Download Blood-Spinal Cord Barrier Alterations in Subacute and Chronic

Journal of Neuropathology & Experimental Neurology Advance Access published June 9, 2016
J Neuropathol Exp Neurol
Vol. 0, No. 0, 2016, pp. 1–16
doi: 10.1093/jnen/nlw040
ORIGINAL ARTICLE
Blood-Spinal Cord Barrier Alterations in Subacute and
Chronic Stages of a Rat Model of Focal Cerebral Ischemia
Svitlana Garbuzova-Davis, PhD, DSc, Edward Haller, Naoki Tajiri, PhD, Avery Thomson, MS,
Jennifer Barretta, MS, Stephanie N. Williams, BS, Eithan D. Haim, MS, Hua Qin, MS,
Aric Frisina-Deyo, BS, Jerry V. Abraham, BS, Paul R. Sanberg, PhD, DSc,
Harry Van Loveren, MD, and Cesario V. Borlongan, PhD
Abstract
From the Center of Excellence for Aging & Brain Repair, Morsani College
of Medicine, University of South Florida, Tampa, Florida (SG-D, NT,
AT, JB, SNW, EDH, HQ, AF-D, JVA, PRS, CVB); Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of
South Florida, Tampa, Florida (SG-D, PRS, HVL, CVB); Department of
Molecular Pharmacology and Physiology, Morsani College of Medicine,
University of South Florida, Tampa, Florida (SG-D); Department of
Pathology and Cell Biology, Morsani College of Medicine, University of
South Florida, Tampa, Florida (SG-D, PRS); Department of Integrative
Biology, University of South Florida, Tampa, Florida (EH); Department
of Psychiatry, Morsani College of Medicine, University of South Florida,
Tampa, Florida (PRS).
Send correspondence to: Svitlana Garbuzova-Davis, PhD, DSc, Center of
Excellence for Aging and Brain Repair, Department of Neurosurgery and
Brain Repair, University of South Florida, Morsani College of Medicine,
12901 Bruce B. Downs Blvd., MDC 78, Tampa, FL 33612; E-mail:
[email protected]
This study was supported by the Center for Excellence and Brain Repair,
Department of Neurosurgery and Brain Repair at the University of South
Florida.
Key Words: Astrocytes, Autophagosomes, Blood-spinal cord barrier (BSCB), Middle cerebral artery occlusion (MCAO), Stroke,
Subacute and chronic diaschisis, Tract demyelination.
INTRODUCTION
Strokes are the fifth leading cause of death in the United
States (1); they are classified as ischemic, intracerebral hemorrhagic, or subarachnoid hemorrhagic; approximately 87% of
strokes are ischemic (2). Stroke survivors are at risk for longterm disability; 50% suffer hemiparesis and about 30% are dependent in daily living activities (3).
Cerebrovascular ischemic injuries occur in a timedependent manner and are categorized as acute (minutes to
hours), subacute (hours to days), or chronic (days to months).
Numerous studies have identified blood-brain barrier (BBB)
disruption after focal cerebral ischemia (4–8). Increased BBB
permeability has been shown in patients with acute ischemic
stroke (9) and in a rodent model of middle cerebral artery occlusion (MCAO) (10–13), likely due to decreased endothelial
cell (EC) tight junction integrity (7). Moreover, BBB leakage
may persist for 3–5 weeks after the initial insult (14, 15). This
prolonged barrier permeability might extend the ischemic tissue injury in association with brain edema and swelling (16).
Pathophysiological processes such as alterations in blood flow
and metabolism in brain regions contralateral to the initial
ischemic insult have also been identified in patients up
to 14 days after cerebral infarcts (17, 18). Brain injuries
remote from focal ischemic lesions have been termed
“transhemispheric diaschisis” (17, 19–22). Crossed cerebellar,
thalamic, and cortical diaschisis have been demonstrated at
acute, subacute, and chronic post-stroke stages; these types of
diaschisis have been correlated with clinical and recoveryrelated outcomes in patients (23–25). In a rat MCAO model of
transient ischemia (tMCAO), transcortical diaschisis was
identified in the neocortex, suggesting widespread degeneration of corticostriatal connections (26, 27). Ipsilateral diaschisis in connected cortical regions distant from the initial
damage was demonstrated in a focal cortical rat ischemia
C 2016 American Association of Neuropathologists, Inc. All rights reserved.
V
1
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
We previously demonstrated blood-brain barrier impairment in remote contralateral brain areas in rats at 7 and 30 days after transient
middle cerebral artery occlusion (tMCAO), indicating ischemic diaschisis. Here, we focused on effects of subacute and chronic focal
cerebral ischemia on the blood-spinal cord barrier (BSCB). We
observed BSCB damage on both sides of the cervical spinal cord in
rats at 7 and 30 days post-tMCAO. Major BSCB ultrastructural
changes in spinal cord gray and white matter included vacuolated
endothelial cells containing autophagosomes, pericyte degeneration
with enlarged mitochondria, astrocyte end-feet degeneration and
perivascular edema; damaged motor neurons, swollen axons with
unraveled myelin in ascending and descending tracts and astrogliosis
were also observed. Evans Blue dye extravasation was maximal at 7
days. There was immunofluorescence evidence of reduction of
microvascular expression of tight junction occludin, upregulation
of Beclin-1 and LC3B immunoreactivities at 7 days and a reduction
of the latter at 30 days post-ischemia. These novel pathological alterations on the cervical spinal cord microvasculature in rats after
tMCAO suggest pervasive and long-lasting BSCB damage after
focal cerebral ischemia, and that spinal cord ischemic diaschisis
should be considered in the pathophysiology and therapeutic approaches in patients with ischemic cerebral infarction.
Garbuzova-Davis et al
MATERIALS AND METHODS
Ethics Statement
All described procedures were approved by the Institutional Animal Care and Use Committee at USF and were conducted in compliance with the Guide for the Care and Use of
Laboratory Animals.
All animals used in the study were obtained from The
Jackson Laboratory, Bar Harbor, Maine. Fifty-seven Sprague
Dawley adult male rats weighting 262.8 6 2.32 g were randomly assigned to 1 of 2 groups: MCAO (n ¼ 32) or control
(n ¼ 25). All rats were housed in a temperature-controlled
room (23 C), and maintained on a 12:12 hour dark: light cycle
(lights on at 06:00 AM). Food and water were available ad libitum. The study design is schematically represented in
Figure 1.
Middle Cerebral Artery Occlusion
tMCAO was induced using the intraluminal filament
technique previously described in detail (29, 30, 36, 37).
Based on our prior standardization of this model there is at
least 80% reduction in regional cerebral blood flow (as determined by laser Doppler [Perimed, J€arf€alla, Sweden]) in the experimental animals during the occlusion period (38–40).
2
FIGURE 1. Schematic representation of study design.
Briefly, the tip of the filament was customized using dental cement (GC Corporation, Tokyo, Japan). Body temperature was
maintained at 37 6 0.3 C during the surgical procedures.
A midline skin incision was made in the neck with subsequent
exploration of the right common carotid artery (CCA), the external carotid artery, and internal carotid artery. A 4-0 monofilament nylon suture (27.0–28.0 mm) was advanced from the
CCA bifurcation until it blocked the origin of the middle cerebral artery (MCA). Animals were allowed to recover from anesthesia during MCAO. At 60 minutes after MCAO, animals
were reanesthetized with 1%–2% isoflurane in nitrous oxide/
oxygen (69%/30%) using a facemask and reperfused by withdrawal of the nylon thread. A midline incision was made in
the neck and the right CCA was isolated. The incisions were
then closed and the animals were allowed to recover from
anesthesia.
Perfusion and Tissue Preparation
Seven or 30 days after reperfusion, tMCAO rats and
controls were killed under CO2 inhalation and perfused transcardially with 0.1 M phosphate buffer ([PB] pH 7.2), followed
by 4% paraformaldehyde in PB solution under pressure controlled fluid delivery at 85 mm Hg. The tMCAO rats (7 days
post-tMCAO: n ¼ 10; 30 days post-tMCAO: n ¼ 9) and controls (for 7 days post-tMCAO: n ¼ 6; for 30 days posttMCAO: n ¼ 7) were intravenously injected with 1 mL of 2%
Evans Blue dye ([EB] Sigma-Aldrich, St. Louis, MO) in saline
solution via the jugular vein 30 minutes prior to perfusion, as
previously described (29, 30). The surgical procedure was performed in tMCAO and control rats using the same protocol,
including exposure to anesthesia. Rats assayed for Evans Blue
extravasation received only PB solution. After perfusion, rat
cervical spinal cords were rapidly removed from tMCAO rats
and controls for Evans Blue extravasation assay. Rats assayed
for electron microscope analysis were randomly chosen from
the 7 and 30 days post-tMCAO groups (tMCAO, n ¼ 3;
control, n ¼ 2). Rat cervical spinal cords were immediately
removed, labeled on right spinal side, and fixed in 4%
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
model (28). Recently, we showed BBB alterations not only in
the ipsilateral hemisphere, but also in contralateral brain areas,
7 and 30 days after tMCAO in rats, indicating subacute and
chronic ischemic diaschisis (29, 30). Damaged ECs containing
numerous autophagosomes and vascular leakage were identified in the contralateral striatum, motor, and somatosensory
cortices. This pervasive and long-lasting BBB damage could
have major effects on brain functions.
Spinal cord injury may also result from cerebral infarcts.
