Bryostatin Improves Survival and Reduces Ischemic Brain

Injury in Aged Rats After Acute Ischemic Stroke

Zhenjun Tan, MD; Ryan C. Turner, PhD; Rachel L. Leon, MD, PhD; Xinlan Li, MD;
Jarin Hongpaisan, PhD; Wen Zheng, MD; Aric F. Logsdon, BS; Zachary J. Naser, BS;
Daniel L. Alkon, MD; Charles L. Rosen, MD, PhD; Jason D. Huber, PhD

Background and Purpose—Bryostatin, a potent protein kinase C (PKC) activator, has demonstrated therapeutic efficacy
in preclinical models of associative memory, Alzheimer disease, global ischemia, and traumatic brain injury. In this
study, we tested the hypothesis that administration of bryostatin provides a therapeutic benefit in reducing brain injury
and improving stroke outcome using a clinically relevant model of cerebral ischemia with tissue plasminogen activator
reperfusion in aged rats.
Methods—Acute cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery (MCAO) in 18-
to 20-month-old female Sprague–Dawley rats using an autologous blood clot with tissue plasminogen activator–mediated
reperfusion. Bryostatin was administered at 6 hours post-MCAO, then at 3, 6, 9, 12, 15, and 18 days after MCAO.
Functional assessment was conducted at 2, 7, 14, and 21 days after MCAO. Lesion volume and hemispheric swelling/
atrophy were performed at 2, 7, and 21 days post-MCAO. Histological assessment of PKC isozymes was performed at
24 hours post-MCAO.
Results—Bryostatin-treated rats showed improved survival post-MCAO, especially during the first 4 days. Repeated
administration of bryostatin post-MCAO resulted in reduced infarct volume, hemispheric swelling/atrophy, and improved
neurological function at 21 days post-MCAO. Changes in αPKC expression and εPKC expression in neurons were noted
in bryostatin-treated rats at 24 hours post-MCAO.
Conclusions—Repeated bryostatin administration post-MCAO protected the brain from severe neurological injury post-MCAO.
Bryostatin treatment improved survival rate, reduced lesion volume, salvaged tissue in infarcted hemisphere by reducing
necrosis and peri-infarct astrogliosis, and improved functional outcome after MCAO.   (Stroke. 2013;44:3490-3497.)
Key Words: aging ◼ blood-brain barrier ◼ infarction ◼ protein kinase C ◼ tissue plasminogen activator

T hrombolytic treatment with recombinant tissue plasmino-

gen activator (tPA) remains the only US Food and Drug
Administration–approved drug for treatment of acute isch-
emic stroke. However, <3% of people with ischemic stroke
receive tPA, because of increased risk of secondary cerebral
hemorrhage and edema formation.1 Based on the aging popu-
lation and increased stroke burden, an unmet need exists to
develop alternative approaches to treat acute ischemic stroke,
either independently of tPA or in an effort to increase the num-
ber of patients eligible for tPA.
Preclinical studies of proposed therapeutics for ischemic
stroke have largely failed to consider the greatest risk fac-
tor for stroke—age.2 Previous studies suggest that aged rats
represent a more clinically relevant model of ischemic stroke
as compared with younger animals.3,4 These studies illustrate
an increased severity of ischemic injury and altered neural
injury progression.3–6 Thus, use of aged animals may increase
clinical relevance and the likelihood of bench-to-bedside ther-
apeutic translation.
Protein kinase C (PKC) plays a critical role in storage of
associative memory and related synaptogenesis in normal
animals7; reducing the accumulation of β-amyloid, synaptic
loss, cognitive deficits, and neurodegeneration in preclini-
cal models of Alzheimer disease8,9; and protecting neurons
against ischemic pathology10,11 and traumatic brain injury in
rodents.12 PKC isozymes α and ε regulate synaptogenic and
antiapoptotic signaling pathways,13 as well as critical func-
tional pathways in the multicellular response to ischemia/
reperfusion injury.14 Individual PKC isozymes play differ-
ing, and sometimes opposing, roles in injury response that
often depend on cell types and degree of pathophysiology.15,16
Observations that PKC activation mediates both protective and
harmful messages results from different PKC isoforms being
activated by different signals that play concentration- and

Received June 7, 2013; accepted September 13, 2013.

