IschemiRs MicroRNAs in Ischemic

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Rajanikant G. K.
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Rajanikant G. K. · Pierre Gressens
Sreekala S. Nampoothiri
Gokul Surendran · Cindy Bokobza

IschemiRs:
MicroRNAs
in Ischemic
Stroke
From Basics to Clinics
IschemiRs: MicroRNAs in Ischemic Stroke
Rajanikant G. K. • Pierre Gressens •
Sreekala S. Nampoothiri •
Gokul Surendran • Cindy Bokobza

IschemiRs: MicroRNAs
in Ischemic Stroke
From Basics to Clinics
Rajanikant G. K. Pierre Gressens
School of Biotechnology NeuroDiderot, Inserm
National Institute of Technology Calicut Université de Paris
Calicut, India Paris, France

Sreekala S. Nampoothiri Gokul Surendran

Development and Plasticity School of Biotechnology
of the Neuroendocrine Brain National Institute of Technology Calicut
Lille Neuroscience & Cognition Calicut, India
INSERM 1172
Lille, France

Cindy Bokobza
NeuroDiderot, Inserm
Université de Paris
Paris, France

ISBN 978-981-15-4797-3 ISBN 978-981-15-4798-0 (eBook)

https://doi.org/10.1007/978-981-15-4798-0

# Springer Nature Singapore Pte Ltd. 2020

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Preface

MicroRNAs are small endogenous noncoding RNA molecules that function as a

regulator of gene expression at the posttranscriptional level. In recent years, the scientific
community has witnessed a massive increase in the wealth of knowledge that bridges the
microRNA and its strategic role as a biomarker and therapeutic in a wide range of
diseases, including ischemic stroke. However, there is still a lack of comprehensive
details linking this small RNA molecule to an ischemic stroke, the more preponderant
among the two main types of stroke. This monograph is intended as a medium to
examine the emerging role of microRNAs in cerebral ischemia. The book addresses
topics related to the activity of microRNAs in multiple pathological manifestations of
strokes such as oxidative stress, excitotoxicity, cell death trends, their use as a biomarker,
diagnostic agents, and currently practiced therapeutic interventions. It also highlights the
latest research on how microRNAs help in neuroregeneration during poststroke events,
the myriad of computational tools and databases used in microRNA research, and how
they modulate other cerebrovascular diseases. The book concludes with a special insight
into the potentials of developing microRNA-based therapies for ischemic stroke.
This book is the first of its kind to bring together pieces of puzzle concerning the
effect of the microRNAs on the ischemic stroke. Given the vast literature available in
public libraries, our book seeks to provide readers with cutting-edge knowledge on
microRNA covering various aspects of stroke. This alone makes our book, one that
researchers in this area would be pleased to have in their bookshelves. This book can
serve as a comprehensive tool for researchers, doctoral and graduate students, and
clinicians in the field of miRNA and ischemic stroke and aid in the development of
reliable biomarkers and neurotherapeutics by identifying suitable microRNAs.
We would like to thank the Springer team, especially Madhurima Kahali and
Selvakumar Rajendran, for their patient, professional, and constant support through-
out the production of this book.

Calicut, India Rajanikant G. K.

Paris, France Pierre Gressens
Lille, France Sreekala S. Nampoothiri
Calicut, India Gokul Surendran
Paris, France Cindy Bokobza

v
Contents

Part I microRNAs and Ischemic Stroke: Back to Basics

1 microRNAs in Normal Brain Physiology . . . . . . . . . . . . . . . . . . . . . 3
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 microRNA Biogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 miRNA in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3.1 miRNA in Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3.2 miRNA in Oligodendrocytes . . . . . . . . . . . . . . . . . . . . 7
1.3.3 miRNA in Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.4 miRNA in Microglia . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
2 Ischemic Stroke: An Imperative Need for Effective Therapy . . . . . . 15
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
2.2 Current Treatment Choices for Ischemic Stroke Victims . . . . . . 16
2.2.1 IV Pharmacological Thrombolysis . . . . . . . . . . . . . . . . 16
2.2.2 Endovascular Recanalization Strategies . . . . . . . . . . . . 18
2.3 Other Reperfusion Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.4 Post-Ischemic Neuroprotective Agents . . . . . . . . . . . . . . . . . . . 24
2.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Part II microRNAs in Ischemic Stroke Pathophysiology: A Bird’s

