Hyperglycemia in stroke and possible treatments

Abstract

Hyperglycemia affects approximately one-third of acute ischemic stroke patients and is associated with poor clinical outcomes. In experimental and clinical stroke studies, hyperglycemia has been shown to be detrimental to the penumbral tissue for several reasons. First, hyperglycemia exacerbates both calcium imbalance and the accumulation of reactive oxygen species (ROS) in neurons, leading to increased apoptosis. Second, hyperglycemia fuels anaerobic energy production, causing lactic acidosis, which further stresses neurons in the penumbral regions. Third, hyperglycemia decreases blood perfusion after ischemic stroke by lowering the availability of nitric oxide (NO), which is a crucial mediator of vasodilation. Lastly, hyperglycemia intensifies the inflammatory response after stroke, causing edema, and hemorrhage through disruption of the blood brain barrier and degradation of white matter, which leads to a worsening of functional outcomes. Many neuroprotective treatments addressing hyperglycemia in stroke have been implemented in the past decade. Early clinical use of insulin provided mixed results due to insufficiently controlled glucose levels and heterogeneity of patient population. Recently, however, the latest Stroke Hyperglycemia Insulin Network Effort trial has addressed the shortcomings of insulin therapy. While glucagon-like protein-1 administration, hyperbaric oxygen preconditioning, and ethanol therapy appear promising, these treatments remain in their infancy and more research is needed to better understand the mechanisms underlying hyperglycemia-induced injuries. Elucidation of these mechanistic pathways could lead to the development of rational treatments that reduce hyperglycemia-associated injuries and improve functional outcomes for ischemic stroke patients.

Full text

Review
Hyperglycemia in stroke and possible
treatments
William A. Li
1
, Shannon Moore-Langston
1
, Tia Chakraborty
1
, Jose A. Rafols
2
,
Alana C. Conti
1,3
, Yuchuan Ding
1
1
Department of Neurological Surgery, Wayne State University School of Medicine, Detroit, MI, USA,
2
Department
of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, MI, USA,
3
Research Service,
John D. Dingell VA Medical Center, Detroit, MI, USA
Hyperglycemia affects approximately one-third of acute ischemic stroke patients and is associated with
poor clinical outcomes. In experimental and clinical stroke studies, hyperglycemia has been shown to be
detrimental to the penumbral tissue for several reasons. First, hyperglycemia exacerbates both calcium
imbalance and the accumulation of reactive oxygen species (ROS) in neurons, leading to increased
apoptosis. Second, hyperglycemia fuels anaerobic energy production, causing lactic acidosis, which
further stresses neurons in the penumbral regions. Third, hyperglycemia decreases blood perfusion after
ischemic stroke by lowering the availability of nitric oxide (NO), which is a crucial mediator of vasodilation.
Lastly, hyperglycemia intensifies the inflammatory response after stroke, causing edema, and hemorrhage
through disruption of the blood brain barrier and degradation of white matter, which leads to a worsening of
functional outcomes. Many neuroprotective treatments addressing hyperglycemia in stroke have been
implemented in the past decade. Early clinical use of insulin provided mixed results due to insufficiently
controlled glucose levels and heterogeneity of patient population. Recently, however, the latest Stroke
Hyperglycemia Insulin Network Effort trial has addressed the shortcomings of insulin therapy. While
glucagon-like protein-1 administration, hyperbaric oxygen preconditioning, and ethanol therapy appear
promising, these treatments remain in their infancy and more research is needed to better understand the
mechanisms underlying hyperglycemia-induced injuries. Elucidation of these mechanistic pathways could
lead to the development of rational treatments that reduce hyperglycemia-associated injuries and improve
functional outcomes for ischemic stroke patients.
Keywords: Stroke, Hyperglycemia, Reactive oxygen species, Metabolism, Blood–brain barrier, Ethanol, Insulin, NADPH oxidase
Introduction
Stroke is a major cause of mortality and morbidity in
America, with an annual combined direct and
indirect cost exceeding 68 billion dollars.
1
In recent
years, accumulating literature supports the notion
that persisting hyperglycemia leads to an unfavorable
prognosis in stroke patients. While diabetes mellitus
is a well-known risk factor contributing to poor
prognosis after ischemic strokes, hyperglycemia with-
out pre-existing diabetes mellitus is also linked to
increased mortality and morbidity after stroke. In
fact, epidemiologic meta-analysis of 30-day post-
stroke mortality revealed a greater than three-fold
increase for non-diabetic hyperglycemic patients,
compared with a two-fold increase for hyperglycemic
patients with diabetes mellitus.
2
Elevated blood glucose, defined as levels greater
than 6.0 mmol/l (108 mg/dl), has been observed in
over one-third of all ischemic stroke patients.
Furthermore, evidence in experimental animal stroke
models supports the observation that hyperglycemia
not only exacerbates stroke related injury but that it
also adversely affects overall functional outcome.
3,4
In this review, the mechanisms by which hyperglyce-
mia exacerbates negative outcomes in ischemic stroke
and several potential treatments are discussed.