From the Department of Neurosurgery, School of Medicine (Z.T., R.C.T., R.L.L., X.L., Z.J.N., C.L.R.), Blanchette Rockefeller Neuroscience Institute
(J.H., W.Z., D.L.A.), and Department of Basic Pharmaceutical Science, School of Pharmacy (A.F.L., J.D.H.), West Virginia University Health Sciences
Center, Morgantown, WV.
Correspondence to Jason D. Huber, PhD, Basic Pharmaceutical Sciences, West Virginia University Health Sciences Center, Morgantown, WV 26506-9530.
E-mail jdhuber@hsc.wvu.edu
© 2013 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.113.002411

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 Tan et al   Bryostatin Reduces Ischemic Brain Injury in Aged Rat    3491
time-dependent roles in the development of neuronal damage
and regeneration.7,17
Bryostatin, an ultrapotent PKC activator, may provide
substantial benefit in the treatment of acute ischemic stroke.
Studied extensively as an antitumorogenic agent, recent stud-
ies demonstrate that administration of bryostatin after global
ischemic insult resulted in curative neurogenesis, synaptogen-
esis, and cognitive enhancement.11 The purpose of this study
was to investigate the pharmacological potential of repeated
administration of bryostatin to improve outcome after acute
ischemic stroke in aged rats.
Materials and Methods
Animals and Treatment Protocol
Sixty-six female Sprague–Dawley rats (18–20 months old) were
purchased from Hilltop Laboratories (Scottdale, PA) and housed un-
der 12-hour light–12-hour dark conditions with food and water ad
libitum. All work involving rats was approved by the West Virginia
University Animal Care and Use Committee. For study 1, rats were
randomly divided into 2 treatment groups (N=56). Group 1 under-
went a 2-hour middle cerebral artery occlusion (MCAO) with tPA-
mediated (5 mg/kg, i.v.) reperfusion as previously described2,18 and
at 6 hours was administered bryostatin (2.5 mg/kg, i.v.) with subse-
quent doses every 3 days for a total of 7 doses over 21 days. Animals
in group 2 served as the control and underwent the same procedures
except they were administered 0.9% saline, instead of bryostatin, at
6 hours post-MCAO. Rats were assayed at 2, 7, and 21 days after
MCAO. A second set of rats (N=10; 6 saline and 4 bryostatin-treated
rats) underwent MCAO with bryostatin/saline treatment at 6 hours
and were then euthanized at 24 hours to visualize changes in PKC
isozyme (α and ε) expression in neurons, endothelial cells, and as-
trocytes. Ischemia and reperfusion were defined as a minimum of
80% decrease in cerebral blood flow at onset of ischemia and return
to 80% of baseline blood flow measurement, respectively. Rats not
achieving these standards were excluded from the study.2,18
Neurological Functional Assessment
The modified Neurological Severity Scores (mNSS) was used to as-
sess neurological function at 2, 7, 14, and 21 days after MCAO (n=7
saline and 10 bryostatin-treated rats). mNSS is a composite score of
motor, sensory, balance, and reflex measures ranging from scores of
1 to 17, with higher scores indicating greater neurological injury.19
TUNEL Staining, Immunohistochemistry, and
Infarct Volume Measurement
TUNEL staining was performed at 7 (n=5 rats per treatment) and 21
(n=7 saline and 10 bryostatin-treated rats) days after MCAO accord-
ing to manufacturer’s instructions (Roche Applied Science). After
mounting and nuclear staining with DAPI, slices were visualized for
apoptotic tissue.
Using alternating slices from the same brain, immunohistochemis-
try using a modified ABC procedure was performed with anti-GFAP
(1:1000) for evaluation of astrocyte morphology (n=7 saline and 10
bryostatin-treated rats). At 2 (n=4 rats per treatment), 7 (n=5 rats per
treatment), and 21 (n=7 saline and 10 bryostatin-treated rats) days af-
ter MCAO, lesion volumes were determined using cresyl violet stain-
ing. Degree of cerebral hemispheric swelling/atrophy at 2, 7, and 21