Eye View
3 MicroRNAs in Ischemic Stroke Pathophysiology: Special
Emphasis on Early Molecular Events . . . . . . . . . . . . . . . . . . . . . . . 33
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Post-Ischemic Glutamate Excitotoxicity and Ionic Imbalance . . . 34
3.2.1 miRNAs Targeting Glutamate Receptors . . . . . . . . . . . 36
3.2.2 miRNAs and Glutamate Transporters . . . . . . . . . . . . . . 37
3.2.3 miRNA Regulation of Post-Ischemic Calcium Overload . 37
3.3 miRNAs in Post-Ischemic Oxidative Stress Regulation . . . . . . . 39

vii
viii Contents

3.3.1 Endogenous Antioxidant Defense Mechanisms . . . . . . . 39

3.3.2 Oxidative Stress-Induced Mitochondrial Dysfunction . . 41
3.3.3 miRNAs and the Master Regulator Nrf2 . . . . . . . . . . . 41
3.3.4 Endothelial Integrity and Blood–Brain Barrier . . . . . . . 42
3.4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4 microRNA Regulation of Ischemic Stroke Inflammatory
and Immune Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4.2 Stroke Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.3 microRNAs Associated with Ischemic Neuro-Inflammation . . . . 51
4.4 microRNAs and Innate Immune Cells in Ischemic Stroke . . . . . 53
4.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5 Regulatory Role of microRNAs in Ischemic Cell Death . . . . . . . . . . 59
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
5.2 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5.2.1 Intrinsic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.2.2 Extrinsic Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.3 Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
5.4 MicroRNAs and Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5.5 MicroRNAs and Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6 The Emerging Role of microRNAs in Post-ischemic Angiogenesis
and Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.2 Post-Stroke Induced Neurogenesis . . . . . . . . . . . . . . . . . . . . . . 68
6.2.1 miRNA Regulation of Early Stages of Neurogenesis . . . 68
6.2.2 Neuronal Maturation and Integration . . . . . . . . . . . . . . 71
6.2.3 miRNAs Regulating Neurotrophic Factors . . . . . . . . . . 73
6.2.4 miRNAs and Neurogenesis Signaling Pathways . . . . . . 74
6.3 miRNA Regulation of Post-Ischemic Angiogenesis . . . . . . . . . . 75
6.3.1 EC-Specific miRNAs in Angiogenesis . . . . . . . . . . . . . 76
6.3.2 Post-Ischemic miRNA Regulation of Angiogenic
Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
6.4 Coupling Angiogenesis and Neurogenesis Post-Ischemia . . . . . . 79
6.5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
7 MicroRNAs as Potential Diagnostic, Prognostic, and Therapeutic
Biomarkers in Ischemic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
7.2 Important Circulating microRNAs in Ischemic Stroke . . . . . . . . 89
7.2.1 Let 7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Contents ix

7.2.2 MiRNA-125a-5p/miR-125b-5p/miR-125b-2
. . . . . . . . 90
7.3 Embolic Stroke Versus Thrombotic Stroke . . . . . . . . . . . . . . . . 90
7.4 Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
7.5 Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
7.6 Neurogenesis and Angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . 91
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
8 Interplay Between microRNAs and Other Cerebrovascular
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
8.2 Hemorrhagic Stroke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
8.3 Intracerebral Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
8.3.1 microRNAs as ICH Biomarkers . . . . . . . . . . . . . . . . . 96
8.3.2 Regulatory Role of microRNAs in ICH . . . . . . . . . . . . 97
8.4 Subarachnoid Hemorrhage . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
8.4.1 microRNAs as SAH Biomarkers . . . . . . . . . . . . . . . . . 99
8.4.2 Disease Modifying Role of microRNAs in SAH . . . . . . 101
8.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
9 New Insights into the Regulatory Role of lncRNA, circRNA,
piRNAs, and ceRNAs in Ischemic Stroke . . . . . . . . . . . . . . . . . . . . 107
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
9.1.1 Non-coding RNAs at a Glance . . . . . . . . . . . . . . . . . . 107
9.2 New Players in the Game . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
9.2.1 Long Non-coding RNA . . . . . . . . . . . . . . . . . . . . . . . 109
9.2.2 Circular RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
9.2.3 PIWI RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
9.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
9.4 Future Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

