Andrew Qian

IM-6/11-AP
The Neurological Impact of Inhibition of EP3 Signaling after ICH

I. 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. For the hundreds of thousands that survive, they are forced to live with debilitating

injuries, most of which reduce the mobility of the patient. 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,

accounts for only 15% of all stroke cases, but 40% of all stroke deaths. This 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 in

the development of 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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II. Background Section:

A) Stroke is a growing problem in the United States among the elderly
a) ICH, (Intracerebral Hemorrhage), can be caused by many different factors, such
as high blood pressure or external injury. It can lead to many detrimental chronic
issues besides death.
i) In the present day, stroke rates have been rising steadily since 1995, with
hospitalizations of men for strokes nearly doubled from 1995 through
2012 (Naqvi).
ii) Intracerebral hemorrhage (ICH) is caused by bleeding within the brain
tissue itself — a life-threatening type of stroke. It is most commonly
caused by hypertension or head trauma.
iii) 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”).
iv) There are several factors that can cause ICH; head trauma, high blood
pressure, blood or bleeding disorders, blood vessel abnormalities, etc.
v) After ICH, lasting issues can remain, such as paralysis,
numbness/weakness in the body, personality changes/emotional issues,
and loss of certain motor functions (Wu 4) .
b) There is a lack of an effective treatment for ICH.
i) ICH remains the most deadly for of stroke, with the highest mortality rate
as well as lacking an effective treatment (Mayer 4).
ii) Only about half of all people will survive intracerebral hemorrhage, and
most of those patients will suffer from a significant disability.
iii) 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).
iv) 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.
v) Often, surgery becomes necessary as a form of treatment for ICH in order
to reduce neuronal bleeding (Stroke Treatments).
B) Microglia
a) Microglia are the immune cells of the brain, and play the role of a macrophage in
the brain.
i) Microglia 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.
ii) At the site of the injury, microglia are key for the phagocytosis of
pathogens as well as remove damaged cells.
iii) 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).
b) Microglia Change Shape after injury, a process known as polarization
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i) During a normal period of time, Microglia are described as in a resting

state. Despite their name, they are still constantly alert, constantly
extending and retracting their “limbs” to inspect the environment. This is
how they are able to detect injury.
ii) After 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.).
iii) The microglia reach their peak of activation within 2-3 days, but will
sustain overall activation for a few weeks (Kim and Cho).
iv) The level of the change in shape of the microglia can be determined by its
distance to the area of injury/stimulus.
v) Microglia closer to the lesion were more ameboid, while microglia further
from the injury maintained their original shape (Heindl et al.).
c) The two major types of microglia polarization, which have beneficial and
detrimental effects
i) Microglia are known to become polarized into two different phenotypes,
M1 and M2.
ii) 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.
iii) The M2 Phenotype is also involved in repair and regeneration processes,
as they can attenuate the inflammatory response and stimulate tissue repair
(Guruswamy and ElAli).

III. 1st Main Idea: PGE2 Receptor Signaling

A) PGE2 receptors, also known as prostaglandin E Receptors
a) These receptors are present in all mammalian tissues, and they exert a wide
variety of actions through the usage of G-Protein coupled receptors (GCPRs).
b) There are 4 types of PGE Receptors: EP1, EP2, EP3, and EP4.
c) While EP receptors are found all throughout the brain, they are abundant through
the brain (Bhattacharya et al.).
d) PGE Receptor signaling is a valuable signaling pathway in the brain that
modulates many important physiological processes such as the CNS, the
cardiovascular, gastrointestinal, and immune systems, among others.
e) In certain cases, PGE receptors appear to have pro-inflammatory properties, while
in others cases they exhibit anti-inflammatory properties (Hata and Breyer).
B) PGE Receptor signaling is involved in a lot of neurological processes, with both benefits
and detriments.
a) Experimental results reveal that activation of the EP2 receptor in neurons
increased cyclic AMP (cAMP) and protein kinase A signaling.
b) This increase in cAMP and protein kinase A signaling, in turn, improved
neuroprotection against hemin neurotoxicity in vitro.
c) Activation of EP2 receptor then could be used to minimize neuronal damage after
certain neural disorders such as hemin (Mohan et al.).
d) Prostaglandin receptors have also been shown to have a protective impact against
cerebral ischemia.
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e) In vitro studies revealed that activation of the EP2 receptor had neuroprotective
impacts NMDA toxicity and oxygen glucose deprivation ( McCullough et al.).