Mechanisms of Hyperglycemia-Mediated
Damage in Ischemic Stroke
Following experimental ischemic stroke, hyperglyce-
mic animals generally tend to have higher incidence
of brain edema, cell death, hemorrhagic transforma-
tion, and larger infarct volume.
3,5
Possible mechan-
isms include intracellular acidification, reactive
oxygen species (ROS) accumulation, blood-brain
barrier (BBB) disruption, inflammatory response
induction, and axonal degradation.
6–9
Correspondence to: Y. C. Ding, Department of Neurological Surgery,
Wayne State University School of Medicine, 550 E Canfield, Detroit, MI
48201, USA. Email: yding@med.wayne.edu
ßW. S. Maney & Son Ltd 2013
DOI 10.1179/1743132813Y.0000000209 Neurological Research 2013 VOL.35 NO.5 479

Figure 1 Elevated cell apoptosis due to calcium imbalance. Excess glucose causes calcium imbalance and exacerbates
cytochrome c leakage into the cytosol. Cytochrome cthen interacts with dATP to activate downstream apoptogenic proteins
that ultimately trigger apoptosis.
Figure 2 Elevated cell stress due to lactate accumulation. Ischemic stroke causes ATP depletion and a decrease in blood
perfusion. To compensate for decreased ATP, anaerobic metabolism of glucose produces lactic acid as a by-product. Owing to
poor blood perfusion, lactic acid accumulates and exacerbates neuronal apoptosis.
Li et al. Hyperglycemia in stroke and possible treatments
480 Neurological Research 2013 VOL.35 NO.5

Hyperglycemia-mediated metabolic alteration
Metabolic pathways play a crucial role in regulating
ischemic pathogenesis. Ischemic stroke in the nor-
moglycemic brain obstructs the supply of substrates
such as glucose and oxygen to the brain, thereby
slowing the synthesis of ATP.
10
As oxygen is an
irreplaceable substrate of mitochondrial respiration,
disruption in oxygen delivery leads to an immediate
mismatch between ATP production and consump-
tion. In an oxygen deprivation (OGD) brain slice
culture model, we previously reported
11
that the ADP
to ATP ratio may increase by as much as 72% after
8 hours of hypoxia. In vivo investigations reported
similar results.
12–14
Another metabolic issue related
to ATP metabolism after stroke relates to calcium
homeostasis. Considerable evidence suggests that N-
methyl-D-aspartate (NMDA) receptor hyperactiva-
tion during and after ischemic insult triggers a massive
intracellular calcium influx. Without a sufficient
energy supply, ATPase-dependent ion transport sys-
tems become dysfunctional and are unable to
maintain intracellular ion homeostasis, resulting in
a massive accumulation of intracellular and mito-
chondrial calcium.
15,16
These pathophysiological
events then trigger several deleterious developments
in the cell, including cellular swelling, organelle dege-
neration, loss of membrane integrity, and eventual
cell death.
17
The increase in intracellular and mitochondrial
calcium that occurs during hyperglycemia may indir-
ectly trigger apoptosis in the stroke brain via an
interaction with the electron transport chain compo-
nent cytochrome c. Elevated intracellular calcium
levels could lead to mitochondrial depolarization and
loss of cytochrome cinto the cytosol.
18
Once in the
cytosol, cytochrome cinteracts with procaspase-9,
apoptotic protease-activating factor-1, and dATP to
form an activated complex.
19
This complex then
activates caspase-3, the irreversible trigger of apopto-
sis. Caspase-3 in turn proteolyzes an inhibitor of
caspase-activated DNase. In apoptosis, the degrada-
tion of this inhibitor results in the translocation of
caspase-activated DNase from the cytoplasm to the
nucleus, where it catalyses internucleosomal DNA
degradation and subsequent cell apoptosis.
20
Hyperglycemia has been shown to amplify intra-
cellular calcium imbalance after ischemic stroke by
upregulating neuronal NMDA receptors and exacer-
bating the aforementioned apoptotic pathway.
Li et al.
21
demonstrated that hyperglycemia increases
extracellular glutamate concentration following fore-
brain ischemia, this excessive glutamate stimulating
Figure 3 Hyperglycemia-mediated increases in ROS gen-
eration. Glucose fuels the production of NADPH, which
donates an electron to oxygen and generates superoxides
via NADPH oxidase. Hyperglycemia also exacerbates cal-
cium and proton imbalance, causing the mitochondrial
membrane to hyperpolarize and increase ROS leakage into
the cytosol.
Figure 4 Hyperglycemia-induced vasoconstriction by
decreased nitric oxide (NO) production and increased NO
elimination. Hyperglycemia amplifies the production of ROS,
whichinteractswithNOtoproduce peroxynitrite and
depletes NO bioavailability. Hyperglycemia also decreases
the activity of eNOS through the PI3K/AKT pathway to
diminish NO production.
Li et al. Hyperglycemiainstrokeandpossibletreatments
Neurological Research 2013 VOL.35 NO.5 481

NMDA receptors which further increases influx of
calcium.
22
Elevated intracellular calcium then pro-
motes the release of cytochrome cinto the cytoplasm,
thus upregulating caspase-3 activity and subsequent
DNA fragmentation (Fig. 1). Indeed, the role of
hyperglycemia and calcium in apoptotic cell death
after stroke has been further elaborated in two more
studies.
