Andrew Qian

2/21/19

The Neurological Impact of PGE2 Signaling Inhibition after ICH

Introduction:

Ranking 5th among all causes of death in the US, stroke has long been a major public

health issue, killing approximately 133,000 Americans and 6.2 million people worldwide

annually. The 59% that survive after a year are forced to live with debilitating injuries, most of

which reduce the mobility of the patient (Sacco et al.) . Since most stroke victims are over the

age of 65, these chronic issues are highly damaging to the elderly (“Stroke Facts”). The main

focus of this paper is Intracerebral Hemorrhage (ICH), otherwise known as Hemorrhagic stroke,

which accounts for only 15% of all stroke cases, but 40% of all stroke deaths. This mortality rate

is primarily due to a lack of an effective treatment method for ICH, resulting in half of all ICH

deaths coming within the first two days. (“Hemorrhagic stroke”). In this subtype of stroke, high

blood pressure or external brain trauma causes thin walled arteries in the brain to tear, resulting

in a ruptured artery within the brain. As blood spills into the brain, the area that the artery

originally circulated is deprived of oxygen-rich blood, leading to neuronal death. This damage

can be furthered by the release of toxic molecules by microglia and other brain cells. The

lethality of ICH, coupled with a lack of a potent treatment, only exacerbates the need for the

development of an effective treatment (Awasthi). This paper discusses the potential of the PGE2

receptor/signaling family to develop a novel therapeutic treatment for ICH. As a result of the

numerous roles that PGE2 receptors play in the brain after ICH, inhibition or activation of certain

receptor pathways could prove to be the key in developing a novel therapeutic treatment for ICH.
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Background:

In the United States, Intracerebral Hemorrhage (ICH) is becoming a growing problem.

Stroke rates have been rising steadily since 1995, with hospitalizations of men for strokes nearly

doubled from 1995 through 2012 (Naqvi). ICH is the result of bleeding within the brain tissue

itself — a life-threatening type of stroke. It is most commonly caused by hypertension or head

trauma, as well as many other different factors, such as high blood pressure or external injury,

blood or bleeding disorders, blood vessel abnormalities, etc. Specifically, High blood pressure

can cause thin-walled arteries to rupture, releasing blood into the brain tissue. Clotted blood and

fluid buildup increases the pressure, which can crush the brain against the bone or cause it to

shift and herniate (“Intracerebral Hemorrhage”). After ICH, lasting issues can remain, including

many detrimental chronic issues besides death such as paralysis, numbness/weakness in the

body, personality changes/emotional issues, and loss of certain motor functions (Wu 4).

Currently, ICH remains the most deadly form of strokes, with the highest mortality rate as well

as lacking an effective treatment (Mayer 4). As a result, Only about half of all patients will

survive intracerebral hemorrhage, and most of those survival patients will suffer from a

significant disability. Although early clinical investigations have traditionally focused on

mortality and its reduction, more recent efforts have begun to attend to disability in survivors

(Saulle, Shambra 2). Survivability of stroke is currently dependent on how quickly treatment is

administered. Immediate treatment greatly reduces the risk of death, and can mitigate possible

long-term detriments. Often, surgery becomes necessary as a form of treatment for ICH in order

to reduce neuronal bleeding (Stroke Treatments).

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Microglia are the immune cells of the brain, and play the role of a macrophage in the

brain. They are the major inflammatory cell type in the brain; after injury, they respond by

becoming activated. This causes them to change shape and migrate to the area of injury. At the

site of the injury, microglia are the key for the phagocytosis of pathogens as well as the removal

of damaged cells. Additionally, they are instrumental in the resolution of the inflammatory

response, through the production of anti-inflammatory cytokines such as Il-10 (Microglia

Function in the Healthy Brain). After injury, microglia change shape, a process known as

polarization. During normal circumstances, microglia are described as in a resting state. Despite

their name, they are still constantly alert, extending and retracting their “limbs” to inspect the

environment. This is how they are able to detect injury. After detection of injury, microglia will

quickly become activated and move to the area of injury. This activation includes a change in

shape, where the microglia become noticeably bigger (Zhao et al.). The microglia reach their

peak activation within 2-3 days, but will sustain overall activation for a few weeks (Kim and

Cho). The level of the change in shape of the microglia can be determined by its distance to the

area of injury/stimulus. Microglia closer to the lesion are more ameboid, while microglia further

from the injury maintain their original shape (Heindl et al.).