days after MCAO was calculated using the formula: cerebral hemi-
spheric swelling/atrophy=(LV−RV)/LV×100%, where LV was volume
(mm3) of left hemisphere and RV was volume of right hemisphere.
Trilabeled confocal microscopy was performed to determine immuno-
localization of εPKC (1:100) and αPKC (1:100) with neurons (1:150),
endothelial cells (1:100), and astrocytes (1:100) at 24 hours after MCAO
(n=4 rats per treatment). Nuclear staining was performed on sections us-
ing DAPI, and secondary antibodies were incubated at 1:200 dilution.
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Expression levels of DNA damage (TUNEL), εPKC, and αPKC
were evaluated based on immunofluorescence intensity by an observ-
er blinded to treatment group. Confocal images were quantified using
ImageJ as previously described.9
Water Maze Test
Spatial acquisition was determined using 6 training days with a
probe test on day 7. Training began the 15th day post-MCAO and
the probe test was conducted at 21 days post-MCAO (n=3 saline
and 4 ­bryostatin-treated rats). Learning trials were conducted over
6 consecutive days with 4 trials per day. Time interval between tri-
als was 60 seconds, and maximum swim time to find platform was
120 seconds. Start location used N, S, E, and W designations for the
start of each trial. Start locations for each trial were semirandomly
selected so that all directions were used for each rat on each training
day. Each training session and probe test was conducted by investiga-
tors blinded to treatment. During the training period and probe test,
latency speed, as well as distance traveled to platform and 300% of
platform, was measured.
Statistical Analysis
All data were compiled and analyzed by an investigator blinded to
treatment and presented as mean±SEM. Physiological parameters,
functional data, and immunofluorescence intensity were compared
by Student t test or ANOVA grouped by treatment (saline versus
bryostatin) or days post-MCAO (2, 7, and 21 days). Mortality data
were compared using Fisher exact test. P<0.05 was considered sta-
tistically significant.
Results
Administration of Bryostatin After MCAO
in Aged Rats Improved Survival Rate and
Neurological Function
A total of 66 rats were used in this study. Fifty-six rats were
evaluated for effects of bryostatin on ischemic brain injury over
21 days after MCAO. Two rats from this cohort, both saline-
treated, were excluded from study for not meeting ischemia/
reperfusion criteria. Survival rates were documented daily
from 1 to 21 days (Figure 1A). Administration of bryostatin
post-MCAO improved survival rates through 21 days with
positive effects most evident during the first 4 days and a sig-
nificant increase in survival achieved from 2 to 17 days post-
MCAO. Although survival decreased in bryostatin-treated rats
after day 4, improved survival was demonstrated at 7 days
(68% bryostatin, n=13, versus 41% saline-treated rats, n=7),
14 days (59% bryostatin, n=11, versus 41% saline-treated
rats, n=7), and 21 days (53% bryostatin, n=10, versus 41%
saline-treated rats, n=7) post-MCAO. The dosing schedule
used in this study was selected based on previous preclinical
studies showing cognitive enhancement in rats,7 therapeutic
efficacy in young rats after global ischemia,11 and neuropro-
tection in transgenic mice displaying Alzheimer disease–like
symptoms. Based on the results of this study, future studies
will need to use dose–response measurements to determine
the optimal dosing schedule for acute ischemic stroke in aged
rats. Functional assessment revealed a significant improvement
in mNSS scores at 21 days post-MCAO in bryostatin-treated
rats (2.6±0.3; n=10) compared with saline-treated rats (3.7±0.2;
n=7; Figure 1B). No difference (P>0.05) in mNSS scores was
observed at 2 (n=15 saline and 18 bryostatin-treated rats),
7 (n=7 saline and 13 bryostatin-treated rats), and 14 (n=7 saline
3492  Stroke  December 2013