23,24
In summary, the above studies demon-
strate that the compounding deleterious effect of
hyperglycemia in stroke-related injuries may be
caused in part by glutamate release, upregulated
intracellular calcium levels, and increased cytoplas-
mic cytochrome c, all of which are known to induce
neuronal death.
Hyperglycemia-mediated lactate accumulation
and acidosis in the penumbral region
Intracellular acidification as a result of persistent
glycolysis increases proton and lactate levels, which
stresses the penumbral tissue, making it more suscep-
tible to pro-apoptotic signals. After ischemic stroke,
cerebral blood perfusion rates decrease to less than
20% of the normal rate in the penumbral region.
25
The
availability of oxygen is even more limited in the
ischemic core. Glucose is metabolized to pyruvate via
glycolysis, which anaerobically undergoes fermenta-
tion to produce lactic acid, causing a decrease in
intracellular pH.
26
In the penumbral tissue, lactate accumulates at a
level many times its normal concentration. This shift
to anaerobic metabolism in cells causes diminished
mitochondrial ATP production which creates condi-
tions that lead to tissue necrosis. Using an animal
middle cerebral artery occlusion (MCAO) model,
Parsons et el.
3
demonstrated acute hyperglycemia to
be associated with higher acute and subacute lactate
production. Although the exact mechanism is not
well elucidated, it is conceivable that high intracel-
lular glucose levels may drive the metabolic process
forward as a result of increased pyruvate availability
for lactic acid formation (Fig. 2). However, several
studies have highlighted the fact that hyperglycemia
can be beneficial to the energy starved brain in
specific situations.
27,28
For example, hyperglycemia
may be beneficial in situations where lactate can be
quickly flushed away by uninterrupted blood flow. By
contrast, blood flow in ischemia is stagnant, allowing
lactate to accumulate. The combination of stagnant
blood and lactate build-up may decrease penumbral
salvage, increase final infarct volume, and ultimately
worsen functional outcome.
3
Hyperglycemia-mediated oxidative stress and
ROS accumulation in penumbral neurons
Normally mitochondria produce ATP with remark-
able efficiency through the reduction of oxygen to
water by cytochrome coxidase. Under normal
conditions, only approximately 1–2% of the oxygen
reduced by mitochondria becomes ROS, such as
superoxide (O{
2).
29
The two known locations in the
Figure 5 Hyperglycemia-mediated increase in leukocyte transmigration. Hyperglycemia upregulates the PKC pathway, which
in turn increases calpain expression. Calpain in turn upregulates the expression of ICAM-1 on endothelial cell surface.
Alternatively, hyperglycemia also increases the translocation of NF-kappaB into the nucleus by upregulating IkappaB
proteosomal degradation. Activated NF-kappaB then translocates to the nucleus and binds DNA at specific kappaB sites in
promoter regions to activate the transcription of ICAM-1 and VCAM-1.
Li et al. Hyperglycemia in stroke and possible treatments
482 Neurological Research 2013 VOL.35 NO.5

oxidative phosphorylation chain at which superoxides
are produced are the nicotinamide adenine dinucleo-
tide dehydrogenase and ubiquinone–cytochrome b–c1
regions.
30
Oxidative stress has a profound effect on
stroke pathogenesis due to the high susceptibility of
the brain to ROS-induced injuries. The brain has high
concentrations of unsaturated lipids, low levels of
antioxidants, high levels of oxygen consumption, and
high levels of iron that acts as an oxidizing agent under
pathological conditions.
31–33
All of these conditions
favor lipid peroxidation.
There are two known pathways by which ROS may
be produced following ischemia, one as a consequence
of mitochondrial membrane hyperpolarization and the
other through the use of the nicotinamide adenine
dinucleotide phosphate oxidase (NADPH oxidase)
pathway.
34
Both are upregulated by hyperglycemia.
The accumulation of intracellular calcium and protons
during ischemia onset causes hyperpolarization of the
mitochondrial membrane.
35,36
The rate of superoxide
production increases exponentially as the mitochon-
drial membrane potential increases above 140 mV.
37,38
Using an oxygen and glucose deprivation model,
Abramov et al. showed ROS production to be elevated
1.6-fold during the initial onset of hypoxia and 2.6-
fold during reoxygenation.
The NADPH oxidase pathway is an alternative
mechanism for ROS production. NADPH oxidase is a
multi-subunit enzyme that catalyzes oxygen reduction
by using NADPH as the electron donor. The five
subunits of NADPH oxidase consist of p47phox,
p67phox, p40phox, p22phox, and the catalytic subunit
gp91phox.
39
In normal brains, p47phox, p67phox, and
p40phox exist in the cytosol, while p22phox and
gp91phox are attached to the cell membrane.
However, upon stimulation, such as during ischemia,
p47phox is phosphorylated and the cytosolic subunits
form a complex that translocates to the cell membrane,
where it associates with p22phox and gp91phox to
assemble the active oxidase.
40
High intracellular glucose levels could exacerbate
reperfusion-induced injuries by upregulating super-
oxide production. Thus, Suh et al.