Neuropathological changes have been demonstrated in the
lumbar spinal cord, contralateral to focal cerebral ischemia in
rodent models (31–33). The glial cell response in the spinal
cord was accompanied by increased expression of various
proinflammatory cytokines and markers of oxidative stress
24–72 hours after permanent MCAO (32, 34). It has been proposed that inflammatory changes occurring not only in remote
cerebral areas but also in the spinal cord distant from initial
focal ischemic insult might be involved in the pathogenesis of
secondary neuronal damage linked to antero- and retrograde
degeneration (35).
Although the importance of inflammatory processes in
post-cerebral infarct spinal cord pathology has been shown,
the competence of the blood-spinal cord barrier (BSCB) following ischemic cerebral infarcts has not to our knowledge
been investigated. Impairment of any BSCB component may
lead to an increasingly toxic environment in the spinal cord. A
better understanding of the BSCB function could encourage
the development of more effective therapeutics for patients
with strokes. We hypothesized that damage from focal cerebral ischemia might extend farther from the initial insult, that
is, to the spinal cord. The aim of this study was to evaluate spinal cord diaschisis in subacute and chronic rat models of focal
cerebral ischemia.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
paraformaldehyde in 0.1 M PB for 16–24 hours at 4 C. The
next day, spinal cords were cut into 1-mm slices, mapped
against a diagram of the whole section according to a rat spinal
cord atlas (41) and coded from the slices of both spinal cord
sides in gray (ventral horn) and white (lateral funiculus) matter. Tissues were then fixed overnight in 2.5% glutaraldehyde
in 0.1M PB (Electron Microscopy Sciences, Inc., Hatfield,
PA) at 4 C and stored for further electron microscope processing. Remaining rats were perfused and their cervical
spinal cords immediately removed, fixed intact in 4% paraformaldehyde in 0.1 M PB for 24–48 hours and then cryoprotected in 20% sucrose in 0.1 M PB overnight. Coronal spinal
cord tissues were cut at 30 lm in a cryostat, thaw-mounted
onto slides, and stored at 20 C for immunohistochemical
and histological analyses.
BSCB Permeability
Electron Microscopy
Ultrastructural analyses were performed in rats 7 and 30
days after tMCAO. Briefly, spinal cord tissue samples were
postfixed in 1% osmium tetroxide (Electron Microscopy Sciences, Inc.) in 0.1M PB for 1 hour at room temperature (RT)
and then dehydrated in a graded series of acetone dilutions.
Tissues were transferred to a 50:50 mix of acetone and LX112
epoxy resin embedding mix (Ladd Research Industries, Burlington, VT) and infiltrated with the mix for 1 hour. The tissues were then transferred to a 100% LX112 embedding mix
and infiltrated with fresh changes of this embedding mix. The
tissues were further infiltrated overnight in fresh embedding
medium at 4 C. On the following day, the tissues were
embedded in a fresh change of resin in tissue capsules. The
blocks were polymerized at 70 C in an oven overnight. The
blocks were trimmed and then sectioned with a diamond knife
on an LKB Huxley ultramicrotome. Thick sections cut at 0.35
mm were placed on glass slides and stained with 1% toluidine
blue stain. Thin sections were cut at 80–90 nm, placed on copper grids, and stained with uranyl acetate and lead citrate.
For analysis of BSCB ultrastructure, microvessels in the
right and left gray and white matter of cervical spinal cords of
7 and 30 days post-tMCAO rats and controls were examined
by an investigator blinded to the animal groups using mapped/
coded sections and photographed with an AMT ActiveVu
XR 16 digital camera (Advanced Microscopy Techniques,
Woburn, MA) attached to a FEI Morgagni transmission electron microscope (FEI, Inc., Hillsboro, OR) at 60 kV.
Immunohistochemistry
Spinal cord tissues were preincubated with 10% normal
goat serum and 0.3% Triton 100X in phosphate-buffered saline (PBS) for 60 minutes at RT. Rabbit polyclonal antioccludin antibody (occludin, 1:50, Abcam, Cambridge, MA)
was applied on the slides overnight at 4 C. The next day, the
slides were rinsed in PBS and incubated with secondary goat
anti-rabbit antibody conjugated to fluorescein isothiocyanate
(FITC) (1:600, Thermo Fisher Scientific, Waltham, MA)
for 2 hours. After rinsing, slides were coverslipped with Vectashield containing 40 ,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) and examined using an
Olympus BX60 epifluorescence microscope. Immunoexpression of occludin was observed in each image and qualitatively
analyzed.
Double immunohistochemical staining for autophagosomes using Beclin-1 and LC3B biomarkers was performed to
detect an autophagy response within capillary ECs in the ventral horns of both sides of the cervical spinal cords. Briefly,
serial spinal cord tissue sections were preincubated in blocking solution as described above and then incubated overnight
with rabbit polyclonal anti-Beclin-1 antibody (Beclin-1,
1:500, Pierce, Rockford, IL). The next day, the slides
were rinsed in PBS and incubated with secondary goat antirabbit antibody conjugated to FITC (1:500, Thermo Fisher
Scientific) for 2 hours. After rinsing, slides were again preincubated with blocking solution as described above for 60 minutes at RT and rabbit polyclonal anti-LC3B antibody (LC3B,
1:250, Abcam, Cambridge, MA) was applied on the slides
overnight at 4 C. The slides were then rinsed in PBS and incubated with secondary goat anti-rabbit antibody conjugated to
Rhodamine (1:1200, Thermo Fisher Scientific) for 2 hours.
After rinsing, slides were coverslipped with Vectashield containing DAPI (Vector Laboratories) and examined using an
Olympus BX60 epifluorescence microscope. Observation and
quantification of Beclin-1 and LC3B fluorescent intensities
within capillaries was performed in randomly selected cervical
spinal cord sections in both right and left sides of ventral horn.
For consistent results, capillaries of diameters from 15 to
20 lm, as measured using NIH ImageJ (version 1.46) software, were used for fluorescent detection of Beclin-1 and
LC3B immunoreactivities. Microvessels with diameters below
or above this range, as well as longitudinal capillaries, were
excluded. Fluorescent images were taken from 3 to 4 sections
in the gray ventral horn of the cervical spinal cords of both
sides per animal separated by approximately 60–90 lm. Fluorescent Beclin-1 and LC3B intensities were analyzed in same
capillary (10–15 capillaries per examined spinal cord areas)
using NIH ImageJ software.
In a separate set of spinal cord sections, immunohistochemical staining of astrocytes was performed as previously described (29, 30). Briefly, the tissue sections were
3
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
Evans Blue dye, 961 Da, was used as a tracer for assessing BSCB disruption. The Evans Blue extravasation assay was
performed as previously described (29, 30, 42). Briefly, after
perfusion, the cervical spinal cords were weighed and placed
in 50% trichloroacetic acid solution (Sigma). Following homogenization and centrifugation, the supernatant was diluted
with ethanol (1:3) and loaded into a 96 well plate in triplicate.
The dye was measured with a spectrofluorometer (Gemini EM
Microplate Spectrofluorometer, Molecular Devices, Sunnyvale, CA) at excitation of 620 nm and emission of 680 nm
(43). Calculations were based on external standards in the
same solvent. The Evans Blue content in tissue was quantified
from a linear standard curve derived from known amounts of
the dye and was normalized to tissue weight (lg/g). All measurements were performed by two experimenters blinded to the
experiment.
BSCB Damage in Focal Cerebral Ischemia
Garbuzova-Davis et al
Histological Analysis and Stereological Counts
of Motor Neurons
The separate set of cervical spinal cord sections were
stained with 0.1% Luxol fast blue (LFB) (Aldrich Chemical,
Milwaukee, WI) and 0.1% Cresyl violet (Sigma-Aldrich) technique using a standard protocol. To determine motor neuron
numbers in the gray ventral horn of the cervical spinal cords
on both sides, the optical fractionator method of unbiased stereological cell counting techniques (44, 45) was used with a
Nikon Eclipse 600 microscope and quantified using Stereo
Investigator software (MicroBrightField, Williston, VT). The
virtual grid (150 150 mm) and counting frame (75 75 mm)
were optimized to count at least 200 cells per animal with
error coefficients <0.05. Outlines of the anatomical structures
were done using a 10/0.45 objective and cell quantification
was conducted using a 60/1.40 objective. The motor neuron
numbers (20- to 25-lm diameter) were counted in discrete levels of the cervical spinal cord (n ¼ 7 sections/level/animal separated by 60 lm) and presented as average per ventral horn
on both spinal cord sides. Motor neuron morphology and myelin staining were also analyzed.
Statistical Analysis
Data are presented as means 6 SE. One-way ANOVA
with Bonferroni’s Multiple Comparison test using GraphPad
Prism software version 5 (GraphPad Software) was performed
for statistical analysis. Significance was defined as p < 0.05.
4
RESULTS
Cervical Spinal Cord Ultrastructure at 7 Days
Post-tMCAO
Both sides of the cervical spinal cords in control rats
were characterized by normal appearance of capillaries surrounded by astrocyte cell processes and myelinated axons in
gray (Fig. 2A) and white matter (Fig. 2D, F). Capillaries consisted of a single layer of ECs surrounded by a basement
membrane layer, sometimes enclosed by additional pericyte
cytoplasm. Motor neurons and glial cells demonstrated normal
morphology with central nuclei in gray matter (Fig. 2B, C).
Organelles in all cells were well preserved and mitochondria
showed a normal pattern of cristae. White matter oligodendrocytes and astrocytes were morphologically typical with distinct nuclei (Fig. 2E, F). At 7 days after right tMCAO there
was dramatic microvascular damage on both sides of the spinal cord in gray and white matter. On the right side (ipsilateral
to tMCAO), vacuolated ECs containing large autophagosomes
were noted in numerous gray matter (Fig. 2G, H) and white
matter (Fig. 2I) capillaries of the ventral cervical spinal cord.