Part III IschemiRs: The Bench and Beyond

10 Computational Resources for microRNA Research . . . . . . . . . . . . . 117
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
10.2 MiRNA Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
10.3 MicroRNA Prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
10.4 MicroRNA Disease Interaction . . . . . . . . . . . . . . . . . . . . . . . . 120
10.5 MicroRNA-Target Interaction . . . . . . . . . . . . . . . . . . . . . . . . . 120
10.6 MicroRNA Interaction with Transcription Factors . . . . . . . . . . . 121
10.7 Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
x Contents

11 MicroRNA-Targeted Therapeutics for Ischemic Stroke: Status,

Gaps and the Way Forward . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
11.2 Restoration of miRNA Function as a Potential Ischemic
Stroke Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
11.3 Development of Anti-miRNA-Based Ischemic Stroke
Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
11.3.1 Antisense Oligonucleotides . . . . . . . . . . . . . . . . . . . . . 127
11.3.2 Chemically Modified Antisense Oligonucleotides/
Antagomirs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
11.3.3 Locked Nucleic Acid (LNA) . . . . . . . . . . . . . . . . . . . . 128
11.3.4 miRNA Sponge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
11.4 Delivery of miRNA Modulators for Ischemic Stroke Therapy . . 130
11.4.1 Viral-Based miRNA Delivery System . . . . . . . . . . . . . 130
11.4.2 Non-Viral miRNA Delivery Systems . . . . . . . . . . . . . . 131
11.4.3 Exosomal Mediated Delivery of miRNAs . . . . . . . . . . 133
11.5 Pharmacological Agents and Small-Molecules as Therapeutic
Modulators of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
11.6 Challenges, Perspectives, and Way-Forward . . . . . . . . . . . . . . . 134
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
About the Authors

Rajanikant G. K. is currently working as a Professor at the School of Biotechnol-

ogy and served as the Coordinator of DBT—Bioinformatics Infrastructure Facility at
National Institute of Technology Calicut, Calicut, India. Having done his Ph.D. in
Radiation Biology and Toxicology from Division of Radiobiology and Toxicology,
Manipal University, Manipal, India, he was also awarded a Senior Research Fellow-
ship from Indian Council of Medical Research, Government of India, New Delhi, for
the year 2001–2004. Till date, he has worked as a Principal Investigator in various
government-funded research projects and has further guided and supervised several
Doctorate and Post-Doctorate students and researchers. He has also successfully
applied for an Indian Patent in the year 2017 for using a pharmaceutical composition
consisting of 1,4-DI(1H-benzimidazol-2-YL)-1,2,3,4-butanetetrol (BTT) as an
ischemic neuroprotectant. He has also been invited as a speaker to various presti-
gious conferences and has conducted many conferences, workshops, and seminars
successfully. He has several articles in renowned national and international journals,
as well as book chapters and edited volumes, to his credit.

Pierre Gressens is presently holding several prominent positions, including a

Research Officer (DR1 Inserm) and Director, UMR 1141 Inserm-Paris University,
Robert Debré Hospital, Paris, France; Professor of Fetal and Neonatal Neurology,
KCL, London, UK; Consultant, Service of Pediatric Neurology, Robert Debré
Hospital, Paris, France; Director, PremUP Foundation, Paris; Coordinator of the
University-Hospital Department PROTECT, Robert Debré Hospital, Paris; and
Vice-Dean for Research, Paris-Diderot Medical School, Paris, France. He obtained
his M.D. and Ph.D. degrees from University of Louvain Medical School at Brussels,
Belgium. He has received various honors, such as Roger de Spoelberch award for
neurosciences (Switzerland) in 2010, FRM Fondation Guillaumat-Piel award
(France) in 2013, and the Elsie Widdowson Lecture at the Neonatal Society,
London, UK in 2017. He has been invited as a speaker to various prestigious
conferences. He has published his research/review articles in many international
journals of great repute.

