessel

distribution. Images were pre-processed, binarized and processed step by step

as shown in Supplementary Fig. 2A. Newly generated blood vessels were

identified by quantifying the amount of CD31/EdU double-positive cells within the

region of interest. The coverage of CD13/CD31 cells was assessed by the ratio

between the binarized signals from CD13+ to CD31+ cells. Signals from Iba1+

and GFAP+ were assessed by relative fluorescence intensity in the ROI. NeuN+,

interneurons (PV+, SST+, VIP+), Caspase3+, DCX+ EdU+ cells were counted in

the ROI. Synaptic markers (SynP+, vGAT+, vGLUT2+) as well as Nf160+

structures were measured by relative fluorescence intensity.

Enzyme-linked immunosorbent assay

For the determination of anti-Nogo-A antibody titers peripheral blood was

collected by puncturing of the tail vein on day 8, 15, and 22 after stroke (and

pump implantation) and serum was obtained by centrifugation. The titer of anti-

8

Nogo-A antibodies was determined by enzyme-linked immunosorbent assay.

Briefly, rat delta20 fragment was coated on a 96-well EIA/RIA assay microplate

(Corning, USA) at a concentration of 3 µg/ml. After blocking with 5% Rapilait

dissolved in TBS-0.1% Tween20 the plates were incubated with the test serum.

Specific binding was detected with IgG-specific horseradish peroxidase-

conjugated goat anti-mouse antibody. Signal was developed by the addition of

Pierce™ 3,3',5,5' tetramethylbenzidine (TMB) substrate (Thermo Fisher) and the

absorption at 450 nm was measured on a Tecan Spark® plate reader (Tecan).

Between each incubation step the plates were washed thoroughly with TBS

containing 0.1% Tween20.

Cell Culture

Human vascular endothelial cells (HUVECs) were isolated from umbilical veins

as described previously and cultured on fibronectin-coated flasks. The

endothelial-cell growth medium (ECGM, PromoCell) contained a supplemental

mix with 2% FBS (SupplementMix C-39215, Promocell). To embed the cells in

the glycosaminoglycan-polyethylene glycol (GAG-PEG) hydrogel, the cells were

detached using Accutase (Sigma-Aldrich) for 5 min at 37 °C and resuspended in

ECGM medium at a final concentration of 40x106 cells/ml.

Hydrogel formation

Biohybrid GAG-PEG hydrogels for in vitro HUVEC morphogenesis studies were

prepared as described previously with slight modifications. In brief, a degradable

starPEG-MMP conjugate (MW 16,489) was dissolved in HUVEC culture medium

at a final concentration of 0.75 mM and a heparin-maleimide conjugate (MW

15,000) was dissolved in the medium and supplemented with the adhesive

peptide CWGGRGDSP (cRGD, MW 990) to reach a final concentration of 1 mM

and 2 mM, respectively. Thereafter, the heparin-RGD mixture was (non-

reactively) functionalized with VEGF165 (PeproTech, USA), SDF1 (Miltenyi

Biotec, Germany) and FGF2 (Miltenyi Biotec, Germany) each at a final

concentration of 5 µg/ml. The proteins investigated were added individually or in

9

combination at the indicated final concentrations (Δ20 and Δ20 scrambled at 0,8

µg/ml; anti-Nogo-A Ab and rat Myelin at 10 µg/ml) before adding a HUVEC

suspension to generate a cell-heparin conjugate mixture. For the formation of

hydrogels, the cell-heparin conjugate mixture was mixed with the starPEG

conjugate solution in a 1:1 volume ratio to form 15 μl hydrogel droplets which

were casted onto hydrophobic 12 well chamber slides (Ibidi, Germany). Following

the in situ crosslinking, the gels were immediately immersed in cell culture

medium and 70% of the medium was exchanged with fresh medium every other

day. Three to five replicates per condition were produced for all seven conditions

investigated. On day 3 and day 7 the samples were fixed with 4%

paraformaldehyde for 15 min at RT and immunostained thereafter.

Immunocytochemistry for in vitro experiments

After fixation and washing with PBS the samples were permeabilized using 0.1%

TritonX-100 for 10 min. The samples were blocked with 2% BSA and 2% goat

serum in PBS for 3 hours and incubated over night with the primary antibodies

mouse anti-CD31 (BD 555444; 1:100) at 4 °C. After 3 washes with PBS/0.1%

BSA, the secondary antibody Alexa Flour 488 goat anti-mouse (Life

Technologies 1:200) was added and incubated over night at 4 °C. Samples were

washed and incubated with Hoechst 33342 (Life Technologies; 1:200) over night

at 4 °C. Fluorescent images were captured using a Dragonfly Spinning Disc

confocal microscope (Andor). At least 3 images were taken at different positions

within each hydrogel. Fields and samples for imaging and quantification were

chosen randomly for all experiments.

Statistics

Statistical analysis was performed using RStudio. Sample sizes were designed

with adequate power according to our previous studies and to the literature. All

data was tested for normal distribution by using the Shapiro-Wilk test. Normally

distributed data was tested for differences with a two-tailed unpaired one-sample

t-test to compare differences between two groups. Non-normally distributed data

10

was tested with a Mann-Whitney U test. Multiple comparisons were assessed

using ANOVA followed by Dunnett’s post hoc test. Data are expressed as mean

± SEM and statistical significance was defined as *p < 0.05, **p < 0.01, and

***p < 0.001.

11

SI Figures

Fig S1. Measurement of total stroke volume (left panel) and areas at multiple

distances relative to bregma (right panel) in wild type, S1PR2-/-, Nogo-A-/-, Ctrl ab

and anti-Nogo-A Ab mice 28 days after stroke.