There are two major types of microglia polarization that have beneficial and detrimental

effects. Primarily, microglia are known to become polarized into two different phenotypes, M1

and M2. The M1 phenotype, also known as the classical phenotype, is known to be involved

with pro-inflammatory processes, while the M2 phenotype is known to be involved with anti-

inflammatory processes. The M2 Phenotype is also involved in repair and regeneration

processes, as they can attenuate the inflammatory response and stimulate tissue repair
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(Guruswamy and ElAli). This paper will discuss minimizing classical microglia polarization,

which may potentially be incorporated into a treatment for ICH.

PGE2 Receptor Signaling:

The main form of signaling that will be focused on in this paper are PGE2 receptors, also

known as Prostaglandin E Receptors. These receptors are present in all mammalian tissues and

use G Protein coupled receptors to exert a wide variety of actions throughout the body.s(?)

(GPCRs), meaning that when these receptors are activated, they stimulate the activation of an

attached G-protein and the subsequent signaling cascade. While EP receptors are found all

throughout the body, they are abundant through the brain (Bhattacharya et al.). There are

currently nine prostanoid receptors(DP, EP1–4, FP, IP, and TP). Three of them, EP1-3, will be

discussed in this review.

PGE Receptor signaling is a valuable signaling pathway within the body because it plays

a major role in modulation of many important physiological processes such as the CNS, the

cardiovascular, gastrointestinal, and immune systems, among others. In addition, prostaglandins

are also involved in a variety of diseases including inflammation, cancer, hypertension, and

cardiovascular disease. The uniqueness of prostaglandin receptors is their ability to have

differing, and often completely opposite, effects, particularly in the immune system. In certain

cases, PGE receptors appear to have pro-inflammatory properties, while in others cases they

exhibit anti-inflammatory properties (Hata and Breyer). Matching each response with the

receptor will be vital to the development of an effective treatment.

Within the brain, PGE Receptor signaling is involved in a lot of neurological processes,

with both benefits and detriments. Past experimental results reveal that activation of the EP2
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receptor in neurons increased cyclic AMP (cAMP) and protein kinase A signaling. This increase

in cAMP and protein kinase A signaling, in turn, improves neuroprotection against hemin

neurotoxicity in vitro. These results indicate that activation of EP2 receptor could potentially be

used to minimize neuronal damage due to their neuroprotective abilities (Mohan et al.).

Prostaglandin receptors have also been shown to have a protective impact against cerebral

ischemia. In vitro studies revealed that activation of the EP2 receptor had a neuroprotective

effect on NMDA toxicity and oxygen glucose deprivation, while inhibition of the Ep2 receptor

through blockage of protein kinase A reversed this effect ( McCullough et al.). Both of these

studies serve to confirm that EP2 serves as a protective mechanism against neuronal injury. This

indicates that this signaling pathway may also be able to serve a neuroprotective role after other

forms of brain trauma, such as stroke.

In contrast, some forms of PGE2 signaling can have a detrimental effect on the brain after

injury. An example of this would be EP3 receptor signaling, which has been shown to exacerbate

neuronal injury. In one study conducted, the researchers determined that deletion of the EP3

receptor reduced the overall neuronal damage in the CA1 region of the hippocampus after

ischemia stroke, suggesting that EP3 receptor contributes to overall acute ischemia injury

(Saleem et al.). The results from these studies all suggest that PGE2 EP1-4 signaling contributes

greatly to overall brain injury, and further research into the effects of these receptor pathways

would allow for better understanding of the factors behind other forms of neuronal trauma.

Neuroprotective Signaling after Stroke:

In regards to stroke, PGE Receptor EP2 has been shown to have neuroprotective effects

after ICH. Recent experimentation has revealed that it has the potential to reduce the overall
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amount of neuronal damage that is suffered. In one experiment, ICH was induced in mice

through intrastriatal injection of collagenase as well as the injection of arterial whole blood.

Within a few days, the mice were afflicted with increased inflammatory responses, oxidative

stress, and matrix metalloproteinase (MMP)-2/9 activity, among other typical stroke symptoms.

Deletion or inhibition of PGE Receptor EP2 exacerbated brain swelling/edema, neuronal death,

and neurobehavioral deficits. Activation of PGE Receptor EP2 through the agonist AE1-259-01,

however, mitigated, and in some cases, reversed this damage. This allowed the researchers to

conclude that the PGE Receptor EP2 plays a major role in protection of the brain after ICH, and

warrants further investigation for potential use in ICH treatment (Wu et al.). Another study,

which focused on a specific agonist of EP2, was shown to have neuroprotective impacts as well.