Some ECs were necrotic with condensed cytoplasm and detached from the capillary lumen (Fig. 2G). Edematous spaces
near capillaries or motor neurons were seen. Also, some axons
with myelin disruption and unraveling were apparent (Fig. 2J).
On the left (contralateral) side there were also severely damaged vessels in both gray and white matter including EC
degeneration and necrosis, endothelial autophagosome accumulation, pericyte degeneration with enlarged mitochondria,
astrocyte end-feet degeneration, and extensive perivascular
edema (Fig. 2K–M). Dilated endoplasmic reticulum in motor
neurons near damaged capillaries with perivascular edema
was also noted in addition to some degree of nuclear fragmentation, suggesting apoptotic processes. Numerous swollen
axons with and without myelin were surrounded by capillaries
in white matter (Fig. 2M, N). Importantly, markedly swollen
axons were seen mainly in white matter of the left side compared with the right side at 7 days post-tMCAO.
Chronic (30 Days) Post-tMCAO Stage
Similar to controls for 7 days post-tMCAO, control cervical spinal cords for 30 days after tMCAO demonstrated normal ultrastructural morphology in gray matter (Fig. 3A–C)
and white matter (Fig. 3D–F) on both sides of the cord. Capillary walls showed healthy-appearing ECs and adjacent
pericytes (Fig. 3A, D). Motor neurons, astrocytes, oligodendrocytes, and microglia all showed well-defined central
nuclei with normal morphology. Myelinated axons of high
density were present in white matter on both right and left
sides (Fig. 3E, F).
At 30 days after tMCAO, numerous capillaries in gray
matter on the right side of the cord revealed swollen ECs containing autophagosomes and enlarged mitochondria (Fig. 3G).
Similar to 7 days post-tMCAO, damaged ECs characterized
by expanded mitochondria and vacuolization and edematous
pericytes were detected in white matter capillaries on the right
side (Fig. 3I). Perivascular edema and detachment of ECs
from capillary lumen were seen in both gray and white matter.
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
preincubated in blocking solution as described above and then
incubated overnight with rabbit polyclonal anti-glial fibrillary
acid protein (GFAP) primary antibody (1:500, Dako, Glostrup,
Denmark) at 4 C. The next day, secondary goat anti-rabbit
antibody conjugated to FITC (1:500, Thermo Fisher Scientific) was applied for 2 hours. After washing, slides were
coverslipped with Vectashield containing DAPI (Vector Laboratories) and examined using an Olympus BX60 epifluorescence microscope. Fluorescent images were taken from 3
sections in the gray ventral horn of the cervical spinal cords of
both sides per animal separated by approximately 90 lm and
fluorescent intensity was analyzed as described above.
Immunohistochemical images of all assessments were
analyzed by an investigator blinded to the experiments and
animal codes were removed prior to analysis. To avoid bias in
the analysis of fluorescence images, the gray matter cervical
spinal cord areas in both sides were identified in a section
using a 10/0.30 numerical aperture lens, and then areas of
interest were photographed with a 20/0.50 numerical aperture lens, photographing the slide in a random raster pattern.
All image analyses for Beclin-1/LC3B and GFAP were performed by measuring intensity of fluorescent expression (%/
lm2) using NIH ImageJ software. For Beclin-1/LC3B immunoexpressions, fluorescent intensity was measured relative to
capillary area. For GFAP, fluorescent intensity was measured
in the gray matter images. Thresholds for detection of Beclin1/LC3B and GFAP expressions were adjusted for each image
to eliminate background noise.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
BSCB Damage in Focal Cerebral Ischemia
Also, motor neurons with dilated endoplasmic reticulum (Fig.
3H) and many axons with damaged myelin (Fig. 3J) were seen
on the right side of the cord; there was also considerable
edema. On the left side of cervical spinal cord, large areas of
edema between capillary and degenerated motor neurons were
apparent in gray matter (Fig. 3K). Some motor neurons demonstrated dilated endoplasmic reticulum (Fig. 3L). Capillaries
in both gray matter (Fig. 3K) and white matter (Fig. 3M, N)
contained large autophagosomes or vacuoles. Some myelin injury and swollen axons near capillaries were also seen on the
left side of the cord (Fig. 3N).
Oligodendrocytes with dark condensed cytoplasm were
also detected proximal to edematous spaces near damaged
capillaries in gray matter on the right side of the cervical spi-
5
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 2. Electron microscope examination of microvasculature in the rat cervical spinal cord at 7 days after tMCAO. (A–F)
Representative areas of gray matter (A–C) and white matter (D–F) of control rat cervical spinal cord demonstrate normal
ultrastructural appearances of capillaries, neurons, myelinated axons, and glial cells. (G–N) Seven days post-tMCAO, numerous
gray matter capillaries on the right side displayed damaged ECs containing large autophagosomes (G, H). Necrotic EC with
condensed cytoplasm was detached from capillary lumen (#) (G). Extracellular edema surrounding the capillary and motor
neuron was seen. Similarly to gray matter, white matter capillaries demonstrated EC alterations including vacuolization or
necrosis (I). Some demyelinated axons were also seen (J). The left side capillaries appeared more damaged in gray matter (K, L)
and white matter (M), as indicated by degenerated and necrotic ECs with autophagosome accumulation, degenerated pericytes
with enlarged mitochondria, astrocyte end-feet degeneration, dilated endoplasmic reticulum in motor neurons, and extensive
perivascular edema. Numerous swollen axons with and without myelin are surrounded by capillaries with perivascular edema in
white matter (M, N). Abbreviations: En, endothelial cell; BM, basement membrane; Ast, astrocyte; m, mitochondrion; c,
capillary; A, axon; P, pericyte; Nu, nucleus; Aph, autophagosome; Oligo. Oligodendrocyte; ER, endoplasmic reticulum.
#Separation of EC from BM. Asterisks in panels G–M indicate edema. Scale bars: A–F, H–J, L, N ¼ 2 lm; G, K, M ¼ 500 nm.
Garbuzova-Davis et al
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
nal cord 30 days after tMCAO (Fig. 4A). Also, a capillary
with 3 layers of ECs and astrocyte end-feet with normal mitochondria was observed on the left cord side of gray matter at
same post-tMCAO stage (Fig. 4B). Of note, there was less
perivascular edema in this field.
In summary, ultrastructural examination of microvasculature in the cervical spinal cord of rats after 7 (subacute) and
30 (chronic) days right tMCAO insult revealed severe damage
of capillary endothelium and pericytes, degeneration of astrocyte end-feet, and extensive perivascular edema in gray and
6
white matter on both sides of the cord. Damaged motor neurons and numerous swollen axons were also observed.
Microvascular Permeability
Tissue measurements showed significantly higher EB
levels at 7 days (8.30 6 0.32 lg/g, p < 0.001) and 30 days
(5.54 6 0.20 lg/g, p < 0.001) post-tMCAO versus controls
(1.58 6 0.16 lg/g) (Fig. 5A). Because there were no significant
differences in EB extravasation between control rats at 7 days
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 3. Electron microscope examination of microvasculature in the rat cervical gray and white matters of spinal cord at 30
days after tMCAO. (A–F) Representative areas of gray matter (A–C) and white matter (D–F) of a control rat cervical spinal cord
are characterized by normal ultrastructural morphology of capillaries, neurons, myelinated axons, and glial cells. (G–J) Thirty
days post-tMCAO, numerous capillaries in gray matter on the right side showed swollen ECs containing autophagosomes and
enlarged mitochondria (G). White matter capillaries have expanded mitochondria, vacuolization in ECs and a pericyte (I).
Perivascular edema and detachment of ECs from capillary lumen were seen in both gray (G) and white (I) matter. Motor
neurons with dilated endoplasmic reticulum in gray matter (H) and many axons with unraveling myelin in white matter (J) were
also found on the right side of the cord. Edema surrounding the swollen axons is noted in the white matter (J). (K–N) On the left
side of the cord there is edema between a capillary and a degenerated motor neuron (K), and around a motor neuron with
dilated endoplasmic reticulum (L) in gray matter. Capillaries in both gray matter (K) and white matter (M, N) contain large
autophagosomes or vacuoles. As on the right side, axons with unraveling myelin are seen near capillaries on the left side of the
cord (N). Abbreviations: En, endothelial cell; BM, basement membrane; Ast, astrocyte; m, mitochondrion; c, capillary; A, axon;
P, pericyte; Nu, nucleus; Aph, autophagosome; Oligo, oligodendrocyte; ER, endoplasmic reticulum; #, separation of EC from
BM. Asterisks in panels G, I–N indicate extracellular edema. Scale bars: B, C, E, F, H, J, L ¼ 2 lm; A, D, G, I, K, M, N ¼ 500 nm.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
BSCB Damage in Focal Cerebral Ischemia
(1.40 6 0.30 lg/g) and 30 days (1.79 6 0.16 lg/g), data for
controls were pooled. Greater parenchymal EB levels were
found at subacute (7 days) versus chronic (30 days) postischemic stages (p < 0.001). These results correlated with the
EM findings showing ultrastructural abnormalities in capillary
endothelia in gray and white matter on both sides of the spinal
cord possibly leading to microvascular leakage. Thus, significant vascular leakage in the spinal cord at subacute and chronic
ischemia might indicate prolonged BSCB impairment.