41
demonstrated
that glucose is central to ROS generation after OGD
and reoxygenation, and that it acts independent of
lactic acidosis in neurons. Glucose sustains the hexose
monophosphate shunt, allowing it to regenerate
NADPH, which serves as a substrate for ROS
production (Fig. 3).
42
Superoxide production is
significantly downregulated in the absence of glucose
as well as when the hexose monophosphate shunt is
inhibited after OGD.
43
Furthermore, the increase in
superoxide production during hyperglycemia is abol-
ished in p47 knockout mice and after NADPH
oxidase inhibition with apocynin.
42,44
Additionally,
2-deoxyglucose, which slows glucose transport and
impairs glucose utilization via hexokinase, may
reverse the accumulation of superoxide effectuated
as a result of hyperglycemia.
45
Hyperglycemia-mediated BBB disruption and
perfusion reduction
The BBB is formed by endothelial cells, tight junction
proteins, pericytes, and astrocytes. The BBB plays a
critical role both in maintaining brain homeostasis and
restricting blood-borne substances from entering the
parenchyma. Hyperglycemia is a risk factor for aug-
mented BBB injury during ischemia. Hyperglycemia-
induced cerebral acidosis and superoxide production
are generally considered the mechanisms of enhanced
brain injury, particularly as pertains to neurons.
46,47
However, recent reports suggest that damage to brain
endothelial cells and the BBB has a profound impact
on overall functional outcome and may even exceed
the impact on neuronal damage.
First, hyperglycemia has been shown to disrupt the
stability of the BBB by indirectly upregulating the
phosphorylation of occludin fragments, leading to
their ubiquitination and subsequent BBB permeabil-
ity. Short-term hyperglycemia enhances de novo
synthesis of diacylglycerol (DAG) in capillary en-
dothelial cells from glycolytic intermediates.
48
DAG
is then responsible for activating protein kinase C-
beta II, one of nine PKC family proteins altered by
DAG during hyperglycemia.
49
Activated PKC-beta
II then phosphorylates occludin fragments at the
Ser490 position, which leads to the ubiquitination
and redistribution of tight junction proteins in
endothelial cells.
50
The end result of these molecular
changes is increased BBB permeability. This mechan-
ism occurs within minutes of reperfusion, which
concurs with the time course observed in in vivo
models.
51
Together, the evidence from human and
animal studies supports the notion that hyperglyce-
mia worsens cerebrovascular reperfusion injuries
through the PKC-beta II pathway.
Second, hyperglycemia has been shown to signifi-
cantly reduce blood flow in the penumbral region. In
animal studies, induction of hyperglycemia by acute
glucose injection caused changes in cerebral blood
flow after either 2 or 4 hours of occlusion and
2 hours of reperfusion. Cerebral blood flow in the
ischemic hemisphere of hyperglycemic animals was
approximately 40% of that in the contralateral non-
ischemic hemisphere, compared to 100% in the
normoglycemic controls.
52
Additionally, induction
of acute hyperglycemia by glucose infusion decreased
penumbral blood flow to 60% of pre-ischemic values
in the hyperglycemic group, compared to 89% in the
normoglycemic group.
53
Another mechanism for the
reduced rate of perfusion in hyperglycemia has been
attributed to decreased bioavailability of nitric oxide
(NO), which is a potent vasodilator and inhibitor of
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Neurological Research 2013 VOL.35 NO.5 483

platelet aggregation.
54,55
Although this mechanism is
not well delineated, it has been suggested that
hyperglycemia could upregulate the dephosphoryla-
tion of endothelial nitric oxide synthase (eNOS) at
the Ser1177 position and its phosphorylation at the
Thr495 position, both of which decrease eNOS
activity.
55,56
Indeed, hyperglycemia has been shown
to downregulate eNOS activity by upregulating the
hexosamine and PKC pathways, thereby downregu-
lating the phosphoinositide 3-kinase/protein kinase B
(PI3K/AKT) pathway.
57,58
This process inhibits
vasodilation, thus decreasing blood flow to the
ischemic regions of the brain (Fig. 4).
Finally, ROS are known to interact with NO to
produce the highly reactive peroxynitrite radical
which decreases the availability of NO and further
impairs the blood vessels’ ability to dilate.
56
Even
mild acute hyperglycemia (y150 mg/dl) significantly
reduces blood flow to the penumbral region and
causes red blood cells to be trapped within areas of
the ischemic vasculature. The end result of these
hyperglycemic sequelae is the diminishing or absence
of oxygen delivery to a portion of the penumbra.
59
These oxygen deprived areas exhibited marked
increases in BBB permeability after reperfusion.
59
In summary, vascular injury appears to be an
important factor associated with hyperglycemia-
induced derangements and the consequences of vascu-
lar damage may significantly limit the benefit derived
from available protective neuronal treatments. As later
mentioned in this Review, more effective treatments
may emerge from a better understanding of hypergly-
cemia’s effects on the cerebrovasculature after ischemia.
Hyperglycemia-mediated inflammatory response
The brain’s inflammatory responses to ischemia are
characterized by the rapid activation of resident cells.
Beginning two hours after stroke onset, microglia are
activated via CD14 receptors and subsequently by
toll-like receptor 4.