IV. 2nd Main Idea: Neuroprotective Signaling after Stroke

A) PGE Receptor EP2 has been shown to have neuroprotective effects after ICH.
a) In one experiment, ICH was induced in mice through intrastriatal injection of
collagenase as well as the injection of arterial whole blood.
i) The ICH that resulted from this was associated with increased
inflammatory responses, oxidative stress, etc.
ii) Deletion/inhibition of PGE Receptor EP2 exacerbated brain
swelling/edema, neuronal death, and neurobehavioral deficits.
iii) Activation of PGE Receptor EP2 through the agonist AE1-259-01,
however, mitigated, and in some cases, reversed this damage.
iv) This allows for the conclusion 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.).
b) Another study, which focused on a specific agonist of EP2, was shown to have
neuroprotective impacts as well.
i) Misoprostol is a synthetic PGE2 agonist, while recent studies have shown
that misoprostol also has some protective effects after cerebral ischemia as
well.
ii) In the brain, macrophages/microglia secrete matrix metalloproteinases,
neuronal inflammation modulators, through a PGE2 dependent
mechanism.
iii) Inhibition of these matrix metalloproteinases have been shown to reduce
neuronal injury after ICH (Gao et al.).
iv) 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.
v) These results support the idea that misoprostol plays a protective role in
the brain after ICH (Hua et al.).

V. 3rd Main Idea: Neurologically Damaging Signaling after Stroke.

A. While PGE Receptors EP2 and EP4 may exhibit beneficial impacts, PGE receptors EP3
and EP1 both have neurotoxic effects.
a. EP1 Receptor is a major factor in neuronal toxicity after ICH.
i. A study conducted at Johns Hopkins University analyzed the role of the
EP1 receptor after ICH.
ii. Primarily, they studied the interaction between the Ep1 receptor and the
Src pathway.
iii. 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
iv. The results of these studies revealed that activation of the EP1 receptor
elicits neural toxicity through the Src kinase pathway (Heng et al.).
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v. This form of reception also acted through the MMP-9 signaling pathway, a
matrix metalloproteinase pathway which has been linked to
neuroinflammation after ICH.
vi. These results indicate that inhibition of the PGE2 receptor EP1 could be
used to protect against secondary brain injury after ICH (Zhao et al.).
b. EP3 reception has also been shown to be detrimental after ICH
i. PGE2 receptor EP3 (EP3R) is expressed mainly by neurons and activated
glial cells in brain, and is associated with neuronal death.
ii. 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.).
iii. In this experiment, the role of the EP3 receptor in thrombin toxicity, a
major contributor to ICH Injury, was investigated
iv. The results of this experiment show that in vivo, EP3 inhibition reduced
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.).
v. 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.
vi. Overall, these results indicate that the the PGE2 receptor EP3 contributes
to thrombin induced neuronal damage after ICH (Han et al.).

VI. Conclusion:

Intracerebral hemorrhage is a major problem 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
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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
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.

Works Cited

Awasthi, D. (n.d.). Spontaneous Intracerebral Hemorrhage. Retrieved December 31, 2018,

from https://www.medschool.lsuhsc.edu/neurosurgery/nervecenter/spontICH.html
Bhattacharya, M., Peri, K. G., Almazan, G., Ribeiro-da-Silva, A., Shichi, H., Durocher, Y.,
… Chemtob, S. (1998). Nuclear localization of prostaglandin E2 receptors. Proceedings
of the National Academy of Sciences, 95(26), 15792–15797.
https://doi.org/10.1073/pnas.95.26.15792
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Chen, Y., Won, S. J., Xu, Y., & Swanson, R. A. (2014). Targeting Microglial Activation in
Stroke Therapy: Pharmacological Tools and Gender Effects. Current Medicinal
Chemistry, 21(19), 2146–2155.

Guruswamy, R., & ElAli, A. (2017). Complex Roles of Microglial Cells in Ischemic Stroke
Pathobiology: New Insights and Future Directions. International Journal of Molecular
Sciences, 18(3). https://doi.org/10.3390/ijms18030496

Han, X., Lan, X., Li, Q., Gao, Y., Zhu, W., Cheng, T., … Wang, J. (2016). Inhibition of
prostaglandin E2 receptor EP3 mitigates thrombin-induced brain injury. Journal of
Cerebral Blood Flow & Metabolism, 36(6), 1059–1074.
https://doi.org/10.1177/0271678X15606462

Hata, A. N., & Breyer, R. M. (2004). Pharmacology and signaling of prostaglandin

receptors: multiple roles in inflammation and immune modulation. Pharmacology &
Therapeutics, 103(2), 147–166. https://doi.org/10.1016/j.pharmthera.2004.06.003

Heindl, S., Gesierich, B., Benakis, C., Llovera, G., Duering, M., & Liesz, A. (2018).
Automated Morphological Analysis of Microglia After Stroke. Frontiers in Cellular
Neuroscience, 12. https://doi.org/10.3389/fncel.2018.00106

Hemorrhagic stroke – National Stroke Association. (n.d.). Retrieved December 31, 2018,
from https://www.stroke.org/understand-stroke/what-is-stroke/hemorrhagic-stroke/
How to Write a Lab Proposal | Synonym. (n.d.). Retrieved October 1, 2018, from
https://classroom.synonym.com/write-lab-proposal-7738594.html