Sreekala S. Nampoothiri is currently a postdoctoral researcher at INSERM 1172,

Development and plasticity of the neuroendocrine brain, Lille Neuroscience &

xi
xii About the Authors

Cognition, Université de Lille, Lille, France. She has a PhD in Biotechnology from
National Institute of Technology Calicut, Kerala, on the thesis entitled “microRNA-
9 Upregulation Integrates Postischemic Neuronal Survival and Regeneration: A
Systems Biology Approach.” She is a recipient of Raman-Charpak Fellowship
2015 and performed a part of her doctoral thesis research at INSERM 1141,
Université Paris Diderot, Hôpital Robert Debré, Paris. She was the University topper
for her Masters (M.Tech) program in Biotechnology and Biochemical Engineering
(specialization in Molecular Medicine) at Sree Buddha College of Engineering,
University of Kerala, Thiruvananthapuram, India, as well as for her Bachelors
(B.E.) in Biomedical Engineering, from Anna University, Tamil Nadu, India. She
was also selected by Indian Academy of Sciences as Summer Junior Research
Fellow (SJRF) in May 2012. She has published articles in journals of good repute
and has also presented her research at various conferences and seminars.

Gokul Surendran is currently pursuing his PhD. from National Institute of Tech-
nology Calicut, Kerala, India. He did his M.S. (Pharm.) Biotechnology from
National Institute of Pharmaceutical Education and Research, Hajipur, India. He
was also awarded a Fellowship during his M.S. (Pharm.) from Ministry of Chemicals
and Fertilizers (Government of India) and has further qualified the National Institute
of Pharmaceutical Education and Research (NIPER) Joint Entrance Examination
(NIPER-JEE-2014) with All India Rank of 400 and Graduate Pharmacy Aptitude
Test (GPAT) Examination (GPAT 2014) with All India Rank of 204. He has also
presented his research at various conferences and seminars, and published articles in
good journals.

Cindy Bokobza holds a Master’s degree from the European Master’s in Genetics
(Magistère) Program at the Université de Paris and from the University of Trieste,
Italy. She worked as an intern at the Center for Human Disease Modeling (CHDM)
at Duke University, USA. She obtained her PhD in Neuroscience from the
Université de Paris (Ecole Doctorale BioSPC) for her studies on the impact of
perinatal inflammation on neurodevelopment in the team of Pierre Gressens at the
Hôpital Robert Debré, Paris. She was a G.E.N.E. Fellow for postdoctoral research,
funded by the EUR G.E.N.E. Graduate School (#ANR-17-EURE-0013). She also
served as a Student Representative at PhD School Bio Sorbonne Paris Cité.
Abbreviations

20 -OMe 20 -O-methyl
AAV Adeno-associated viruses
AGO2 Argonaute 2
AMPA α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
aNPCs Amplifying neural progenitor cells
APAF-1 Apoptotic protease-activating factor 1
ARE Antioxidant response element
aSAH Aneurysmal subarachnoid hemorrhage
ATP Adenosine triphosphate
AuNPs Gold nanoparticles
BBB Blood–brain barrier
BCL2 B-cell lymphoma 2
Bcl-2 B-cell lymphoma 2
Bcl2l11 BCL2 like 11
BDNF Brain-derived neurotrophic factor
bFGF Basic fibroblast growth factor
BIM BCL2 interacting mediator of cell death
Ca2+ Calcium ion
CALM1 Calmodulin 1
CaM Calmodulin
CaMKIIδ Calcium/calmodulin-dependent protein kinase II δ
CaMKK Calcium/calmodulin-dependent protein kinase kinase
CaMKs Calcium/calmodulin-dependent protein kinases
CAT Catalase
CEBPb CCAAT enhancer binding protein beta
circRNA DLGAP4 circDLGAP4
CMV Cytomegalovirus
CNS Central nervous system
CypD Cyclophilin D
DAMP Damage-associated molecular pattern
DGCR8 DiGeorge syndrome chromosome region 8
DISC Death-inducing signaling complex
DRG Dorsal root ganglion