60–62
Activated microglia undergo
morphological transformation into phagocytes and
act as cytotoxin-producing factories releasing proin-
flammatory cytokines such as tumor-necrosis factor-
alpha (TNF-alpha), IL-1, and IL-6 in addition to NO
and ROS.
63,64
Similar to microglia, astrocytes are
also activated during the acute phase to produce pro-
inflammatory cytokines, chemokines, and inducible
nitric oxide synthase.
65
Elevated levels of the afore-
mentioned molecules initiate the second step of the
post-ischemic inflammatory response, which includes
circulating inflammatory cell chemotaxis and trans-
migration into the injured brain.
66–68
Lymphocytes,
normally excluded from the neuronal compartment
under quiescent conditions, can be visualized by
immunohistochemistry in the same compartment in
the post-ischemic brain. Recent work suggests that
components of the immune system are not only
intimately involved in the ischemic cascade, but that
they interact with tight junction proteins and the basal
lamina to disrupt the BBB and degrade white matter to
cause parenchymal injuries.
69–72
Hyperglycemia has
been shown to have a tremendous effect on the
inflammatory pathway post ischemia, elevating several
transcription pathways to increase leukocyte migra-
tion to the brain and their ability to cross into the
injured tissue.
Elevated intracellular glucose results in an increase
in nuclear factor-kappaB (NF-kappaB) and a de-
crease in IkappaB expressions in endothelial cells.
73,74
Under quiescent conditions, NF-kappaB is bound to
IkappaB in the cytosol. However during IkappaB
degradation, NF-kappaB translocates into the nucleus
and stimulates the expression of pro-inflammatory
cytokines such as TNF-alpha, interleukin (IL)-1 and
IL-6 and chemokines, such as monocyte chemoattrac-
tant protein.
74–77
These cytokines and chemokines
attract mononuclear cells to the site of injury and
upregulate endothelial cell surface receptors to facil-
itate mononuclear cell attachment and their transmi-
gration into the parenchyma.
78,79
Indeed, Morigi
et al.
80
found that maintaining a blood glucose level
of 174 mg/dl for 24 hours significantly increased levels
of NF-kappaB in endothelial cells and adhesion
molecules including intercellular cell adhesion mole-
cule-1 (ICAM-1) and vascular cell adhesion molecule-
1. The inhibition of NF-kappaB with pyrrolidine
dithiocarbamate and N-tosyl-L-phenylalanine chloro-
methyl ketone reversed hyperglycemia-induced
increases in NF-kappaB expression and subsequent
mononuclear cell adhesion to endothelial cells.
80
The second major pathway in hyperglycemia media-
tion of the immune response was elucidated by
Smolock et al.,
81
who demonstrated that elevated
glucose levels activated the PKC pathway. In turn,
PKC upregulated its downstream molecule calpain in a
calcium-dependent manner.
81
This upregulation of
calpain ultimately resulted in the overexpression of
ICAM-1 on the surface of endothelial cells.
81
However,
there is controversy over whether elevated glucose
concentrations alone cause the increase in ICAM-1
expression. Supporting the direct role of hyperglyce-
mia, Kado et al.
82
demonstrated that incubating
endothelial cells with 30 mM of glucose for 24 hours
significantly increased ICAM-1 levels (Fig. 5). Several
other studies have confirmed the above results.
80,83
In contrast, studies conducted by Rasmussen
et al.
84
suggested that the induction of cellular
adhesion molecules in human endothelial cells could
be associated with the upregulation of pro-inflamma-
tory cytokines. Esposito et al.
85
further demonstrated
that acute hyperglycemia could induce an increase in
the concentrations of plasma IL-6 and TNF-alpha.
Li et al. Hyperglycemia in stroke and possible treatments
484 Neurological Research 2013 VOL.35 NO.5

This last finding has been substantiated by in vitro
studies.
86–89
Taken together, the above observations
suggest multiple mechanisms by which hyperglycemia
orchestrates pro-inflammatory phenotypes.
Inflammatory mechanisms intrinsic to the brain
and its vasculature are crucial mediators of increased
BBB permeability during ischemia. Hyperglycemia
may upregulate activator protein-1, which then
stimulates the transcription of matrix metalloprotei-
nases -2 (MMP-2) and -9 (MMP-9) in leukocytes.
90
Furthermore, hyperglycemia promotes the recruit-
ment of blood-borne leukocytes, mainly neutrophils,
which further increase the levels of MMP-9.
91
Expression of MMP-2 and MMP-9 has been inver-
sely correlated with BBB tight junction protein
(zonula occludens-1) expression and basal laminin
levels, both of which are integral to the integrity of
the BBB.
92
Decreases in the latter two protein
concentrations are thought to affect the integrity of
the BBB and increase its permeability, resulting in
edema and hemorrhagic transformation.
The source of MMP-9 is still a matter of debate.
Gasche et al.
93
found active MMP-9 to be colocalized
with endothelial cells and astrocytic processes. In
contrast, our initial results show OGD significantly
increased the expression of MMP-9 by resident micro-
glial cells in the brain. Recent studies using chimeric mice
with GFP bone marrow cells provided the opportunity
to distinguish brain versus blood macrophages and their
contribution to vascular injury after ischemic stroke.
