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ONLINE SUPPLEMENT
SUPPLEMENTARY METHODS
Drug storage and intranasal dosing. LM22A-4 was stored as a 10x solution (5 mg/ml in saline)
at -80 °C. Aliquots were diluted with saline within 1 hour prior to administration. Each mouse
was weighed weekly and the week’s dosage adjusted accordingly. For intranasal dosing, mice
were manually restrained and inverted, and a P20 pipetman (Gilson) was used to place one drop
at a time on one nostril until the liquid level fell beneath the external nares, and nostrils
alternated each day of dosing.
Hypoxic-ischemic stroke. Mice were anesthetized using 2% isoflurane in 100% oxygen and
body temperature was maintained at 37oC. To induce hypoxic-ischemic stroke the right common
carotid artery was permanently ligated. Mice were then placed in a 37°C chamber containing 8%
oxygen and 92% nitrogen for 25 minutes.
Western blots. Mice underwent hypoxic-ischemic stroke and those exhibiting >17% error on the
horizontal ladder test were treated with daily LM22A-4 from days 3 to 10 after stroke, then
sacrificed at 1 hour after the last dose of LM22A-4 or vehicle. Ipsilateral striatum was isolated,
homogenized in cell lysis buffer, sonicated, and equalized as described.1 Protein extracts were
subjected to Western blot analysis using standard techniques. Blots were probed with antibodies
recognizing pTrkB-Y817 (rabbit mAb 1:30,000, Epitomics), TrkB (rabbit polyclonal antibody
1:2500, Millipore), and HRP goat anti-rabbit (1:10,000, Pierce Biotechnology). Optical densities
were measured using UN-Scan-it Gel & Graph Digitizing Software Ver. 6.14. Ratios were
calculated as pTrkB over full length TrkB receptor and data was normalized to saline-treated
stroked mice.
Mass spectroscopy. Mice underwent hypoxic-ischemic stroke and those exhibiting >17% error
on the horizontal ladder test were treated with daily LM22A-4 from days 3 to 10 after stroke,
then sacrificed at 1 hour after the last dose of LM22A-4 or vehicle. Ipsilateral hemispheres were
obtained and immediately frozen on dry ice. The tissue extraction and LC-MS/MS analysis were
performed by Absorption Systems (Exton, PA) as described.2 Brain homogenate from an
untreated C57BL/6J mouse was used for the standard curve.
Behavioral tests. Prior to behavior training mice were handled three times, for 5 minutes each
day. Behavioral apparatuses were cleaned in between mice with Simple Green. The same
individual performed each behavioral test for the duration of each study, and this was never an
individual who was also dosing mice. All individuals dosing and testing mice were blinded
throughout the study, and dosing was performed after behavioral testing had been completed for
the day.
Ladder test. The hypoxic-ischemic stroke model produces a deficit in this test in mice.3 A
horizontal ladder was custom made from plexiglass and measured 30 inches across with rungs
spaced 0.25 inches apart.4 It was supported on clean mouse cages and runs were filmed from
underneath. To run each trial mice were removed from their home cage and placed at the end of
the ladder, next to a bright light. They then walked across the ladder back towards their home
cage. Mice underwent four training sessions on the ladder in the four weeks prior to stroke. For
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the first session mice were sent across the ladder 3 times, then twice for the second training
session, and only once for third and fourth training sessions. Baseline was obtained from the last
training run. Post stroke testing was performed with one run per mouse. Ladder test performance
was scored as the percent correct steps and the total number of left front foot missteps.
Automated gait analysis. Mice were trained on the automated gait analysis apparatus (Noldus
Catwalk). This test was evaluated in a prior study (Pollak and Buckwalter, unpublished data),
and limb swing speed and stride length were pre-specified variables for this study based on their
high correlation with stroke size in the prior study. Mice were trained once a week for three
weeks prior to stroke, and then tested weekly on day 5, 12, 19, 26, and 33 after stroke. For
analysis, each mouse had to complete the run within 8 seconds and have at least 4 consecutive
strides without pausing. Runs where the animal went backwards, reared, or paused excessively
were disregarded. Two successful runs per mouse were obtained for each session.
Rotarod. This test has been previously used to follow deficits in the hypoxic-ischemic stroke
model.5 Pre-stroke training was performed three times a week for four weeks prior to stroke on a
mouse rotarod (Rotor-RodTM, San Diego Instruments). For training day 1 each mouse was placed
on the stationary rotarod for 1 minute to allow it to get acclimated to the rotarod and the testing
environment. For training day 2 the rotarod was turned on and set to run at a constant 5 rpm.