Figure 1. A, Administration of bryostatin increased the survival rate of aged rats from 2 to 17 days after middle cerebral artery occlusion
(MCAO) and tissue plasminogen activator reperfusion. B, Neurological function was improved in bryostatin-treated aged rats at 21 days
post-MCAO. No difference in neurological score between saline- and bryostatin-treated rats was measured at 2, 7, and 14 days post-
MCAO. Latency speed (C) and distance traveled (D) were measured at 21 days post-MCAO. Results showed a reduction in latency speed
and distance traveled in bryostatin-treated rats as compared with saline-treated rats. Both treatment groups demonstrated a reduction in
distance traveled when using the 300% platform. Data presented as means±SEM.

and 11 bryostatin-treated rats) days post-MCAO. Spatial acqui-
sition was measured using the Morris water maze test. Results
showed no difference (P>0.05) between treatment groups in
latency speed or distance traveled to platform during the 6 train-
ing days. However, a significant improvement in latency speed
(200±10 cm/s; n=3) and distance traveled (13.2±0.8 m; n=3) to
platform was measured in bryostatin-treated rats as compared
with saline-treated rats (300±10 cm/s and 15.9±0.4 m, respec-
tively; n=4) on probe day (Figure 1C and 1D). When the plat-
form was increased to 300% of the test platform, no difference
(P>0.05) in latency speed and distance traveled was observed
between treatment groups (Figure 1C and 1D). Both saline- and
bryostatin-treated rats demonstrated a significant decrease in
distance traveled to find the 300% platform as compared with
the test platform (Figure 1D). No difference (P>0.05) in latency
speed between treatment groups was measured (Figure 1C).
Bryostatin Resulted in Diminished Lesion
Volume Post-MCAO
Quantification of lesion volume for cortex, striatum, and total
cerebral hemisphere was measured at 2, 7, and 21 days post-
MCAO using cresyl violet–stained coronal sections. No dif-
ference (P>0.05) in lesion volume was observed at 2 days
post-MCAO (Figure 2A). At 7 days, bryostatin administra-
tion resulted in a significant reduction in lesion volume in the
striatum and total hemisphere as compared with saline-treated
rats (Figure 2A). At 21 days, a significant decrease in lesion vol-
ume was observed in cortex, striatum, and total hemisphere as
compared with saline-treated rats (Figure 2A). Measurements
of cortical hemispheric swelling or atrophy revealed a signifi-
cant decrease in hemispheric swelling at 2 and 7 days after
MCAO (Figure 2B). At 21 days after MCAO, the swelling
phase of ischemic brain injury had subsided and atrophy of
the saline-treated rats was visually evident by cavitation and
necrosis of the infarcted hemisphere (Figure 3B and 3D).
A significant decrease in atrophy was measured in bryostatin-
treated rats at 21 days after MCAO (Figure 2C).
Administration of Bryostatin Decreases Astrogliosis
and Reduces Cellular Apoptosis in Aged Rats at 7
and 21 Days After MCAO
A significant reduction in TUNEL-positive cells, representing
those cells undergoing apoptosis because of DNA fragmenta-
tion, was detected in the peri-infarct region surrounding the
infarct in bryostatin-treated rats at 7 (75±6% of control) and 21
(43±3% of control) days after MCAO (Figure 4). This finding
was reinforced by a profound reduction in reactive astrocytes,
identified by GFAP expression, observed in bryostatin-treated
rats, in which not only was astrogliosis reduced, but also
the degree of tissue destruction was markedly attenuated

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 Tan et al   Bryostatin Reduces Ischemic Brain Injury in Aged Rat    3493

Figure 2. A, Repeated administration of bryostatin decreased lesion volume in striatum and total hemisphere at 7 days after middle cere-
bral artery occlusion (MCAO). At 21 days after MCAO, lesion volume was reduced in the cortex, striatum, and total hemisphere. No dif-
ference in lesion volume was measured at 2 days post-MCAO. B, Bryostatin reduced hemispheric swelling at 2 and 7 days after MCAO.
C, Bryostatin reduced cerebral atrophy in the ischemic hemisphere as compared with saline-treated rats at 21 days post-MCAO. Data
presented as means±SEM.