Microvascular Occludin
Microvascular occludin immunostaining demonstrated
strong expression of occludin in capillary lumens of ventral
horns (Fig. 5B, a, a0 ) and in numerous microvessels of right
and left lateral funiculi (Fig. 5B, b, b0 ) in controls. EB was visible within microvessels in a white matter lateral funiculus
(Fig. 5B, b, asterisk). At 7 days post-tMCAO, weak occludin
immunoexpression was observed in portions of numerous ventral horn capillaries showing perivascular EB leakage
(Fig. 5B, c, c0 , asterisks). In the lateral funiculi of both sides of
the spinal cord in these animals, occludin immunostaining was
identified in only a few microvessels (Fig. 5B, d, d0 ). At 30
days after tMCAO, limited occludin immunostaining was seen
in ventral horn capillaries, some of which showed EB leakage,
similar to 7 days post–infarct findings (Fig. 5B, e, e0 ). Interestingly, microvessels with enhanced occludin immunostaining
in both sides of lateral funiculi areas were noted in these animals, similar to controls (Fig. 5B, f, f0 ). Thus, the extensive
vascular leakage determined via EB extravasation into parenchyma of the cervical spinal cord at subacute and chronic
ischemia might result from reduction of occludin “tightness”
between ECs, mainly in animals at 7 days post-tMCAO.
Autophagosomes in Spinal Cord Endothelium
Because autophagosome accumulations were observed
by EM in numerous EC capillaries at 7 and 30 days after
tMCAO, immunohistochemical analysis of capillary Beclin-1
and LC3B expression and intensity measures were performed.
There was a significant increase in capillary Beclin-1 immunoreactivity on the right (7 days: 5.74% 6 0.6%; 30 days:
5.36% 6 0.32%) and left (7 days: 6.09% 6 0.46%; 30 days:
6.91% 6 0.33%) side of the spinal cord (Fig. 6A, c–f, B) versus controls (right side: 3.36% 6 0.24%; left side:
3.48% 6 0.26%) (Fig. 6A, a, b, B) (p < 0.001). Importantly,
significantly higher Beclin-1 immunostaining was noted in
capillaries on the left versus the right side at 30 days posttMCAO (p < 0.05). By contrast, significant upregulation of
capillary LC3B immunostaining was seen only at 7 days
post-tMCAO on the right (p < 0.05, 4.27% 6 0.42%) and left
(p < 0.01, 5.21% 6 0.63%) sides versus controls (right:
2.82% 6 0.29%; left: 2.79% 6 0.48%) (Fig. 6A, c0 , d0 , B).
Moreover, higher LC3B fluorescent expression was observed
on the left side of ventral horn at 7 days versus 30 days posttMCAO (p < 0.05). In control animals, LC3B and Beclin-1
expression on both sides of the spinal cord were similar. Comparative reduction of LC3B versus Beclin-1 was identified
after 7 and 30 days tMCAO (Fig. 6B); at 30 days post-infarct,
LC3B immunostaining was less than half of Beclin-1 on the
left side of the cervical spinal cord. In merged Beclin-1/LC3B
images representing in controls, double immunostaining demonstrates overlap between these autophagic biomarkers in capillary endothelium in both sides of the cervical spinal cord
(Fig. 6A, a00 , b00 ), whereas some capillaries show lack of
Beclin-1/LC3B overlay immunoexpression after 7 days (Fig.
6A, c00 , d00 ) and 30 days (Fig. 6A, e00 , f00 ) post-tMCAO. In these
merged images, Beclin-1 protein expression is apparent without overlap with LC3B. Because Beclin-1 is an essential
protein inducing the autophagy process by autophagosome
formation, and LC3B is required for autophagy activity (leading to autolysosome formation for removal of various intracellular components), reduced LC3B processing might indicate
impaired autophagy in the capillary endothelium resulting in
increased numbers of nonprocessing autophagosomes.
7
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 4. Electron microscope examination of potential repair of microvasculature in the rat cervical spinal cord gray matter at
30 days after tMCAO. (A) Activated-appearing oligodendrocytes characterized by dark condensed cytoplasm are seen near an
edematous space and damaged capillary on the right side of cord in gray matter. (B) A capillary with 3 layers of ECs and
astrocyte end-feet with normal mitochondria is seen on the left cord side of gray matter. Abbreviations: En, endothelial cell; BM,
basement membrane; Ast, astrocyte; m, mitochondrion; c, capillary; A, axon; Oligo, oligodendrocyte. Asterisks in A and B
indicate extracellular edema. Scale bars: A ¼ 2 lm; B ¼ 500 nm.
Garbuzova-Davis et al
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
These novel data correlate with the EM observations
and suggest that extensive EC autophagosome accumulations
in spinal cord capillaries likely affect cell integrity and thus
contribute to the BSCB dysfunction.
Spinal Cord Astrocyte Reactivity in tMCAO Rats
FIGURE 5. Quantitative analysis of Evans blue (EB) parenchymal
extravasation and occludin microvascular immunostaining
patterns in the rat cervical spinal cord at 7 days and 30 days
after tMCAO. (A) Quantitative measurement of parenchymal
EB content showed higher extravasated EB levels at 7 days
and 30 days post-tMCAO versus control (p < 0.001). Levels at 7
days were greater than at 30 days (***p < 0.001). (B)
Immunofluorescence staining for occludin (a, a0 ) within ventral
horn capillaries (green, arrowheads) and (b, b0 ) in lateral
funiculus of both sides of the cervical spinal cord (green,
arrowheads) show typical tight junction expression patterns in
control rats. Capillaries with strong occludin immunostaining
were noted near motor neurons (mn) in ventral horn (a).
The EB is within microvessels in white matter lateral funiculus
(b, light red, asterisk). At 7 days post-tMCAO, weak occludin
immunoreactivity was observed in (c, c0 , green, arrowheads)
portions of numerous ventral horn capillaries with detection
of perivascular EB leakage (light red, asterisks). Occludin
immunostaining was identified in only a few microvessels in the
lateral funiculus of both sides of the spinal cord in these animals
(d, d0 , green, arrowheads). At 30 days after tMCAO, limited
occludin immunoexpression (e, e0 , green, arrowheads) was
determined in ventral horn capillaries, which some of them are
leaky for EB (light red, asterisks), similar to 7 days post-stroke.
Microvessels with enhanced occludin immunostaining in both
sides of lateral funiculus areas were noted in these animals
similar to controls (f, f00 , green, arrowheads). Scale bars: a, b,
b0 , c, d, d0 , e, f, f0 ¼ 50 mm; a0 , c0 , e0 ¼ 25 mm.
8
Spinal Cord Histology and Stereological Counts
of Motor Neurons
The projections from the brain to the spinal cord or from
the spinal cord to the brain consist mainly of 3 descending
tracts: dorsal corticospinal tract (dCST), reticulospinal tract
(ReST), and the rubrospinal Tract (RST), and 2 ascending
tracts: fasciculus cuneatus (FC, tract of Burdach) and the spinothalamic tract (STT) (46, 47) (Fig. 8A). The zones of the
descending (motor) tracts indicated on the right side and the
ascending (sensory) tracts on the left side of the cervical spinal
cord in control rats demonstrated normal myelin staining. In
rats at 7 and 30 days after tMCAO, pale areas in white matter
spinal cord (ie, weakly stained by LFB) were identified
through C3–C6 cervical levels (Fig. 8A). However, at the C8
level, white matter myelin abnormalities were not identified.
At 7 days post-tMCAO, there was distinct myelin pallor primarily on the left side of the spinal cord (ie, contralateral to
the tMCAO insult) in areas of the descending tracts, that is, in
C3, ReST; in C5, C6, RST and the ascending tracts; in C3,
STT; in C5, FC, STT; and in C6, STT (Fig. 8A). Some degree
of myelin pallor was seen in dCST at C3 level on the right side
of the cervical spinal cord, ipsilateral to tMCAO.
At 30 days post-tMCAO rats, myelin pallor areas were
noted in the descending tracts as ReST (C3 and C5) and RST
(C6) and the ascending tracts as FC (C5) and STT (C3 and C6)
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
In 7 day post-tMCAO rats, significantly elevated
GFAP fluorescence expression was identified in right (5.26%
6 0.67%, p < 0.01) and left (6.77% 6 0.73%, p < 0.001) sides
of ventral horn gray matter of the cervical spinal cord versus
control animals (right: 2.92% 6 0.48%; left: 2.79% 6 0.43%)
(Fig. 7A, a–c, B). Although there was higher GFAP immunoreactivity on the left side of the spinal cord, there were no statistical differences compared to the right side. GFAP-positive
cells were mostly distinguished proximal to motor neurons in
both sides of the cervical spinal cord (Fig. 7A, b, c), likely
indicating spinal cord astrocyte reactivity at the subacute
stage.