Although it is generally thought that both resident and
blood-derived macrophages play significant roles in
the inflammatory pathway, resident microglial cells
predominate.
94
This conclusion is supported by our
studies using an in vitro OGD-reoxygenation brain
slice culture method in which blood-borne macro-
phages are eliminated from the preparation.
Compelling evidence suggests that hyperglycemia-
induced MMP-9 overexpression may also enhance
white matter degeneration following ischemic stroke,
leading to poorer outcomes.
95
Patients with inade-
quately regulated serum glucose levels are more
susceptible to white matter lesions.
96
Similarly, dis-
ruption of the BBB as a result of ischemic onset
exposes the brain parenchyma to blood-borne macro-
phages and has been associated with white matter
lesions.
97,98
Asahi et al.
99
showed that MMP-9 knock-
out mice have reduced myelin degradation after
ischemic stroke. Given the critical role of white matter
cells in ischemic outcomes, further investigation is
needed to better understand the effects of the
inflammatory response on white matter degeneration.
Treatments
Despite the ongoing effort to better understand
the mechanisms underlying hyperglycemia-enhanced
ischemic injuries, the translation of knowledge gath-
ered from experimental studies into effective treat-
ments has been uniformly disappointing. While tissue
plasminogen activator (tPA) administration after
stroke is considered the gold standard in re-establish-
ing blood flow to the brain, co-administration of
neuroprotective agents that interrupt pathways of
ischemic cell death could improve stroke outcomes. A
therapy that would both salvage ischemic tissue and
protect the brain from hyperglycemia-induced injuries
post-ischemia could benefit millions, yet this medical
need remains unmet.
Insulin therapy
There is an emerging body of evidence supporting the
beneficial effects of normalizing serum glucose levels
within 48 hours of ischemic stroke onset.
100
In animal
models, insulin therapy has been shown to reduce
infarct volume and enhance neuroprotection during
acute focal ischemia.
101
However, the translation of
these findings from the laboratory to the bedside has
yielded mixed results.
102,103
Evidence supports the notion that hyperglyce-
mia can have a significantly deleterious effect on the
neurovasculature. In rodent MCAO models, insulin
exerts a neuroprotective effect on the brain vasculature
through the upregulation of NO via the PI3K/AKT
pathway.
104
The activation of the PI3K/AKT path-
way negatively modulates genes that promote vascular
permeability and inflammation, thereby protecting
vascular integrity.
105
eNOS, a molecule downstream
of AKT, is a known catalyst of NO. As previously
discussed, NO has a crucial role in regulating vascular
tone and integrity. Generation of NO could potentially
increase vasodilation and improve blood flow to the
penumbra, thus diminishing platelet aggregation.
106
In
addition to increasing NO synthesis, insulin therapy
also decreases ROS accumulation by downregulating
NADPH oxidase activity. This change in NADPH
oxidase increases the bioavailability of NO.
73
In addition to lowering glucose levels, insulin may
also have a direct neuroprotective effect on the CNS
parenchyma by reducing inflammation.
107
In patients
who received intensive insulin therapy (target glucose
level between 80 and 110 mg/dl), the activity of the
transcription regulator NF-kappaB and its down-
stream targets, ICAM-1 and E-selectin, were signifi-
cantly reduced.
107
As ICAM-1 and E-selectin are
cardinal mediators of blood macrophage transmigra-
tion across the endothelium after stroke onset,
decreased expression of both receptors produces an
oppositional effect to that of hyperglycemia on the
vascular endothelium.
However, a broad-spectrum reduction in the
inflammatory response may paradoxically limit recov-
ery since immune cells and inflammation also play a
Li et al. Hyperglycemiainstrokeandpossibletreatments
Neurological Research 2013 VOL.35 NO.5 485

crucial role in tissue repair. Although there is evidence
to support the beneficial effects of the anti-inflamma-
tory response during the acute phase in ameliorating
tissue damage, counteracting this response may
actually worsen long-term outcomes by reducing
overall tissue repair and reorganization.
108–113
More
work in the area of the brain’s immune response after
injury would likely lead to effective novel therapies.
Early insulin based clinical trails have failed to
show significant results. The Glucose Insulin in
Stroke Trial (GIST) conducted in the UK reported
no evidence of benefit from insulin therapy.
102
There
are several possible explanations for this result. First,
the difference in mean glucose levels between the
control group and the experimental group was only
10 mg/dl after insulin treatment.
114
As a result, there
were no significant differences in clinical outcomes
as measured by mortality rate and neurological
deficits.
102
Second, the optimal range of serum
glucose was not well defined and a heterogeneous
group of patients was accepted into the study.
Nonetheless, the GIST study helped establish a 24-
hour glucose-insulin infusion safety parameter com-
prised of maintaining glucose levels between 72 and
126 mg/dl in order to avoid the significant risk of
hypoglycemia or mortality in excess of 40 weeks.
Currently, the Stroke Hyperglycemia Insulin Net-
work Effort study, a phase III single-blinded ran-
domized control trial, is looking for a correlation
between normalized serum glucose levels and a
reduction in thrombolysis-induced intracranial hemor-
rhage associated with hyperglycemia.