(Figure 3). At both 7 and 21 days, astrocytic hypertrophy
with enlarged processes was evident in tissue surrounding
the necrotic zone around the infarct in saline-treated aged
rats, consistent with the astrocytic response. Bryostatin
attenuated this response and reduced tissue damage in the
peri-infarct area (Figure 3).
αPKC Expression Was Reduced in Neurons in the
Penumbral Region of Bryostatin-Treated Rats at 24
Hours After MCAO
Colocalization of αPKC with neurons (NeuN), endothelial
cells (CD31), and astrocytes (GFAP) was investigated in
penumbra surrounding the infarct in aged rats at 24 hours
post-MCAO (n=4 rats per treatment group). αPKC immu-
noreactivity was observed in neurons and endothelial cells
(Figures 5A and 6A). Expression of αPKC was diffusely
localized throughout the cytoplasm of the cell. No colocaliza-
tion of αPKC was shown with GFAP-positive astrocytes (data
not shown). Rats treated with bryostatin showed a signifi-
cant decrease in immunoreactivity of αPKC in neurons at 24
hours after MCAO (Figure 5A and 5C). No change (P=0.23)
in αPKC expression was observed in endothelial cells at 24
hours after MCAO (Figure 6A and 6C).
Bryostatin Increased εPKC Within Neurons
in the Penumbra at 24 Hours After MCAO
and tPA Reperfusion
Colocalization of εPKC with NeuN and CD31 in the penum-
bral area at 24 hours after MCAO was investigated (n=4 rats
per treatment group). εPKC immunoreactivity was observed in
neurons and endothelial cells (Figures 5B and 6B). Expression
of εPKC was punctate along cytoplasmic edge of the plasma
membrane of the cell. No colocalization of εPKC was shown
with GFAP-positive astrocytes. Rats treated with bryostatin
showed a significant increase in immunoreactivity of εPKC in
neurons at 24 hours after MCAO (Figure 5B and 5C).
Discussion
The primary finding of this study was that administration of
bryostatin after MCAO with tPA reperfusion decreased mor-
tality over 21 days, with marked improvement during the first
4 days. Additionally, to the best of our knowledge, we are the
first to report that bryostatin administration improved recov-
ery from ischemic brain injury and functional outcome up
to 21 days after MCAO in aged animals. Use of bryostatin
attenuated both necrotic and apoptotic cell death. The reduced
hemispheric swelling/atrophy observed at 2 and 7 days after

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3494  Stroke  December 2013

Figure 3. A, Representative photomicro-

graphs of GFAP-positive cells at 7 and 21
days after middle cerebral artery occlusion
(MCAO). Astrogliosis was observed in saline-
treated brain at 7 and 21 days after MCAO;
bryostatin mitigated the degree of astroglio-
sis. B, At 21 days after MCAO, cavitation and
necrosis was evident in saline-treated rats.
Administration of bryostatin decreased lesion
volume and reduced atrophy as compared
with saline-treated rats. Cell nuclei were
stained using DAPI.

MCAO and the significant attenuation of ipsilateral hemi-
spheric atrophy seen in aged rats treated with bryostatin at
21 days suggests the potential for decreased inflammation
and reduced cellular death after MCAO and tPA reperfusion.
A marked improvement in neurological function was observed
in bryostatin-treated rats at 21 days after MCAO. In this study,
we also evaluated changes in poststroke cognition using the
Morris water maze test. Results showed that bryostatin-treated
rats had improved spatial cognition as compared with saline-
treated rats at 21 days after MCAO. Extinguishment of the
cognitive deficit in saline-treated rats in the 300% platform
trial suggests that discrimination of environment is improved
in aged rats treated with bryostatin.

Figure 4. Comparison of TUNEL staining in aged rats showed a marked reduction in TUNEL-positive cells in bryostatin-treated rats at 7
and 21 days after middle cerebral artery occlusion as compared with saline-treated rats. Cell nuclei were stained using DAPI.

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 Tan et al   Bryostatin Reduces Ischemic Brain Injury in Aged Rat    3495

Figure 5. Representative images of tricolor confocal immunofluorescent microscopy demonstrating colocalization of NeuN with αPKC (A)
and εPKC (B) in the penumbra of the infarcted hemisphere of saline- and bryostatin-treated aged rats at 24 hours after middle cerebral
artery occlusion (MCAO). C, In neurons, a 68% decrease in αPKC expression and a 202% increase in εPKC expression were measured at
24 hours after MCAO in bryostatin-treated rats as compared with saline-treated rats. Data expressed as means±SEM; *P<0.05. Cell nuclei
were stained using DAPI.