Misoprostol is a synthetic PGE2 agonist, while recent studies have shown that

misoprostol also has some protective effects after cerebral ischemia as well. In the brain,

macrophages/microglia secrete matrix metalloproteinases, neuronal inflammation modulators,

through a PGE2 dependent mechanism. Inhibition of these matrix metalloproteinases have been

shown to reduce neuronal injury after ICH (Gao et al.). The result of this experiment revealed

that treatment with misoprostol decreased brain lesion volume, edema, and brain atrophy and

improved long-term functional outcome, a result that is likely attributed to the decrease in matrix

metalloproteinase expression. These results support the idea that misoprostol plays a protective

role in the brain after ICH (Hua et al.).

Neurologically Damaging Signaling after Stroke:

While PGE Receptors EP2 and EP4 may exhibit beneficial impacts, PGE receptors EP3

and EP1 both have neurotoxic effects. EP1 Receptor is a major factor in neuronal toxicity after

ICH. A study conducted at Johns Hopkins University analyzed the role of the EP1 receptor after
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ICH. Primarily, they studied the interaction between the Ep1 receptor and the Src pathway.

Previous evidence has already revealed that Src Kinase activation mediates thrombin-induced

blood-brain barrier disruption, while inhibition of Src kinase activation reduces blood toxicity.

The results of these studies revealed that activation of the EP1 receptor elicits neural toxicity

through the Src kinase pathway (Heng et al.). This form of reception also acted through the

MMP-9 signaling pathway, a matrix metalloproteinase pathway which has been linked to

neuroinflammation after ICH. These results indicate that inhibition of the PGE2 receptor EP1

could be used to protect against secondary brain injury after ICH (Zhao et al.).

EP3 reception has also been shown to be detrimental after ICH. PGE2 receptor EP3

(EP3R) is expressed mainly by neurons and activated glial cells in brain, and is associated with

neuronal death. Thrombin is a serine protease that converts fibrinogen into fibrin in blood

coagulation. Past evidence has confirmed that thrombin acts as a major contributor to acute ICH

injury by promoting blood–brain barrier disruption, brain edema, and neuroinflammation (Li et

al.). In this experiment, the role of the EP3 receptor in thrombin toxicity, a major contributor to

ICH Injury, was investigated. The results of this experiment show that in vivo, EP3 inhibition

reduced lesion volume, neurologic deficit, cell death, and matrix metalloproteinase-9 activity. In

vitro, EP3 inhibition reduced thrombin-induced hippocampal CA1 cell death (Zhu et al.).

Additionally, RhoA-Rho kinase levels were increased after thrombin injection and were

decreased by EP3 receptor inhibition, suggesting that the Rho-ROCK signaling pathway is

involved in the neuronal damage sustained after ICH. Overall, these results indicate that the the

PGE2 receptor EP3 contributes to thrombin induced neuronal damage after ICH (Han et al.).

Conclusion:
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Intracerebral hemorrhage is a major problem in today’s world, and remains incredibly

deadly. Even for those who are lucky to survive, they are plagued with irreversible disabilities

for the rest of their lives. With stroke rates rising within the last few decades, it becomes

imperative that a novel, effective treatment for ICH is discovered. (“Stroke Facts”). This may be

able to be accomplished through modulation of neuronal signaling pathways, which are able to

stimulate the activation of microglia, the immune cells of the brain. This, in turn, would help to

mitigate the detrimental impacts of the immune response while simultaneously promoting the

beneficial aspects of the immune response. A major source of potential for developing a better

treatment for ICH lies in PGE2 signaling. As a result of the major role that it plays in the brain

after ICH, it can be assumed that modulation of all PGE2 signaling would allow for better access

into certain neuronal processes that are necessary to target after ICH. As discussed in this paper,

experimentation has revealed that EP1 and EP3 receptor signaling, specifically, are detrimental,

while EP2 signaling is actually beneficial and has a neuroprotective role (Wu et al.) (Li et al.

(Hua et al.). Further research should be conducted in order to determine how PGE2 signaling can

be manipulated to develop an effective treatment for ICH.

Appendix:

In Vitro - A term that indicates that a procedure that was conducted outside of a living organism.

Agonist - A substance that initiates a physiological response when combined with a receptor.

Matrix Metalloproteinase - A group of enzymes that are responsible for the degradation of most

extracellular matrix proteins

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Signaling Pathway - A group of molecules in a cell that work together to control one or more cell

functions. After the first molecule in a pathway receives a signal, it activates another molecule.

Polarization - Change in the spatial differences in shape, structure, and function within a cell.

Oxidative Stress - An imbalance between the production of free radicals and the ability of the

body to counteract or detoxify their harmful effects through neutralization by antioxidants.

Brain Edema - Excess accumulation of fluid in the intracellular or extracellular spaces of the

brain.

Brain Atrophy - Deterioration and breakdown of the cells within the brain.

Hemin - An iron-containing porphyrin with chlorine that can be formed from a haem group
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