115
A stratified
result from this trial has the potential to alter future
treatment decisions for hyperglycemic stroke patients.
Furthermore, the Continuous Glucose Monitoring
and Insulin Pump Therapy in Diabetic Gastroparesis
clinical trial, currently conducted at the University of
Michigan, uses continuous glucose monitoring and an
insulin pump to precisely maintain the patient’s
glucose levels. The validated end point has the
potential to reduce the resulting complications of
hypoglycemia and would allow physicians to maintain
patients’ glucose at a more consistent level.
Insulin-like growth factor 1
Insulin-like growth factor I (IGF-1) is structurally
similar to insulin and thus has many analogous
functions. In animal models, it was found that IGF-1,
when injected into the ventricles, reduced ischemic
damage.
116
IGF-1 displays strong anti-apoptotic cell
survival properties and is a potent neuroprotective
agent when administered after stroke induction.
117
Cheng et al.
117
showed neuroprotective properties of
IGF-1 are mediated by the activation of PI3K/AKT
as well as FKHRL, both of which improve cell
survival. While both insulin and IGF-1 can directly
increase neuronal survival by interacting with the
IGF-1 receptor, IGF-1 binds to the receptor with
much higher affinity.
118
Thus there is reason to
believe that IGF-1 treatment may be effective in
ameliorating the negative effects of hyperglycemia
during stroke onset.
Glucagon-like peptide-1 therapy
Glucagon-like peptide-1 (GLP-1) and artificial GLP-
1 receptor agonists such as exendin-4 (Ex-4) are well-
established diabetes medications.
119
In recent years,
several studies have shown GLP-1 receptor (GLP-
1R) stimulation to ameliorate the adverse effects of
ischemic stroke.
119,120
GLP-1 and Ex-4 have a wide
range of physiological actions in glucose metabolism,
including the stimulation of insulin secretion, inhibi-
tion of glucagon release, and reduction of appetite
and food intake.
120,121
GLP-1R is expressed through-
out the brain, and GLP-1 and Ex-4 are able to cross
the BBB due to high lipophilicity.
122
GLP-1 admin-
istration elicits the combined outcomes of decreased
intracerebral glucose content and increased cerebral
metabolic rate.
123
Models of transient MCAO stroke
in rodent have shown that treatment with Ex-4
provides marked benefits on locomotor activity and
infarct volume that are absent in GLP-1R knock out
mice.
124
GLP-1R stimulation has also been shown to reduce
the synthesis of pro-inflammatory cytokine IL-1beta
in activated astrocytes via stimulation of adenylate
cyclase and the accumulation of cAMP.
125
The
mechanism by which GLP-1R stimulation drives
neuroprotection is unclear, and future research is
necessary to investigate the potential of GLP-1 as a
treatment.
Hyperbaric oxygen preconditioning
Accumulating evidence indicates that hyperbaric
oxygen therapy (HBO) may have beneficial effects in
stroke. Qin et al.
126
demonstrated that HBO precon-
ditioning decreased hyperglycemia-induced hemorrha-
gic transformation. While the exact mechanism by
which HBO limits hemorrhagic transformation is not
well-defined, recent reports suggest that HBO pre-
conditioning may ameliorate hyperglycemia-induced
injuries by reducing BBB disruption. Veltkamp
et al.
127
showed that HBO therapy reduces basal
lamina degradation and MMP-9 levels, which are
associated with hemorrhagic transformation.
HBO preconditioning also protects against brain
ischemia–reperfusion injury by upregulating antiox-
idant species.
128,129
Evidence suggests that several
antioxidant genes may act synergistically to remove
ROS through sequential enzymatic reactions. It is
believed that nuclear factor erythroid 2 mediates the
expression of these genes. The relevant enzymes
include heme oxygenase-1, glutathione S-transferase,
Li et al. Hyperglycemia in stroke and possible treatments
486 Neurological Research 2013 VOL.35 NO.5

and NADPH quinone oxidoreductase.
130
Soejima
et al.
128
found that HBO preconditioning significantly
attenuated transient MCAO-induced neurological
deficits, infarct volume, and BBB permeability in
hyperglycemic rodents.
Hypothermia
Hypothermia has long been suspected to have
neuroprotective effects when administered immedi-
ately after stroke and has been a topic of intense
research. In temporary ischemic stroke models,
hypothermia has been shown to reduce infarct size
and improve functional outcome.
131
The physiological
effects of hypothermia are multifaceted. Hypothermia
decreases inflammation,
132–134
neuronal apoptosis,
135
mitochondrial dysfunction,
136
and excitotoxicity.
137
Mild hypothermia (33C) significantly reduces cyto-
chrome ctranslocation into the cytoplasm and Bax
and Bcl-2 levels.
135
Furthermore, hypothermia re-
stores elevated calcium levels after stroke outcome and
reduces calcium dependent degradative pathways
leading to cell death.
138
Ethanol
The efficacy of ethanol therapy as a treatment for
hyperglycemia-induced brain injury post-ischemic
stroke has yet to be tested. However, there is evidence
to suggest that a moderate concentration of etha-
nol treatment may be neuroprotective and can re-
duce hyperglycemia-associated injuries. Previously
we showed that acute ethanol administration re-
duces both infarct lesion volume and hemorrhagic
transformation.