Effects measured in this study were associated with changes
in expression and localization of PKC isozymes in neurons
and endothelial cells within the peri-infarct region.
Regulation of PKC activity is the primary reported mech-
anism of action for bryostatin. Inhibition of PKC isozymes
blocked the synaptogenic and cognitive-enhancing effects
of bryostatin on normal animals.7 Therefore, it is plausible
that pathological mechanisms initiated after ischemic brain
injury involve PKC-mediated pathways.20,21 Results from this
study clearly show increased εPKC expression and a modest
decrease in αPKC expression in neurons of bryostatin-treated
rats at 24 hours post-MCAO. Roles of PKC activity after isch-
emic stroke are complex and poorly defined. What is known is
that PKC activity mediates protective and damaging signaling
cascades during the injury and reparative phases of ischemic
stroke,15,16 suggesting that timing, localization, and the PKC
isozyme activated play critical, and sometimes contrasting,
roles in injury response and progression of neuronal dam-
age and regeneration after stroke.7,17 Previous studies report
that increased εPKC activity initiates proliferative and cell
survival signaling pathways after ischemic stroke and reper-
fusion,14,22,23 whereas increased αPKC activity exacerbates
neuronal damage after poststroke brain injury through ini-
tiation of antiproliferative and proapoptotic pathways24; thus,
although still inconclusive, our results suggest that changes
in PKC activity in endothelial cells and neurons may play a
vital role in the acute neuroprotective actions of bryostatin.
It should be noted that although bryostatin initially activates
both αPKC and εPKC, it only causes prolonged binding of
εPKC to the critically required anchoring protein, RACK 1,
that is required for prolonged activation (unpublished data).
Of particular interest especially during the acute phase of
stroke injury is the role different PKC isozymes play in altera-
tions of endothelial cell function during pathophysiological
conditions. Increased PKC activity has been shown to affect
structural and functional integrity of the blood-brain barrier
after ischemic brain injury25 with αPKC and βPKC increasing
blood-brain barrier opening through alterations in tight junc-
tion protein localization, and εPKC enhancing blood-brain
barrier integrity by upregulating the expression of claudin
5.26–28 Our study showed that αPKC and εPKC were localized
to both neurons and cerebral endothelial cells. No colocal-
ization of εPKC or αPKC was observed with GFAP-positive
astrocytes; however, that does not mean that changes in PKC
activity do not have a significant influence on astrocyte reac-
tivity as demonstrated by our study, which showed a reduction
in astrogliosis and tissue damage in bryostatin-treated rats.
In fact, in relation to chronic brain response after ischemic
injury, attenuation of glial response may end up being the
most significant finding of this study. A recent study shows
that PKC activation leads to a significant decrease in calcium
wave propagation between astrocytes by attenuating astroglial
gap junction communication.29 Gaining a better understanding
of the critical mediators and timing of this response will be a
primary focus of follow-up studies. A limitation of this study
and immunofluorescent imaging, in general, was the effect of
bryostatin on PKC activation. Future studies will probe for
changes in PKC activity by measuring cytosolic to membrane

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3496  Stroke  December 2013

Figure 6. Representative images of tricolor confocal immunofluorescent microscopy demonstrating colocalization of CD31 (endothelial
cells) with αPKC (A) and εPKC (B) in the penumbra of the infarcted hemisphere of saline- and bryostatin-treated aged rats at 24 hours
after middle cerebral artery occlusion (MCAO). C, In endothelial cells, no difference (P>0.05) in αPKC and εPKC expression was observed
at 24 hours after MCAO between bryostatin- and saline-treated rats. Data expressed as means±SEM; *P<0.05. Cell nuclei were stained
using DAPI.