Hua, W., Dong, X., Gao, Y., Zhao, X., Chen, W., Cao, W., … Wu, T. (2015). PGE2
receptor agonist misoprostol protects brain against intracerebral hemorrhage in mice.
Neurobiology of Aging, 36(3), 1439–1450.
https://doi.org/10.1016/j.neurobiolaging.2014.12.029

Intracerebral hemorrhage (ICH), hemorrhagic stroke, stroke | Cincinnati, OH Mayfield Brain

& Spine. (n.d.). Retrieved January 2, 2019, from https://mayfieldclinic.com/pe-ich.htm

Kim, E., & Cho, S. (2016). Microglia and Monocyte-Derived Macrophages in Stroke.
Neurotherapeutics, 13(4), 702–718. https://doi.org/10.1007/s13311-016-0463-1

McCullough, L., Wu, L., Haughey, N., Liang, X., Hand, T., Wang, Q., … Andreasson, K.
(2004). Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia.
Journal of Neuroscience, 24(1), 257–268. https://doi.org/10.1523/JNEUROSCI.4485-
03.2004

Microglial function in the Healthy Brain. (n.d.). Retrieved September 17, 2018, from
https://faculty.sites.uci.edu/kimgreen/bio/microglia-in-the-healthy-brain/
 Qian 7

Mohan, S., Narumiya, S., & Dore, S. (n.d.). Neuroprotective Role of Prostaglandin PGE2
EP2 Receptor in Hemin-mediated Toxicity. Retrieved January 6, 2019, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681391/

Stroke Facts | cdc.gov. (2018, October 10). Retrieved December 31, 2018, from
https://www.cdc.gov/stroke/facts.htm

Wu, B. (n.d.). Brain hemorrhage: Causes, symptoms, and treatments. Retrieved January 2,
2019, from https://www.medicalnewstoday.com/articles/317080.php

Wu, H., Wu, T., Han, X., Wan, J., Jiang, C., Chen, W., … Wang, J. (2017).
Cerebroprotection by the neuronal PGE2 receptor EP2 after intracerebral hemorrhage in
middle-aged mice. Journal of Cerebral Blood Flow & Metabolism, 37(1), 39–51.
https://doi.org/10.1177/0271678X15625351

Zhao, S., Ma, L., Chu, Z., Xu, H., Wu, W., & Liu, F. (2017). Regulation of microglial
activation in stroke. Acta Pharmacologica Sinica, 38(4), 445–458.
https://doi.org/10.1038/aps.2016.162

Zhao, X., Wu, T., Chang, C.-F., Wu, H., Han, X., Li, Q., … Wang, J. (2015). Toxic Role of
Prostaglandin E2 Receptor Ep1 After Intracerebral Hemorrhage in Mice. Brain,
Behavior, and Immunity, 46, 293–310. https://doi.org/10.1016/j.bbi.2015.02.011
 Qian 8

Works Consulted

Deb, P., Sharma, S., & Hassan, K. (n.d.). Pathophysiologic mechanisms of acute ischemic
stroke: An overview with emphasis on therapeutic significance beyond thrombolysis -
Pathophysiology. Retrieved September 10, 2018, from
https://www.pathophysiologyjournal.com/article/S0928-4680(09)00136-9/fulltext

Kanazawa, M., Ninomiya, I., Hatakeyama, M., Takahashi, T., & Shimohata, T. (2017).
Microglia and Monocytes/Macrophages Polarization Reveal Novel Therapeutic
Mechanism against Stroke. International Journal of Molecular Sciences, 18(10).
https://doi.org/10.3390/ijms18102135

Mayer, S. A. (2003). Ultra-early hemostatic therapy for intracerebral hemorrhage. Stroke,

34(1), 224–229.

Naqvi, J. (n.d.). Stroke rates appear to be rising steadily in young adults - The Washington
Post. Retrieved December 31, 2018, from https://www.washingtonpost.com/news/to-
your-health/wp/2017/04/15/stroke-rates-appear-to-be-rising-steadily-in-young-
adults/?noredirect=on&utm_term=.8a15b60d28b7

Overview of Immunohistochemistry - US. (n.d.). Retrieved September 22, 2018, from

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-
biology-learning-center/protein-biology-resource-library/pierce-protein-
methods/overview-immunohistochemistry.html

Saulle, M. F., & Schambra, H. M. (2016). Recovery and Rehabilitation after Intracerebral
Hemorrhage. Seminars in Neurology, 36(3), 306–312. https://doi.org/10.1055/s-0036-
1581995

Wang, N., Liang, H., & Zen, K. (2014). Molecular Mechanisms That Influence the
Macrophage M1–M2 Polarization Balance. Frontiers in Immunology, 5.
https://doi.org/10.3389/fimmu.2014.00614