12

Fig S2: (A) Schematic representation of the image analysis procedure in ImageJ.

(B) Quantification of vascular area fraction, blood vessel length, branches nearest

neighbor distance and variability in intact and lesioned cortex in wildtype animals

three weeks following stroke. (C) Quantification of vascular area fraction, blood

13

vessel length, branches, nearest neighbor distance and variability in the

contralesional hemisphere between wildtype, S1PR2-/-, Nogo-A-/-, Crtl-Ab and a-

Nogo-A antibody treated animals. Representative fluorescent images of the

contralesional hemisphere, Scale bar 100 µm. (C, D) Representative fluorescence

images of GFAP and Iba1 immunostainings to assess scar formation and

inflammation. Scale bars 100 µm.

Fig. S3: Quantification of (A) vascular area fraction and (B) number of vascular

branches 10 days following stroke injury between Ctrl-Ab and a-Nogo-A antibody

treated animals.

14

Fig S4: (A) Experimental timeline of antibody administration after stroke. (B)

Quantification of ELISAs coated with Δ20 a functional domain of Nogo-A to detect

anti-Nogo-A antibody titers in serum 0, 8, 15 and 22 days post injury (dpi). Control

antibody did not show any binding to target antigen as expected.

15

Fig. S5: (A) Functional assessment of blood perfusion in different cortical regions

in non-stroked animals. (B) Wildtype (N= 4) and Nogo-A–/– (N= 4) mice were tested

in nine sensorimotor regions for differences in naïve cerebral blood flow. Data are

mean ± SEM (Student’s t test). Asterisks indicate significance: *P < 0.05, **P <

0.01, ***P < 0.001.

16

Fig. S6: (A, B) Motor performance in sham operated animals tested at baseline,

3, 7, 14, 21 days after sham-surgery (arrows) in the horizontal ladder and cylinder

test. (C) Ratios of paw preference, dragging and symmetry in the cylinder test. (D)

Ratio of forelimb and hindlimb errors per total number of steps in the horizontal

ladder test. Data are mean ± SEM (1-way ANOVA with Dunnett’s post hoc test).

Asterisks indicate significance: *P < 0.05, **P < 0.01, ***P < 0.001.

17

Fig. S7: Measurement of stroke volume (left panel) and area (right panel) of Nogo-

A and wildtype animals either treated with anti-VEGF (Avastin) antibodies or Ctrl

Ab.

18

Fig S8: Quantification of (A) vascular area fraction and (B) number of vascular

branches in male wildtype and Nogo-A-/- mice three weeks following injury.

19

Fig S9. Graphical abstract. The neurite outgrowth inhibitor Nogo-A has been

previously shown to be a negative regulator of developmental CNS angiogenesis.

In this study, we identify its inhibitory role in post-stroke angiogenesis. Blocking the

Nogo-A pathway results in increased vascular repair in the peri-infarct zone with

improved tissue recovery and motor function following stroke. These results may

form a basis for future studies on clinical applications to improve the functionality

of affected CNS tissue after ischemia.

20

SI Tables

Table S1: List of primary antibodies

Antigen Target Host Dilution Company

CD13 Pericytes Goat 1:200 R&D Systems

CD31 Vascular ECs Rat 1:50 BD

Biosciences

Cleaved Caspase

3

Apoptotic cells Rabbit 1:200 Abcam

Glial fibrillary

acidic protein

(GFAP)

Astrocytes Rabbit 1:200 Dako

Iba1 Microglia Rabbit 1:500 Wako

NeuN Mature neurons Mouse 1:200 EMD Millipore

Neurofilament

160 (NF160)

Axons Mouse 1:200 Life

Technologies

Laura Nogo-A Rabbit 1:200 Schwab Group

Parvalbumin (PV) Ca2+ binding protein

(GABAergic

interneurons)

Mouse 1:1000 Swant

EDG-5 S1PR2 Mouse 1:200 Santa Cruz

Somatostatin

(SST)

Inhibitory peptide

hormone

(GABAergic

interneurons)

Rat 1:200 EMD Millipore

Synaptophysin Presynaptic

membrane protein

Rabbit 1:100 Synaptic

Systems

Vasoactive

intestinal peptide

(VIP)

GABAergic

interneurons

Rabbit 1:200 Abcam

21

vGat Vesicular GABA

transporter

Mouse 1:500 Synaptic

Systems

vGlut2 Vesicular glutamate

transporter 2

Rabbit 1:500 Synaptic

Systems

5-HT Serotonin Rabbit 1:200 Immunostar

TH Tyrosinhydroxylase/

Dopamine

Mouse 1:200 Immunostar

22

Table S2: List of secondary antibodies.

Reactivity Host Conjugate Dilution Company

Anti-mouse Goat Alexa Fluor 488 1:500 ThermoFischer

Anti-mouse Goat Cy3 1:500 ThermoFischer

Anti-rabbit Goat Cy 3 1:500 ThermoFischer

Anti-rat Goat Cy 3 1:500 ThermoFischer

Anti-rat Goat Alexa Fluor 647 1:500 ThermoFischer

Anti-goat Donkey Alexa Fluor 488 1:500 ThermoFischer

Anti-rat Donkey Cy 3 1:500 ThermoFischer