xiii
xiv Abbreviations

Drp-1 Dynamin-related protein-1

EAAT1–5 Excitatory amino acid transporters 1–5
EC Endothelial cell
eEF2 Eukaryotic elongation factor-2 kinase
EGFL7 Epidermal growth factor-like-domain 7 gene
EGR2 Early growth response 2
eNOS Endothelial nitric oxide synthase
ePAM-R Polyamidoamine ester
EPAR Endovascular photoacoustic recanalization
ER Endoplasmic reticulum
FADD Fas-associated death domain protein
FasIII Fasciclin III
FDA Food and Drug Administration
GDNF Glial-derived neurotrophic factor
GLAST/EAAT1 Glutamate-aspartate transporter
GLT-1/EAAT2 Glutamate transporter-1
GluR2 Glutamate receptor-2
GSHPx Glutathione peroxidase
H2O2 Hydrogen peroxide
HI Hypoxic-ischemic
HO-1 Heme oxygenase-1
HSPs Heat shock proteins
I/R Ischemia/reperfusion
IA Intra-arterial
IA Intracranial aneurysm
IAT Intra-arterial thrombolysis
ICAM-1 Intercellular adhesion molecule-1
ICH Intracerebral hemorrhage
ICV Intracerebroventricular
IGF-1 Insulin-like growth factor 1
IL Interleukin
IMS Interventional management of stroke
IV Intravenous
JAG1 Jagged-1
Keap1 Kelch-like erythroid cell-derived protein
LimK1 LIM domain kinase 1
LNA Locked nucleic acid
lncRNA Long noncoding RNA
LSD1 Lysine demethylase 1A
Map1b Microtubule-associated protein 1b
MAPK/ERK1/2 Mitogen-activated protein kinase/extracellular regulated
kinase 1/2
Mash1 Achaete-scute homolog 1
MCA Middle cerebral artery
MDA Methane dicarboxylic aldehyde
Abbreviations xv

MeCP2 Methyl CpG binding protein 2

Mib1 Mind bomb one
miRNA microRNA
miRNAs microRNAs
miRs MicroRNAs
MMP Matrix metalloproteinase
MPTP Mitochondrial permeability transition pore
MSC Mesenchymal stem cell
Na+ Sodium
ncRNAs Noncoding RNAs
NCX Sodium–calcium exchanger
NeuroD1 Neuronal differentiation 1
NF-kB Nuclear factor kappa b
NGF Nerve growth factor
NIHSS National Institutes of Health Stroke Scale
NINDS National Institute of Neurological Disorders and Stroke
NMDA N-methyl-D-aspartate
NO Nitric oxide
NOX Nicotinamide adenine dinucleotide phosphate-oxidase
NPCs Neural progenitor cells
Nrf2/NFE2L2 Nuclear factor erythroid 2-related factor 2
Nrg Neuroglian
NSC/NSPC Neural stem/progenitor cell
NSCs Neural stem cells
Numbl NUMB like endocytic adaptor protein
O2 Oxygen
OGD Oxygen–glucose deprivation
OLs Oligodendrocytes
OPCs Oligodendrocytes progenitor cells
PAMAM Polyamidoamine
PAMAM-Arg PAMAM conjugated with arginine
PAMP Pathogen-associated molecular pattern molecule
PDGF Platelet-derived growth factor
PEI Polyethylenimine
PI3K/mTOR Phosphatidylinositol-3-kinase/mammalian target of rapamycin
PLGA Polylactic-co-glycolic acid
pMCAO Permanent middle cerebral artery occlusion
Pol II RNA polymerase II
PRDXs Peroxiredoxins
PS Phosphorothioate
PTA Percutaneous transluminal angioplasty
PTEN Phosphatase and tensin homolog
PUMA p53 upregulated modulator of apoptosis
qHTS Quantitative high-throughput screening
xvi Abbreviations