139
One of the pathways by which
ethanol exerts its neuroprotective effects may be
through a reduction of ROS. Thus, in a MCAO
model, we found that a moderate ethanol treatment
(1.5 g/kg) administered immediately upon reperfu-
sion significantly reduced NADPH oxidase and
gp91(phox) in the brain.
140
Furthermore, acute ethanol treatment reduces
disruption of the BBB caused by reperfusion. In the
same model, we showed that ischemic rodents gained
neuroprotection as exhibited by decreased expression
of aquaporins 4 and 9, two important mediators of
cerebral edema post-ischemic stroke, when adminis-
tered moderate concentrations of ethanol immedi-
ately following reperfusion.
92
In addition, using an in
vitro oxygen and glucose deprivation and reoxygena-
tion model, we found that ethanol administration
at a moderate concentration of 30 mM significantly
reduced the levels of MMP-2 and MMP-9 and
increased basal lamina and tight-junction zonula
occludins-1 levels.
92
These effects likely helped to
stabilize the BBB after stroke onset.
Ethanol-derived neuroprotection may constitute one
method of combating hyperglycemiainduced post-
ischemic injuries. Evidence that ethanol treatment
interferes with pathways of ischemic hemorrhagic
transformation, neuronal cell death, and increased
blood vessel permeability shows promise. However,
further studies are needed to determine whether
ethanol’s effect on these post-ischemic injuries could
be clinically significant.
Conclusion
Acutely elevated glucose levels are associated with
poorer outcomes in ischemic stroke patients.
141
Substantial evidence from experimental work and
clinical studies indicate that hyperglycemia further
complicates the metabolic state and mitochondrial
function in the ischemic penumbra, increases both
infarct volume and hemorrhagic transformation, and
worsens outcomes even after thrombolytic therapy.
Using in vivo (e.g. MCAO models) and in vitro (e.g.
OGD) models, investigators have shown hyperglyce-
mia to exacerbate intracellular calcium imbalance
and the accumulation of ROS in endothelial cells.
These intracellular changes are thought to cause
detrimental effects in the penumbral region and the
neurovascular system. In addition, the activation of
both microglia and macrophages in the neuronal
compartment after ischemic stroke aggravate the
extent of injury by disrupting the BBB and causing
degeneration of the white matter.
Although experimental studies have elucidated
several mechanisms by which hyperglycemia influ-
ences the survival of ischemic penumbral tissue,
studies bridging the gap between clinical stroke and
experimental models have been limited. Recently,
MRI data have shown a strong correlation between
loss of penumbral tissue and elevated blood glucose.
3
Although several treatments have been proposed
to ameliorate hyperglycemia-induced injury after
ischemic stroke, their translation from the laboratory
to the bedside has been disappointing thus far. While
insulin therapy to normalize glucose levels has shown
promise in animal models, clinical application of this
strategy in the GIST trial failed to show significant
improvement. Furthermore, the GIST trial revealed
the difficulty with managing patient’s glucose levels in
a clinical setting. Lowering the patient’s glucose levels
using insulin has the potential to induce hypoglyce-
mia, causing more harm than good. Anti-inflamma-
tory therapies have shown paradoxical outcomes; as a
result, non-specific anti-inflammatory therapy has yet
to fulfill its promise. Research is needed to delineate
specific inflammatory pathways and their effects on
ischemia-induced injuries. A better understanding of
the inflammatory pathways involved in ischemic
stroke will allow physicians to inhibit destructive
pathways while promoting those that are neuro-
restorative. Finally, though in its infancy, ethanol ther-
apy is promising, as it exerts significant depressive
Li et al. Hyperglycemiainstrokeandpossibletreatments
Neurological Research 2013 VOL.35 NO.5 487

effect on glucose metabolism and influence on the
neurovascular system. Ethanol therapy is one of the
few treatments that impedes both the metabolic
pathway and the inflammatory pathway to protect
the penumbral tissue. However, more research is
necessary to establish ethanol-derived neuroprotection
in hyperglycemic conditions.
The effect of hyperglycemia on ischemic stroke
outcome is profound and may significantly affect
treatment outcome. Accumulating data suggests
diabetic rodents, when treated with tPA, exhibit
negative side effects including increased BBB perme-
ability and an orders of magnitude increase in
intracerebral hemorrhage volumes compared to
non-hyperglycemic rodents treated with tPA.
142,143
A recent study published by Chen et al. showed that
bone marrow stromal cell treatment of stroke in
diabetic rats precipitated adverse effects, including
increased BBB leakage, cerebral artery neointimal
formation, arteriosclerosis, and worse functional
outcome.
144
Traditional methods known to improve
functional outcome after ischemic stroke have shown
adverse effects when used in hyperglycemic animal
models.
As the diabetic population continues to expand in
both developed and developing countries, the effect
of hyperglycemia post-ischemic stroke should be
investigated. Successful therapeutic intervention for
the large population facing the social, financial, and
health consequences of diabetes and ischemic stroke
will depend on understanding the interplay between
glucose and different systems of the brain, including
the cerebrovascular system, the inflammatory system,
and the metabolic system.
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