Each mouse was placed on the moving rotarod until it could stay on the rotating rotarod for 1
minute continuously. For subsequent training and testing days the rotarod was set to accelerate
from 0 to 5 rpm in the first ten seconds, then to 10 rpm over the next 290 seconds. Each mouse
performed the test twice in a row and then the cohort was cycled through and the test repeated,
for a total four trials per training or testing day. The length of time each mouse was able to stay
on the rotarod, up to 300 seconds was recorded. Post stroke rotarod testing was performed
weekly for 10 weeks after stroke, beginning on day 2 after stroke.
Stratification. Stratification was performed by one individual (MB) who assigned the stroked
mice into two groups, A and B, that had day 1 ladder scores that were evenly distributed between
groups, using the rotarod results from day 2 as a tiebreaker where needed. Sham mice were also
assigned to groups A and B. A second individual (TY) prepared the drug and, without knowing
their functional test results, assigned groups A and B to either LM22A-4 or saline. Dosing was
then performed by individuals (JH, KPD, KTL, JZG, EC) who did not know whether A or B was
the drug or the placebo control.
Perfusion and brain processing. Mice were sedated with 3.8% chloral hydrate perfused with
0.9% heparinized saline (10U/mL). The ipsilateral hemisphere was processed in 4%
paraformaldehyde (PFA) in phosphate buffer, while the contralateral hemisphere was processed
for Golgi-Cox impregnation. Ipsilateral hemispheres were fixed in 4% PFA for 24hr, rinsed with
PBS, and sunk in 30% sucrose in PBS. Coronal brain sections 40µM thick were cut with a
freezing sliding microtome (Microm HM430) into 24 sequential tubes, so that each tube
contained every 24th section, and stored in cryoprotective medium at -20°C. The contralateral
hemispheres were impregnated with Modified Golgi-Cox Staining Solution for 8 days at room
temperature in the dark. The brains were then rinsed twice with dH2O and then transferred to
30% sucrose in dH2O for 3 days at 4˚C, with the solutions changed after the first initial 12 hours.
The brains were then sectioned coronally using a vibratome (Leica VT10005) at 150µM in 30%
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sucrose in dH2O and mounted in 0.3% gelatin in dH2O. Once the gelatin solidified, the slide was
immersed in 40% sucrose in dH2O and allowed to dry for 72hrs in the dark. Slides were then
rinsed three times in dH2O for ten minutes, immersed in Developing Solution in for 7 minutes,
and rinsed three times in dH2O for ten minutes each. The slides were then dehydrated through
graded ethanol, followed by Histoclear (HS-200, National Diagnostics) and immediately coverslipped with DPX mounting medium (EMS).
Immunohistochemistry,
Hemisphere
size
quantification,
and
stereology.
Immunohistochemistry was performed on PFA fixed, free-floating coronal brain sections (40µm)
using standard techniques. All analysis was performed in a blinded fashion. The following
primary antibodies were used: anti-NeuN (1:1000, Cat# MAB377, Millipore), anti-BrdU
(1:5000, Cat# AB6326, Abcam), biotinylated anti-BrdU (1:500, Cat# AB2284, Abcam), antidoublecortin (1:500; Cat# SC8066, Santa Cruz Biotechnology); anti-GFAP (1:1000, Cat# Z0334,
DakoCytomation), anti-MHC II (1:500, Cat# 553621, BD Pharmingen), anti-CD68 (1:1000,
Cat#MCA1957S, Serotec); anti-PECAM1/CD31 (1:300, Cat# 550274, BD Pharmingen).
Hemisphere size was determined by tracing the remaining hemisphere size in every 24th section,
spaced 960 µm apart, stained with Cresyl Violet. Stereological estimation of total BrdU+ cells
was done on coronal sections spaced 480 µm apart and spanning the entire cortex and striatum
(Stereo Investigator, MBF Bioscience). To determine the percentage of double-positive
NeuN+/BrdU+ and PECAM+/BrdU+, the same areas were quantified in two sections spaced
960µm apart. From each section, 100 BrdU cells in each of the penumbral cortex, dorsolateral
and ventral striatum were analyzed (40x objective). For doublecortin, the percent area covered in
the entire section was measured in every 12th section spanning the entire brain. For GFAP, MHC
II, and CD68, we quantified percent area covered in one 10x field of the penumbral cortex. For
PECAM, we quantified percent area covered in one 10x field of the penumbral cortex from 3-5
coronal sections spaced 960 µm apart.
Analysis of Golgi stained neurons. Morphological reconstruction and analysis of pyramidal
neurons in cortical layers 5/6 and 2/3, and medium spiny neurons in the dorsolateral striatum
were performed using Neurolucida software (MBF Biosciences). Five neurons per region per
mouse were analyzed. Dendritic spines were identified as protrusions along the dendrite axis and
were traced at 100x objective. The spine densities of five secondary dendrites from layer 2/3
pyramidal neurons were analyzed using NeuroExplorer software (MBF Biosciences).