In rats at 30 days post-tMCAO, no significant differences were found in GFAP expression in either right
(2.77% 6 0.10%) or left (3.39% 6 0.39%) sides of cervical
ventral horn versus controls (right: 2.53% 6 0.25%; left:
2.44% 6 0.30%) (Fig. 7A, d–f, B). However, slightly greater
GFAP immunoreactivity was noted in the left side versus the
right side (Fig. 7A, e, f). Thus, significant upregulation of
GFAP immunoreactivity in the gray matter ventral horn of the
cervical spinal cord in tMCAO rats was mainly demonstrated
in both sides at subacute (7 days) post-infarct phase, with
higher GFAP fluorescent expression in the left side; astrocyte
reactivity was also evident in the left side of the spinal cord in
rats at 30 days after tMCAO.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
BSCB Damage in Focal Cerebral Ischemia
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 6. Autophagosome analysis in capillary endothelium of the ventral horn cervical spinal cord in rats 7 and 30 days after
tMCAO. (A) Immunofluorescence staining of spinal cord in control rats for Beclin-1 (green, arrowheads) (a, b) and LC3B (red,
arrowheads) (a0 , b0 ) showed typical expression patterns in capillary endothelium in both sides of the spinal cord. In merged
Beclin-1/LC3B images, double immunostaining demonstrates overlap between these autophagic biomarkers (orange,
arrowheads) (a00 , b00 ). At 7 days after tMCAO, extensive Beclin-1 (green, arrowheads) (c, d) and LC3B (red, arrowheads) (c0 , d0 )
staining is shown in capillaries in the right and left sides of the spinal cord. Some capillaries showed lack of Beclin-1/LC3B overlay
immunostaining; Beclin-1 protein immunostaining (green, asterisks) is clearly seen in merged images on both sides of the cord
(c00 , d00 ). Similarly, greater Beclin-1 (green, arrowheads) (e, f) and (e0 , f0 ) LC3B (red, arrowheads) immunostaining is seen in the
right and the left side capillaries 30 days after tMCAO versus controls. In merged images, omitted overlap between Beclin-1 and
LC3B immunoexpressions (green, asterisks) was determined in the right (e00 ) and the left (f00 ) side capillaries. Nuclei are stained
with DAPI in the merged images (a00 –f00 ). Scale bar in a–f00 is 50 mm. (B) Quantitative analysis of Beclin-1 and LC3B
immunostaining in capillary endothelium in control rats showed similar patterns on each side. Increases in Beclin-1
immunostaining on the right and left side spinal cord capillaries at 7 and 30 days post-tMCAO was noted versus that in controls
(p < 0.001). Higher Beclin-1 immunostaining was noted in capillaries on the left versus the right side at 30 days post-tMCAO
(p < 0.05). Nevertheless, there was upregulation of LC3B expression only at 7 days post-tMCAO in capillaries on the right
(p < 0.05) and left (p < 0.01) sides versus controls. Higher LC3B expression was detected on the left side of ventral horn at 7 days
versus 30 days post-tMCAO (p < 0.05). *p < 0.05; **p < 0.01; ***p < 0.001.
9
Garbuzova-Davis et al
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
DISCUSSION
This study focused on BSCB status and related pathological processes in the cervical spinal cord remote from an
initial brain ischemic insult at 2 time points following experimental focal cerebral ischemia in rats. BSCB damage was detected not only in the gray matter of the cervical spinal cord
but also in white matter on both sides of the cord at both time
points. The BSCB pathological alterations included: (1)
vacuolated ECs containing large autophagosomes, (2) pericyte
degeneration with enlarged mitochondria, (3) astrocyte endfeet degeneration and extensive perivascular edema, (4)
damaged motor neurons and swollen axons with myelin unraveling, (5) EB extravasation, (6) reduction of tight junction
occludin immunoreactivity, (7) upregulation of microvascular
Beclin-1 and LC3B immunoreactivity at 7 days and reduction
of LC3B versus Beclin-1 immunoreactivity at 30 days, (8) parenchymal astrogliosis, (9) myelin pallor in descending and ascending tracts, and (10) motor neuron loss. These alterations
in the cervical spinal cord indicate pervasive and long-lasting
BSCB damage that likely would severely affect spinal cord
function.
Substantial vascular damage represents a major pathologic feature of subacute and chronic tMCAO. The findings of
vascular damage in the cervical spinal cord remote from the
focal cerebral ischemia site indicate ongoing pathological
vascular changes in association with subacute and chronic ischemic diaschisis. The spinal cord capillary leakage and ultrastructural alterations induced by focal cerebral ischemia
suggest that there is an extended period of microvascular permeability, as we identified in brain regions distant from initial
tMCAO damage (29, 30); it is thus possible that widespread
blood-CNS barrier impairment persists following cerebral ischemic events. Also, EB extravasation may result from
decreased tight junction protein expression, as has been shown
for cerebral microvessels under hypoxic conditions (48–50).
10
Because there are no reports regarding vascular tight junction
integrity in the spinal cord after tMCAO, we analyzed the
immunoreactivity of occludin, one of several tight junction
proteins, and demonstrated reduced immunoexpression within
capillaries in the ventral horn and in microvessels of lateral funiculus of both sides of the cervical spinal cord, mainly in animals at 7 days post-tMCAO. Lost tightness between ECs
might account for paracellular vascular leakage, as determined
by EB extravasation into the spinal cord parenchyma.
Additionally, studies on rabbit spinal cord ischemia
have demonstrated a decline in motor function, edema formation in gray and white matter and increased numbers of necrotic neurons in the lumbar spinal cord during reperfusion after
an initial ischemic insult via aortic occlusion (51–53). Moreover, blood flow reduction and severe vascular permeability
for EB-albumin were noted in this rabbit model, suggesting
progressive BSCB breakdown (52).
In this study, autophagosome accumulation within
spinal cord capillary endothelium was identified at the
ultrastructural level and was confirmed by upregulation of
immunoreactivity of the autophagy biomarker Beclin-1 in
microvessels on both sides of the cervical spinal cord at both
post-tMCAO time points. Importantly, significantly higher
Beclin-1 expression was noted in the left (contralateral) versus
right side of spinal cord in chronic (30 days) tMCAO rats.
Immunoreactivity of another autophagy marker, LC3B, was
increased on both sides of the spinal cord at 7 days posttMCAO compared to controls but there were reductions of
LC3B on both sides of the cord in rats at 30 days after
tMCAO. This was verified via double immunostaining for
both autophagic biomarkers. The lack of overlap between
Beclin-1 and LC3B expression was evident in numerous capillary endothelia at both time points. Beclin-1 is an essential
protein inducing the autophagy process by autophagosome
formation (54, 55). LC3B, as one of several members of the
mammalian LC3 family, which is tightly associated with the
autophagosome membranes, is indispensable for autophagic
activity leading to autolysosome formation for removal of
various intracellular components (56–58). Therefore, it is possible that lower LC3B expression indicates impaired autophagy processing in the endothelium via diminished ability of
the autophagosome to fuse with the lysosome, resulting in
increased numbers of nonprocessing autophagosomes.
Although autophagy plays an important role in cell survival
by degradation of cytoplasmic components through the
autophagosome-lysosomal pathway (59), excessive autophagic processes might induce cellular death (60). Together, our
data demonstrated for the first time upregulation of Beclin-1,
indicating intensified autophagy, and decreased LC3B protein
expression at the later stage, suggesting an impaired autophagy process in post-tMCAO spinal cord capillaries. The endothelial autophagosome accumulation might be a result of
blocking traffic to lysosomes without concomitant changes in
autophagosome biogenesis, resulting in a reduction of the degradative activity of cytoplasmic components needed to maintain cell survival. Together with our EM findings these data
suggest that EC autophagosome accumulations in spinal cord
capillaries likely affect cell integrity that may be impaired in
spinal cord capillaries following focal cerebral ischemia.
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
on the left side of the spinal cord (Fig. 8A). Interestingly,
ReST and STT were also affected on the right side.
Stereological motor neuron counts in both the right and
left sides of discrete levels of the cervical spinal cord demonstrated substantial cell losses in rats at 7 and 30 days after
tMCAO versus controls (Fig. 8B). Motor neuron numbers
were decreased on the left side of the spinal cord primarily at
C5-C6 and C7-C8 levels in animals at subacute (7 days) and
chronic (30 days) tMCAO stages (p < 0.001). The reduced
appearance of motor neurons was observed in each ventral
horn side/level of the cervical spinal cord via Cresyl violet
counterstaining of the Nissl substance (Fig. 8A, inserts).
Thus, histological analysis of the cervical spinal cord
demonstrated myelin pallor in the descending (ReST and
RST) and ascending (STT) tracts, mainly affecting the left
side of the spinal cord, contralateral to the tMCAO insult in
rats at 7 and 30 days post-tMCAO. However, some degree of
myelin pallor was also observed in these animals on the right
side of the upper level cervical spinal cord, ipsilateral to
tMCAO. Additionally, significant motor neuron loss was
determined at different levels of the spinal cord on both sides
at subacute (7 days) and chronic (30 days) tMCAO stages.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
BSCB Damage in Focal Cerebral Ischemia
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 7. Astrocytes in the cervical spinal cord ventral horn in rats 7 and 30 days after tMCAO. (A) Immunohistochemical
analysis of GFAP immunoexpression in the right or left sides of control rats for (a) 7 days and (d) 30 days tMCAO demonstrated
normal appearance of parenchymal GFAP positive cells (green). At 7 days after tMCAO, increased GFAP immunoreactivity
(green, arrowheads), indicating astrogliosis, was determined in ventral horn parenchyma mostly surrounding motor neurons in
both (b) the right and (c) the left side of the spinal cord. At 30 days after tMCAO, greater GFAP fluorescent expression (green,
arrowheads) was observed in the (f) panel versus the right (e) side. The nuclei are stained with DAPI; scale bars, 50 mm. (B)
Quantitative analysis of GFAP immunostaining in the cervical spinal cord parenchyma of control rats showed a similar extent on
both sides. In 7 days post-tMCAO rats, there was an increase of GFAP expression on the right (p < 0.01) and the left (p < 0.001)
sides versus controls. There was greater GFAP immunostaining on the left versus the right side of the cord. There were no
significant differences between 30 days tMCAO and control rats in GFAP immunoexpression on both sides of the spinal cord,
although there was greater GFAP immunostaining on the left side at 7 versus 30 days post-tMCAO. **p < 0.01; ***p < 0.001.