translocation and phosphorylation of specific PKC isozymes Disclosures

at different time points postischemia.
In conclusion, administration of bryostatin after MCAO and
tPA reperfusion has neuroprotective effects on the magnitude
of ischemic brain injury. A notable finding in this study was
the marked improvement in survival observed in bryostatin-
treated rats, especially during the first week after ischemic
insult. Although mortality increased in the bryostatin-treated
group during the 21 days, it must be emphasized that PKC
activator treatment, depending on dose and duration, causes 3
sequential consequences: activation, downregulation (and thus
inhibition), and de novo protein synthesis. Thus, careful selec-
tion of treatment dosing and duration has a profound impact
on PKC-mediated phosphorylation of downstream substrates
and associated therapeutic benefits.30 The beneficial effects of
bryostatin administration after stroke is most likely because
of attenuation of inflammatory cell activity; thus, bryostatin
may act as a potent adjuvant with other proven methods,
such as hypothermia,31 for reduction of inflammatory reac-
tion during the acute phase of ischemia/reperfusion injury. In
summary, bryostatin-treated rats displayed reduced ischemic
brain injury after MCAO, characterized by increased survival,
reduced infarct volume, decreased hemispheric swelling/atro-
phy, and improved neurological function.
Sources of Funding
This study was supported by a grant from National Institutes of
Health’s National Institute of Neurological Disorders and Stroke
(RO1 NS061954 to J.D.H.).
None.
References
1. Katzan IL, Hammer MD, Hixson ED, Furlan AJ, Abou-Chebl A, Nadzam
DM; Cleveland Clinic Health System Stroke Quality Improvement
Team. Utilization of intravenous tissue plasminogen activator for acute
ischemic stroke. Arch Neurol. 2004;61:346–350.
2. Rosen CL, Dinapoli VA, Nagamine T, Crocco T. Influence of age
on stroke outcome following transient focal ischemia. J Neurosurg.
2005;103:687–694.
3. DiNapoli VA, Huber JD, Houser K, Li X, Rosen CL. Early disruptions
of the blood-brain barrier may contribute to exacerbated neuronal dam-
age and prolonged functional recovery following stroke in aged rats.
Neurobiol Aging. 2008;29:753–764.
4. Shlosberg D, Benifla M, Kaufer D, Friedman A. Blood-brain barrier
breakdown as a therapeutic target in traumatic brain injury. Nat Rev
Neurol. 2010;6:393–403.
5. Badan I, Buchhold B, Hamm A, Gratz M, Walker LC, Platt D, et al.
Accelerated glial reactivity to stroke in aged rats correlates with reduced
functional recovery. J Cereb Blood Flow Metab. 2003;23:845–854.
6. Dinapoli VA, Benkovic SA, Li X, Kelly KA, Miller DB, Rosen CL, et al.
Age exaggerates proinflammatory cytokine signaling and truncates sig-
nal transducers and activators of transcription 3 signaling following isch-
emic stroke in the rat. Neuroscience. 2010;170:633–644.
7. Hongpaisan J, Alkon DL. A structural basis for enhancement of long-
term associative memory in single dendritic spines regulated by PKC.
Proc Natl Acad Sci U S A. 2007;104:19571–19576.
8. Etcheberrigaray R, Tan M, Dewachter I, Kuipéri C, Van der Auwera I,
Wera S, et al. Therapeutic effects of PKC activators in Alzheimer’s dis-
ease transgenic mice. Proc Natl Acad Sci U S A. 2004;101:11141–11146.
9. Hongpaisan J, Sun MK, Alkon DL. PKC ε activation prevents synaptic
loss, Aβ elevation, and cognitive deficits in Alzheimer’s disease trans-
genic mice. J Neurosci. 2011;31:630–643.
10. Sun MK, Hongpaisan J, Alkon DL. Postischemic PKC activation res-
cues retrograde and anterograde long-term memory. Proc Natl Acad Sci
U S A. 2009;106:14676–14680.

Downloaded from http://stroke.ahajournals.org/ at CMU Libraries - library.cmich.edu on September 13, 2015