Axonal Sprouting. Mice underwent dMCAO surgery at 5 months of age and were injected with
300nL 10% solution of biotinylated dextran amine (BDA; 10,000MW, Invitrogen) into the barrel
cortex (-1.0mm bregma, 3.5.mm lateral) two weeks later. They were dosed with 0.22mg/kg/day
LM22A-4 from day 3 to 21 after stroke. To measure axonal sprouting, 4 coronal sections that
were 160 µm apart and centered on the injection core were used per animal. Streptavodin-488
(1:200, Cat#86493, Jackson ImmunoResearch) was used to detect BDA-labeled axons. Using a
40x objective, confocal stacks were taken in four areas of interest: contralesional dorsal striatum,
ipsilesional dorsal striatum, penumbra, and the corpus callosum at midline.
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SUPPLEMENTARY FIGURES AND FIGURE LEGENDS
Supplementary Figure 1. Histopathological features of hypoxic-ischemic stroke.
Photomicrographs of Cresyl violet stained sequential coronal sections spaced 960 µm apart from
two mice with typical lesion characteristics, sacrificed 6 weeks after stroke. Four sections are
shown from each mouse. Injury occurred in large areas of cortex, hippocampus, and striatum
ipsilateral to carotid occlusion, producing both areas of frank scarring and marked atrophy at this
timepoint. (A) LM22A-4-treated mouse (B) saline-treated mouse. *, hole made by a needle that
was used to mark the non-stroked side prior to sectioning and after sacrifice.
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Supplementary Figure 2. Horizontal ladder testing on day 1 and rotarod testing on day 2
after surgery correlate significantly with stroke size, and ladder test results are superior.
(A) Left front error on the horizontal ladder test. (B) Rotarod performance compared to baseline.
n = 14 in hypoxic-ischemic stroke group and 6 shams. P values and R2 values are from linear
regression using all mice.
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Supplementary Figure 3. Angiogenesis is not increased by LM22A-4 treatment. (A)
Representative immunostaining for BrdU and PECAM. Scale bar, 20 µm. (B) TrkB pathway
stimulation with LM22A-4 did not significantly alter the total number of BrdU+/PECAM+
double-positive cells in affected (dorsolateral) or unaffected (ventral) regions of the striatum, or
in penumbral cortex at ten weeks after stroke. n = 10 per group (C) Representative
photomicrographs of PECAM immunostaining in penumbral cortex. Scale bar, 200 µm. (D)
Quantification of total PECAM immunostaining in penumbral cortex did not reveal significant
differences in blood vessel density. n = 7-9 per group; Graphs, means ± SEM.
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Supplementary Figure 4. LM22A-4 treatment did not affect markers of astrogliosis or
immune response at 10 weeks after stroke. (A) Representative photomicrograph of peri-infarct
cortex immunostained with GFAP. Graph is percent area covered by GFAP immunostaining. (B)
Representative photomicrograph of peri-infarct cortex immunostained with the activated
microglial/macrophage marker CD68. Graph is percent area covered by CD68 immunostaining.
(C) Representative photomicrograph of peri-infarct cortex immunostained with MHC II. Graph
is percent area covered by MHC II immunostaining. Graphs are mean ± SEM. Scale bars, 100
µm.
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Supplementary Figure 5 - LM22A-4 exerted no effect on neuronal arborization of striatal
medium spiny neurons and layer 2/3 or 5/6 motor cortex pyramidal neurons contralateral
to stroke. Golgi staining was performed on the contralateral hemisphere of mice sacrificed 72
days after stroke or sham surgery. Motor cortex pyramidal neurons in layers 5/6 and 2/3, and
medium spiny neurons in the dorsolateral striatum were traced using Neurolucida software. Five
neurons per region per mouse were traced. No significant differences were observed between
groups in number of primary dendrites, average dendrite nodes, total dendrite length, or average
dendrite ends. n = 6-8 mice per group. Graphs are mean ± SEM.
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Supplementary Figure 6. LM22A-4 treatment did not increase axonal sprouting after
stroke. Mice underwent dMCAO stroke and were then treated with LM22A-4 0.22 mg/kg or
saline from days 3-21 after stroke. BDA was injected into barrel cortex contralateral to the stroke
on day 14, one week before sacrifice. (A,B) Graphs of quantification of BDA-containing axons
in (A) ipsilesional vs. contralesional dorsolateral striatum and (B) penumbral cortex vs. the
central commisure of the corpus callosum. Graphs, mean ± SEM.
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