11
Garbuzova-Davis et al
12
Although the cortex and striatum are the areas most affected by MCAO (62, 63), some studies have shown extensive
neuronal post-ischemic damage in the thalamus, brainstem,
and spinal cord accompanied by degeneration of corticofugal
axonal fibers extending caudally along descending tracts indicating remote changes (31, 64–66). Wu and Ling suggested
that selective lesions of dorsal horn neurons in the lumbar spinal cord in rats with permanent MCAO might be due to their
trans-synaptic de-afferentation, mediated by ischemic degeneration of the descending axons of the corticospinal tract (31).
Another study by Iizuka et al (64) using the Fink-Heimer silver staining method to trace degenerating axons showed
degenerate corticospinal axons in the ventral portion of contralateral dorsal funiculus at low cervical (C7) and lumbar (L6)
segments in rats at 1–6 weeks after MCAO. However, our results demonstrated myelin pallor of descending dCST on the
right (ipsilateral) side of the cervical (C3) spinal cord in rats at
7 days post-tMCAO, which contrasts with results from the
above mentioned study. Possibly, the BBB alterations we
determined at 7 days after tMCAO (29) in contralateral brain
areas (striatum, motor and somatosensory cortices) from
tMCAO insult induce damage of descending corticospinal
fibers, which cross over to ipsilateral spinal cord. However, it
is unclear why demyelinated dCST was not observed on the
contralateral spinal cord side in subacute tMCAO rats or why
there was no evidence of myelin abnormality of this tract in
the cervical spinal cord at 30 days after tMCAO. Although the
crossed corticospinal tract is located in the ventral part of the
dorsal column of the spinal cord of most mammals (67), no
topographical organization of the CST has been noted in the
cervical spinal cord of the adult rat (68). Interestingly, Liu et
al (69) noted that normal rats had predominantly unilateral
innervation of CST and corticorubral tract axons and that bilateral innervation occurred through axonal sprouting of the
uninjured CST and corticorubral tract in rats 28 days after permanent MCAO. The authors suggested that axonal restructuring occurred as compensatory innervation to “rewire the
denervated spinal cord”. Also, anterograde axonal degeneration of different descending tracts might take place over
dCST damage in the cervical spinal cord in rats at 7 and 30
days after tMCAO. Although Iizuka et al (64) showed that no
degenerated axons were detected in other white matter areas
of the spinal cord besides corticospinal axons in 1–6 weeks
post-tMCAO rats, our analysis demonstrated myelin abnormalities in areas of the descending ReST and RST, mainly
affecting the left side of the cord at 7 days and 30 days posttMCAO. Consistent with the RST projections from the brain
to the spinal cord (47, 70, 71), axon degeneration likely moves
from the damaged hemisphere, crossing over at the tegmental
decussation, and permeating the contralateral left side of the
spinal cord as shown here.
Compared to the dCST and RST, ReST connectivity is
more complex. It has been shown that cortical neurons bilaterally target the reticular formation and reticulospinal neurons connect with ipsilateral motor neurons directly or indirectly via segmental interneurons (72–74). Because there is
no evidence of direct corticomotor neuronal synaptic connections in the cervical rat spinal cord (75), Umeda et al
(76) investigated descending pathways involved in func-
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
Because many EC are unable to self-repair through autophagocytosis, they may die and slough off the capillaries, contributing to the impaired BSCB demonstrated by EB extravasation.
Additionally, our results showed a significant increase
of GFAP immunoreactivity proximate to motor neurons on
both sides of the spinal cord in rats at 7 days post-stroke with
higher expression in the left side; astrocyte reactivity was also
present at 30 days post-tMCAO. These results indicating reactive astrogliosis bilaterally in the cervical spinal cord of rats
in subacute tMCAO and unilaterally (left side) in chronic
tMCAO rats likely point to prolonged post-ischemic inflammation. Inflammation has been shown to play a major role in
the pathogenesis of ischemic stroke (4, 61); activated astrocytes (and microglia) contribute to neuroinflammation by the
secretion of various proinflammatory cytokines.
Wu and Ling showed increased activated microglia in
the lumbar spinal cord, specifically in the dorsal and ventral
horns contralateral to focal cerebral ischemia, in rats with permanent MCAO beginning at 24 hours and transforming into
amoeboid form at day 3 post-infarct (31). Activated microglia
emerged near degenerated motoneurons in the dorsal lumbar
spinal cord, while ventral horn motoneurons remained ultrastructurally intact. These authors also showed that hypertrophied astrocytes appeared at 7 days post-MCAO, replacing
perineuronal microglia (32). Similarly, cortical lesions associated with contralateral lumbar spinal cord microglia priming
lacked significant reactive astrogliosis or lower motor neuron
degeneration in a tMCAO mouse model at 24 and 72 hours
post-reperfusion (33). The glial cell response in the spinal
cord was accompanied by increased expression of proinflammatory cytokines and markers of oxidative stress at 24–
72 hours after permanent MCAO (32, 34). Inflammatory
changes in the spinal cord distant from the initial focal ischemic insult might also be involved in the pathogenesis of secondary neuronal damage linked to antero- and retrograde degeneration (35). However, these studies primarily focused on
the lumbar spinal cord at acute or early subacute stages of
MCAO. Although the microglial response was not examined
in this study, our results indicate substantial astrogliosis in the
cervical spinal cord mainly in rats at 7 days after tMCAO;
these might lead to motor neuron damage and BSCB impairment. These data correlated with our EM findings showing
astrocyte end-feet degeneration and extensive perivascular
edema as well as degenerated motor neurons with dilated
endoplasmic reticulum not only at 7 but also at 30 days posttMCAO.
Potentially long-lasting BSCB damage might affect
post-stroke motor neuron survival. We demonstrated significant motor neuron loss at different levels of the cervical spinal cord on both sides of the ventral horn at subacute (7
days) and chronic (30 days) tMCAO stages. Moreover,
histological analysis of the cervical spinal cord demonstrated areas of myelin pallor of the descending (ReST and
RST) and ascending (STT) tracts, mainly affecting the left
side of the spinal cord, contralateral to the tMCAO, in the
post-tMCAO rats. Some degree of myelin pallor was also
observed in these animals on the right side of the upper level
cervical spinal cord, ipsilateral to tMCAO. This may represent secondary demyelination.
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
BSCB Damage in Focal Cerebral Ischemia
tional connections with motor neurons using a hemidecorticate rat model. They showed at least 2 indirect descending
corticomotor pathways: the cortico-reticulo-spinal and cortico-spinal pathways (76). In hemidecorticate rats, CST ori-
ginated from the undamaged sensorimotor cortex, connecting to motor neurons on the opposite spinal cord side in
addition to the normal descending cortical motor pathway.
Also, these connections were mediated via spinal inter-
13
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
FIGURE 8. Histological analysis of the cervical spinal cord in rats 7 and 30 days after tMCAO. (A) Cervical spinal cord sections
were stained with Luxol fast blue (LFB) and Cresyl violet (cv) for analysis of myelin and motor neurons. Zones of the descending
tracts are indicated on the right side and the ascending tracts on the left side of the cervical spinal cord in control rats
demonstrated normal myelin staining (middle part). At 7 days post-tMCAO, areas of myelin pallor in white matter spinal cord,
weakly stained by LFB, were primarily present on the left side of the spinal cord, contralateral to tMCAO insult, of the descending
(ReST and RST) and the ascending (STT) tracts. Some degree of demyelination was seen in dCST at C3 level on the right side of
the cervical spinal cord, ipsilateral to tMCAO. (right part) In rats after 30 days tMCAO, demyelinated areas were similarly
observed in the descending (ReST and RST) and the ascending (FC and STT) tracts on the left side of the spinal cord. Also, ReST
and STT were affected on the right side. Abbreviations: descending tracts: dorsal Corticospinal Tract (dCST), Reticulospinal Tract
(ReST), Rubrospinal Tract (RST); ascending tracts: Fasciculus Cuneatus (FC, tract of Burdach), Spinothalamic Tract (STT), ventral
horn (VH). Mark hole on coronal spinal cord sections indicates the right side. Magnification: coronal spinal cord section is 4,
inserts on both sides of coronal sections are 20. (B) Stereological motor neuron counts on both the right and left sides of
discrete levels of the cervical spinal cord demonstrated substantial cell loss in rats at 7 and 30 days after tMCAO versus controls.
Motor neuron numbers were significantly (p < 0.001) decreased on the left side of the spinal cord primarily at C5-C6 and C7-C8
levels in animals at subacute (7 days) and chronic (30 days) tMCAO stages. C2-C3, C5-C6, and C7-C8 are levels of the cervical
spinal cord. *p < 0.05; **p < 0.01; ***p < 0.001.
Garbuzova-Davis et al
14
correlation with BSCB condition are needed. Additionally, the
early post-ischemic timing of BSCB damage needs to be
defined. These and other questions will be answered in our future studies.
In summary, we show altered BSCB integrity in subacute and chronic post-ischemic rats on both sides of the cervical spinal cord, that is, areas remote from the initial cerebral
ischemic insult, indicating that spinal cord diaschisis occurs at
early and remains extended up to 30 days post-ischemia. The
widespread microvascular impairment in the gray and white
matter of the cervical spinal cord aggravated motor neuron deterioration and could have to motor dysfunction following
cerebral ischemia. More structural capillary alterations were
observed on the left side of the spinal cord, likely accompanied and/or due to denervation by the damaged descending
tracts crossing over from the tMCAO injured hemisphere.