 Tan et al   Bryostatin Reduces Ischemic Brain Injury in Aged Rat    3497
11. Sun MK, Hongpaisan J, Nelson TJ, Alkon DL. Poststroke neuronal res-
cue and synaptogenesis mediated in vivo by protein kinase C in adult
brains. Proc Natl Acad Sci U S A. 2008;105:13620–13625.
12. Zohar O, Lavy R, Zi X, Nelson TJ, Hongpaisan J, Pick CG, et al. PKC
activator therapeutic for mild traumatic brain injury in mice. Neurobiol
Dis. 2011;41:329–337.
13. Quattrone A, Pascale A, Nogues X, Zhao W, Gusev P, Pacini A, et al.
Posttranscriptional regulation of gene expression in learning by the neu-
ronal ELAV-like mRNA-stabilizing proteins. Proc Natl Acad Sci U S A.
2001;98:11668–11673.
14. Bright R, Mochly-Rosen D. The role of protein kinase C in cerebral isch-
emic and reperfusion injury. Stroke. 2005;36:2781–2790.
15. Matsumoto S, Shamloo M, Matsumoto E, Isshiki A, Wieloch T. Protein
kinase C-gamma and calcium/calmodulin-dependent protein kinase
II-alpha are persistently translocated to cell membranes of the rat brain
during and after middle cerebral artery occlusion. J Cereb Blood Flow
Metab. 2004;24:54–61.
16. Skaper SD, Facci L, Strijbos PJ. Neuronal protein kinase signaling cas-
cades and excitotoxic cell death. Ann N Y Acad Sci. 2001;939:11–22.
17. Reshef A, Sperling O, Zoref-Shani E. Activation and inhibition of pro-
tein kinase C protect rat neuronal cultures against ischemia-reperfusion
insult. Neurosci Lett. 1997;238:37–40.
18. Dinapoli VA, Rosen CL, Nagamine T, Crocco T. Selective MCA
occlusion: a precise embolic stroke model. J Neurosci Methods.
2006;154:233–238.
19. Chen J, Li Y, Wang L, Zhang Z, Lu D, Lu M, et al. Therapeutic benefit
of intravenous administration of bone marrow stromal cells after cerebral
ischemia in rats. Stroke. 2001;32:1005–1011.
20. Popa-Wagner A, Buga AM, Kokaia Z. Perturbed cellular response to
brain injury during aging. Ageing Res Rev. 2011;10:71–79.
21. Selvatici R, Marino S, Piubello C, Rodi D, Beani L, Gandini E, et al.
Protein kinase C activity, translocation, and selective isoform subcellular
redistribution in the rat cerebral cortex after in vitro ischemia. J Neurosci
Res. 2003;71:64–71.
Downloaded from http://stroke.ahajournals.org/ at CMU Libraries - library.cmich.edu on September 13, 2015
22. Bright R, Sun GH, Yenari MA, Steinberg GK, Mochly-Rosen D. epsi-
lonPKC confers acute tolerance to cerebral ischemic reperfusion injury.
Neurosci Lett. 2008;441:120–124.
23. Zhao CT, Li K, Li JT, Zheng W, Liang XJ, Geng AQ, et al. PKCdelta
regulates cortical radial migration by stabilizing the Cdk5 activator p35.
Proc Natl Acad Sci U S A. 2009;106:21353–21358.
24. Shirai Y, Adachi N, Saito N. Protein kinase Cepsilon: function in neu-
rons. FEBS J. 2008;275:3988–3994.
25. Willis CL, Meske DS, Davis TP. Protein kinase C activation modulates
reversible increase in cortical blood-brain barrier permeability and tight
junction protein expression during hypoxia and posthypoxic reoxygen-
ation. J Cereb Blood Flow Metab. 2010;30:1847–1859.
26. Fleegal MA, Hom S, Borg LK, Davis TP. Activation of PKC modu-
lates blood-brain barrier endothelial cell permeability changes induced
by hypoxia and posthypoxic reoxygenation. Am J Physiol Heart Circ
Physiol. 2005;289:H2012–H2019.
27. Krizbai I, Szabó G, Deli M, Maderspach K, Lehel C, Oláh Z, et al.
Expression of protein kinase C family members in the cerebral endothe-
lial cells. J Neurochem. 1995;65:459–462.
28. Sonobe Y, Takeuchi H, Kataoka K, Li H, Jin S, Mimuro M, et al.
Interleukin-25 expressed by brain capillary endothelial cells maintains
blood-brain barrier function in a protein kinase Cepsilon-dependent
manner. J Biol Chem. 2009;284:31834–31842.
29. Enkvist MO, McCarthy KD. Activation of protein kinase C blocks astro-
glial gap junction communication and inhibits the spread of calcium
waves. J Neurochem. 1992;59:519–526.
30. Alkon DL, Sun MK, Nelson TJ. PKC signaling deficits: a mechanistic
hypothesis for the origins of Alzheimer’s disease. Trends Pharmacol Sci.
2007;28:51–60.
31. Joseph C, Buga AM, Vintilescu R, Balseanu AT, Moldovan M, Junker
H, et al. Prolonged gaseous hypothermia prevents the upregulation of
phagocytosis-specific protein annexin 1 and causes low-amplitude EEG
activity in the aged rat brain after cerebral ischemia. J Cereb Blood Flow
Metab. 2012;32:1632–1642.
 Bryostatin Improves Survival and Reduces Ischemic Brain Injury in Aged Rats After
Acute Ischemic Stroke
Zhenjun Tan, Ryan C. Turner, Rachel L. Leon, Xinlan Li, Jarin Hongpaisan, Wen Zheng, Aric
F. Logsdon, Zachary J. Naser, Daniel L. Alkon, Charles L. Rosen and Jason D. Huber

Stroke. 2013;44:3490-3497; originally published online October 30, 2013;

doi: 10.1161/STROKEAHA.113.002411
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