Importantly, substantial right side microvessel pathology
could also indicate damaged neuronal connective pathways
from the opposite hemisphere, initially unaffected by tMCAO.
Hence, BSCB impairment in ischemic stroke may have significant input on disease pathology, indicating that spinal cord
diaschisis should be considered in revised disease pathology
and therapeutic approaches targeting BSCB repair in stroke.
ACKNOWLEDGMENTS
We gratefully acknowledge Maria C.O. Rodrigues,
MD, PhD, from the Department of Internal Medicine,
Ribeir~
ao Preto School of Medicine, University of Sao Paulo,
Sao Paulo, Brazil, and Diana G. Hernandez-Ontiveros, PhD.
from USF, for help in performing the EB assay.
REFERENCES
1. Mozaffarian D, Benjamin EJ, Go AS, et al. Heart disease and stroke
statistics–2015 update: A report from the American Heart Association.
Circulation 2015;131:e29–322
2. Go AS, Mozaffarian D, Roger VL, et al. Heart disease and stroke
statistics–2013 update: A report from the American Heart Association.
Circulation 2013;127:e6–245
3. Kelly-Hayes M, Beiser A, Kase CS, et al. The influence of gender and
age on disability following ischemic stroke: The Framingham study.
J Stroke Cerebrovasc Dis 2003;12:119–26
4. del Zoppo GJ, Hallenbeck JM. Advances in the vascular pathophysiology
of ischemic stroke. Thromb Res 2000;98:73–81
5. del Zoppo GJ, Mabuchi T. Cerebral microvessel responses to focal ischemia. J Cereb Blood Flow Metab 2003;23:879–94
6. Persidsky Y, Ramirez SH, Haorah J, et al. Blood-brain barrier: Structural
components and function under physiologic and pathologic conditions.
J Neuroimmune Pharmacol 2006;1:223–36
7. Sandoval KE, Witt KA. Blood-brain barrier tight junction permeability
and ischemic stroke. Neurobiol Dis 2008;32:200–19
8. Jin R, Yang G, Li G. Molecular insights and therapeutic targets for
blood-brain barrier disruption in ischemic stroke: Critical role of matrix
metalloproteinases and tissue-type plasminogen activator. Neurobiol Dis
2010;38:376–85
9. Dankbaar JW, Hom J, Schneider T, et al. Dynamic perfusion-CT assessment of early changes in blood brain barrier permeability of acute ischaemic stroke patients. J Neuroradiol 2011;38:161–6
10. Preston E, Sutherland G, Finsten A. Three openings of the blood-brain
barrier produced by forebrain ischemia in the rat. Neurosci Lett 1993;
149:75–8
11. Yang GY, Betz AL. Reperfusion-induced injury to the blood-brain barrier after middle cerebral artery occlusion in rats. Stroke 1994;25:
1658–64 discussion 1664–5
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
neurons and reticulospinal neurons. As we showed in this
study demyelinated ReST on both contralateral and ipsilateral sides of the cervical spinal cord in 7 and 30 days posttMCAO rats, it is possible that axonal degeneration is
spreading from the tMCAO damaged hemisphere, passing
through both sides of the reticular formation, and ultimately
causing secondary demyelination in both the left and right
ReST. Subacute and chronic cerebral diaschisis likely contributes to the myelin abnormality observed.
With respect to the ascending pathways, it is possible
that axonal degeneration with secondary demyelination
travels retrograde from the cerebral hemispheres towards
the cell bodies of the STT and FC in the spinal cord. Iizuka
et al (65) demonstrated that MCAO of the somatosensory
cortex in rats caused retrograde neuronal degeneration in the
thalamus, possibly as a secondary effect from thalamocortical fiber damage in the infarcted region. Interestingly, the
STT in this study was the most consistently abnormal tract
following tMCAO; the potential cause of this secondary demyelination may originate in the cerebral hemispheres and
travel to cervical STT axons via the thalamus. STT abnormalities were observed on both the contralateral and ipsilateral sides of the cervical (C6) spinal cord at 30 days posttMCAO rats. Right STT myelin loss may indicate ipsilateral
progression of degeneration from the right tMCAO hemisphere (77), whereas left STT demyelination may represent
ipsilateral damage flow from the left hemisphere damaged
via diaschisis (29, 30). However, it is unclear why more
STT abnormalities were observed on the left side of the spinal cord contralateral to the tMCAO hemisphere versus the
ipsilateral side of the cord.
With respect to the FC (tract of Burdach), axonal damage and demyelination may also be progressing in the retrograde direction from the brain to the spinal cord. The FC
contralateral to the tMCAO hemisphere showed myelin pallor
at the C5 level at both time points. Because FC fibers cross to
the opposite side as they ascend to the thalamus (78), it is
possible that a right tMCAO hemisphere triggers axonal degeneration in a retrograde fashion down ascending axons and
decussates, which results in secondary demyelination in the
FC on the left side of the cervical spinal cord. Together,
demyelinated descending and ascending pathways, as indicated by histological analyses, were supported by our ultrastructural examination showing damaged motor neurons and
numerous swollen axons in the cervical spinal cord of rats at
both time points. Future studies are needed to confirm axonal
degeneration projections using anterograde and retrograde
tracing systems.
The appearance of oligodendrocytes with dark cytoplasm and multilayers of ECs in capillary surrounded by morphologically normal astrocyte end-feet were noted at 30 days
post-tMCAO; these findings may indicate spontaneous reparative processes. Because our investigations on post-stroke
pathological microvascular alterations including BSCB have
just been initiated, many questions remain. Specifically, protein expression for autophagosomes and tight junctions identified in our study by immunofluorescence techniques should be
confirmed via additional assays such as Western blot. Also,
behavioral tests of motor function in post-stroke animals in
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016
38. Matsukawa N, Yasuhara T, Hara K, et al. Therapeutic targets and limits
of minocycline neuroprotection in experimental ischemic stroke. BMC
Neurosci 2009;10:126
39. Yasuhara T, Matsukawa N, Hara K, et al. Notch-induced rat and human
bone marrow stromal cell grafts reduce ischemic cell loss and ameliorate
behavioral deficits in chronic stroke animals. Stem Cells Dev 2009;18:
1501–14
40. Borlongan CV, Kaneko Y, Maki M, et al. Menstrual blood cells display
stem cell-like phenotypic markers and exert neuroprotection following
transplantation in experimental stroke. Stem Cells Dev 2010;19:439–52
41. Watson C, Paxinos G, Kayalioglu G, et al. Atlas of the rat spinal cord. In:
Watson C, Paxinos G, Kayalioglu G, editors. The Spinal Cord: A Christopher and Dana Reeve Foundation Text and Atlas. Cambridge, Massachusetts: Academic Press; 2009. p. 238–307
42. Borlongan CV, Lind JG, Dillon-Carter O, et al. Bone marrow grafts restore cerebral blood flow and blood brain barrier in stroke rats. Brain Res
2004;1010:108–16
43. Ay I, Francis JW, Brown RH Jr. VEGF increases blood-brain barrier permeability to Evans blue dye and tetanus toxin fragment C but not adenoassociated virus in ALS mice. Brain Res 2008;1234:198–205
44. West MJ, Slomianka L, Gundersen HJ. Unbiased stereological estimation
of the total number of neurons in thesubdivisions of the rat hippocampus
using the optical fractionator. Anat Rec 1991;231:482–97
45. Morganti JM, Nash KR, Grimmig BA, et al. The soluble isoform of
CX3CL1 is necessary for neuroprotection in a mouse model of Parkinson’s disease. J Neurosci 2012;32:14592–601
46. Kayalioglu G. Projections from the spinal cord to the brain. In: Watson
C, Paxinos G, Kayalioglu G, editors. The Spinal Cord: A Christopher and
Dana Reeve Foundation Text and Atlas. Cambridge, Massachusetts:
Academic Press; 2009. p. 148–67
47. Watson C, Harvey, Alan R. Projections from the brain to the spinal cord.
In: Watson C, Paxinos G, Kayalioglu G, editors. The Spinal Cord: A
Christopher and Dana Reeve Foundation Text and Atlas. Cambridge,
Massachusetts: Academic Press; 2009. p. 168–79
48. Mark KS, Davis TP. Cerebral microvascular changes in permeability and
tight junctions induced by hypoxia-reoxygenation. Am J Physiol Heart
Circ Physiol 2002;282:H1485–94
49. Koto T, Takubo K, Ishida S, et al. Hypoxia disrupts the barrier function
of neural blood vessels through changes in the expression of claudin-5 in
endothelial cells. Am J Pathol 2007;170:1389–97
50. Kago T, Takagi N, Date I, et al. Cerebral ischemia enhances tyrosine
phosphorylation of occludin in brain capillaries. Biochem Biophys Res
Commun 2006;339:1197–203
51. Jacobs TP, Shohami E, Baze W, et al. Deteriorating stroke model: Histopathology, edema, and eicosanoid changes following spinal cord ischemia in rabbits. Stroke 1987;18:741–50
52. Jacobs TP, Kempski O, McKinley D, et al. Blood flow and vascular
permeability during motor dysfunction in a rabbit model of spinal cord
ischemia. Stroke 1992;23:367–73
53. DeGirolami U, Zivin JA. Neuropathology of experimental spinal cord ischemia in the rabbit. J Neuropathol Exp Neurol 1982;41:129–49
54. Hsieh Y-C, Athar M, Chaudry IH. When apoptosis meets autophagy:
Deciding cell fate after trauma and sepsis. Trends Mol Med 2009;15:
129–38
55. Itakura E, Mizushima N. Atg14 and UVRAG: Mutually exclusive subunits of mammalian Beclin 1-PI3K complexes. Autophagy 2009;5:534–6
56. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue
of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 2000;19:5720–8
57. Wu J, Dang Y, Su W, et al. Molecular cloning and characterization of rat
LC3A and LC3B–two novel markers of autophagosome. Biochem Biophys Res Commun 2006;339:437–42
58. Hansen TE, Johansen T. Following autophagy step by step. BMC Biol
2011;9:39
59. Uchiyama Y, Shibata M, Koike M, et al. Autophagy-physiology and
pathophysiology. Histochem Cell Biol 2008;129:407–20
60. Reggiori F, Klionsky DJ. Autophagy in the eukaryotic cell. Eukaryotic
Cell 2002;1:11–21
61. Huang J, Upadhyay UM, Tamargo RJ. Inflammation in stroke and focal
cerebral ischemia. Surg Neurol 2006;66:232–45
15
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
12. Belayev L, Busto R, Zhao W, et al. Quantitative evaluation of bloodbrain barrier permeability following middle cerebral artery occlusion in
rats. Brain Res 1996;739:88–96
13. Kuroiwa T, Ting P, Martinez H, et al. The biphasic opening of the bloodbrain barrier to proteins following temporary middle cerebral artery occlusion. Acta Neuropathol 1985;68:122–9
14. Strbian D, Durukan A, Pitkonen M, et al. The blood-brain barrier is continuously open for several weeks following transient focal cerebral ischemia. J Neurosci 2008;153:175–81
15. Abo-Ramadan U, Durukan A, Pitkonen M, et al. Post-ischemic leakiness
of the blood-brain barrier: A quantitative and systematic assessment by
Patlak plots. Exp Neurol 2009;219:328–33
16. Gerriets T, Walberer M, Ritschel N, et al. Edema formation in the hyperacute phase of ischemic stroke. Laboratory investigation. J Neurosurg
2009;111:1036–42
17. Dobkin JA, Levine RL, Lagreze HL, et al. Evidence for transhemispheric
diaschisis in unilateral stroke. Arch Neurol 1989;46:1333–6
18. Baron JC. Pathophysiology of acute cerebral ischemia: PET studies in
humans. Cerebrovasc Dis 1991;1:22–31
19. Andrews RJ. Transhemispheric diaschisis. A review and comment.
Stroke 1991;22:943–9
20. Gonzalez-Aguado E, Martı-Fabregas J, Martı-Vilalta JL. [The phenomenon of diaschisis in cerebral vascular disease]. Rev Neurol 2000;30:
941–5
21. Meyer JS, Obara K, Muramatsu K. Diaschisis. Neurol Res 1993;15:
362–6
22. Nguyen DK, Botez MI. Diaschisis and neurobehavior. Can J Neurol Sci
1998;25:5–12
23. Takasawa M, Watanabe M, Yamamoto S, et al. Prognostic value of subacute crossed cerebellar diaschisis: Single-photon emission CT study in
patients with middle cerebral artery territory infarct. Am J Neuroradiol
2002;23:189–93
24. Liu Y, Karonen JO, Nuutinen J, et al. Crossed cerebellar diaschisis in
acute ischemic stroke: A study with serial SPECT and MRI. J Cereb
Blood Flow Metab 2007;27:1724–32
25. Seitz RJ, Azari NP, Knorr U, et al. The role of diaschisis in stroke recovery. Stroke 1999;30:1844–50
26. Reinecke S, Lutzenburg M, Hagemann G, et al. Electrophysiological
transcortical diaschisis after middle cerebral artery occlusion (MCAO) in
rats. Neurosci Lett 1999;261:85–8
27. Neumann-Haefelin T, Witte OW. Periinfarct and remote excitability
changes after transient middle cerebral artery occlusion. J Cereb Blood
Flow Metab 2000;20:45–52
28. Carmichael ST, Tatsukawa K, Katsman D, et al. Evolution of diaschisis
in a focal stroke model. Stroke 2004;35:758–63
29. Garbuzova-Davis S, Rodrigues MCO, Hernandez-Ontiveros DG, et al.
Blood-brain barrier alterations provide evidence of subacute diaschisis in
an ischemic stroke rat model. PLoS One 2013;8:e63553
30. Garbuzova-Davis S, Haller E, Williams SN, et al. Compromised bloodbrain barrier competence in remote brain areas in ischemic stroke rats at
chronic stage. J Comp Neurol 2014;522:3120–37
31. Wu YP, Ling EA. Transsynaptic changes of neurons and associated
microglial reaction in the spinal cord of rats following middle cerebral artery occlusion. Neurosci Lett 1998;256:41–4
32. Wu YP, Ling EA. Induction of microglial and astrocytic response in the
adult rat lumbar spinal cord following middle cerebral artery occlusion.
Exp Brain Res 1998;118:235–42
33. Moisse K, Welch I, Hill T, et al. Transient middle cerebral artery occlusion induces microglial priming in the lumbar spinal cord: A novel model
of neuroinflammation. J Neuroinflam 2008;5:29
34. Fu D, Ng Y-K, Gan P, et al. Permanent occlusion of the middle cerebral
artery upregulates expression of cytokines and neuronal nitric oxide synthase in the spinal cord and urinary bladder in the adult rat. Neuroscience
2004;125:819–31
35. Block F, Dihné M, Loos M. Inflammation in areas of remote changes following focal brain lesion. Prog Neurobiol 2005;75:342–65
36. Borlongan CV, Hadman M, Sanberg CD, et al. Central nervous system
entry of peripherally injected umbilical cord blood cells is not required
for neuroprotection in stroke. Stroke 2004;35:2385–9
37. Tajiri N, Acosta S, Glover LE, et al. Intravenous grafts of amniotic fluidderived stem cells induce endogenous cell proliferation and attenuate behavioral deficits in ischemic stroke rats. PLoS One 2012;7:e43779
BSCB Damage in Focal Cerebral Ischemia
Garbuzova-Davis et al
16
71. Raineteau O, Fouad K, Bareyre FM, et al. Reorganization of descending motor tracts in the rat spinal cord. Eur J Neurosci 2002;16:
1761–71
72. Alstermark B, Ogawa J, Isa T. Lack of monosynaptic corticomotoneuronal EPSPs in rats: Disynaptic EPSPs mediated via reticulospinal neurons
and polysynaptic EPSPs via segmental interneurons. J Neurophysiol
2004;91:1832–9
73. Newman DB, Hilleary SK, Ginsberg CY. Nuclear terminations of corticoreticular fiber systems in rats. Brain Behav Evol 1989;34:223–64
74. Jankowska E, Hammar I, Slawinska U, et al. Neuronal basis of crossed
actions from the reticular formation on feline hindlimb motoneurons.
J Neurosci 2003;23:1867–78
75. Yang H-W, Lemon RN. An electron microscopic examination of the corticospinal projection to the cervical spinal cord in the rat: Lack of evidence for cortico-motoneuronal synapses. Exp Brain Res 2003;149:
458–69
76. Umeda T, Takahashi M, Isa K, et al. Formation of descending pathways
mediating cortical command to forelimb motoneurons in neonatally hemidecorticated rats. J Neurophysiol 2010;104:1707–16
77. Namjoshi DR, McErlane SA, Taepavarapruk N, et al. Network actions of
pentobarbital in the rat mesopontine tegmentum on sensory inflow
through the spinothalamic tract. J Neurophysiol 2009;102:700–13
78. Feldman SG, Kruger L. An axonal transport study of the ascending projection of medial lemniscal neurons in the rat. J Comp Neurol 1980;192:
427–54
Downloaded from http://jnen.oxfordjournals.org/ by guest on June 10, 2016
62. Nagasawa H, Kogure K. Correlation between cerebral blood flow and
histologic changes in a new rat model of middle cerebral artery occlusion. Stroke 1989;20:1037–43
63. Popp A, Jaenisch N, Witte OW, et al. Identification of ischemic regions
in a rat model of stroke. PLoS One 2009;4:e4764
64. Iizuka H, Sakatani K, Young W. Corticofugal axonal degeneration in rats
after middle cerebral artery occlusion. Stroke 1989;20:1396–402
65. Iizuka H, Sakatani K, Young W. Neural damage in the rat thalamus after
cortical infarcts. Stroke 1990;21:790–4
66. Dihné M, Grommes C, Lutzenburg M, et al. Different mechanisms of
secondary neuronal damage in thalamic nuclei after focal cerebral ischemia in rats. Stroke 2002;33:3006–11
67. Nudo RJ, Masterton RB. Descending pathways to the spinal cord, III:
Sites of origin of the corticospinal tract. J Comp Neurol 1990;296:559–83
68. Jeffery ND, Fitzgerald M. Lack of topographical organisation of the corticospinal tract in the cervical spinal cord of the adult rat. Brain Res
1999;833:315–8
69. Liu Z, Li Y, Qu R, et al. Axonal sprouting into the denervated spinal cord
and synaptic and postsynaptic protein expression in the spinal cord after
transplantation of bone marrow stromal cell in stroke rats. Brain Res
2007;1149:172–80
70. Antal M, Sholomenko GN, Moschovakis AK, et al. The termination
pattern and postsynaptic targets of rubrospinal fibers in the rat spinal
cord: A light and electron microscopic study. J Comp Neurol 1992;325:
22–37
J Neuropathol Exp Neurol • Volume 0, Number 0, 2016