REST RE1 silencing transcription factor

RGCs Retinal ganglion cells
Rheb Ras homolog, MTORC1 binding
RISC RNA-induced silencing complex
RNA Ribonucleic acid
RNS Reactive nitrogen species
ROS Reactive oxygen species
RTN Retrograde transvenous neuroperfusion
rt-PA Recombinant tissue-plasminogen activator
SAH Subarachnoid hemorrhage
SAINT Stroke-acute ischemic NXY treatment
Sema3A Semaphorin 3A
SESs Self-expanding stents
SGZ Subgranular zone
SHIP-1 Inositol polyphosphate-5-phosphatase D
siRNA Small interfering RNA
SOCS-1 Suppressor of cytokine signaling 1
SODs Superoxide dismutases
SOX9 SRY-box transcription factor
Spred-1 Sprouty-related EVH1 domain containing 1
SPRY2 Sprouty 2
STAIR Stroke Treatment Academic Industry Roundtable
SUMO Small ubiquitin-like modifier
SVZ Subventricular zone
TAp73 Tumor protein P73
TF Tissue factor
TGF Transforming growth factor
TJs Tight junctions
TLR Toll-like receptor
TLR4 Toll-like receptor 4
TNF Tumor necrosis factor
t-PA Tissue-plasminogen activator
Usp14 Ubiquitin specific peptidase 14
UTR Untranslated
VCAM Vascular cell adhesion molecule
VEGF Vascular endothelial growth factor
VEGFR Vascular endothelial growth factor receptor
WHO World Health Organization
XPO-5 Exportin-5
ZO-1 Tight junction protein
 Part I
microRNAs and Ischemic Stroke: Back to Basics
microRNAs in Normal Brain Physiology
1

Abstract

MicroRNAs (miRs) are short single-stranded non-coding RNAs that regulate

protein synthesis by translational repression or degradation by targeting
mRNAs at the post-transcriptional level. Highly conserved across species, they
are known to regulate many genes in critical pathways during the lifetime. Any
dysregulation at the cellular level, but also in the entire organism, is therefore
harmful. The brain is a privileged organ in many ways, and interestingly, the
majority of miRs are expressed in the brain. We describe miRs biogenesis in this
chapter and focus on the role of cell-specific miRs in normal brain physiology and
homeostasis.

Keywords

miRNAs · Brain · Biogenesis · Physiology · Neurons oligodendrocytes ·

Astrocytes · Microglia

1.1 Introduction

In general, gene expression passes through several processes, including transcription

of the genomic DNA sequence in mRNA that then translates to a protein. To
modulate which gene is expressed and at which level (in terms of quantity and
localization), evolutionarily integrated several checkpoints and/or keylocks are
required to fine-tune the regulation of gene expression. All of them are mostly
grouped in epigenetics, which is the study of gene expression modulation without
affecting the nucleotide sequence of the genome. This regroups DNA methylation,
histone modifications, and non-coding RNA.
A non-coding RNAs (ncRNAs) are RNA molecules that are not translated into a
protein. Many of the newly identified ncRNAs have not been validated for their
function. Many ncRNAs are also likely to be non-functional. A microRNA

# Springer Nature Singapore Pte Ltd. 2020 3

R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke,
https://doi.org/10.1007/978-981-15-4798-0_1
4 1 microRNAs in Normal Brain Physiology

(abbreviated miRNA) is a small non-coding RNA molecule (containing approxi-

mately 22 nucleotides) found in plants, animals, and some viruses, that functions in
silencing RNA and post-transcriptional regulation of gene expression [1] (Box 1.1).
The expression of aberrant miRNAs is implicated in various diseases. miRNAs
appear to control the development and function of the nervous system [2].

Box 1.1: miRNAs Discovery

• Historically, the first miRNA was described in 1993 in Caenorhabditis
elegans (C. elegans).
• This non-coding RNA is a sequence of 22 nucleotides that partially
base-to-base matched to multiple sequences in the 3’UTR of lin-14
mRNA sequence; this double-strand demonstrated a reduction of
LIN-14 protein.
• It was only several years after that a second ncRNA was characterized not
only in C. elegans, but also in Drosophila and humans.

Historically, miRs have been linked to numerous cellular processes, including

differentiation, cell proliferation, embryonic development, metabolism, and stress
response [3]. Given the fundamental role of miR in several systems, several studies
indicate that any aberration in miR biogenesis may lead to the ontogeny of human
pathologies such as cancer, diabetes, cardiovascular disease, HIV, obesity, and
nervous system disorders [4]. Besides, it is also proposed that miRs be quantifiable
biomarkers of disease progression and especially in stroke [5, 6]. The advancement
of high-throughput sequencing has allowed an exponential number of miR discovery
since 1993. Most of them are grouped in miRBase catalog (version 22, 2019),
comprising around 49,000 mature miR sequences from 271 organisms [7]. The
sequence of most miRs are conserved, suggesting a preserved role in the regulation
of gene expression at the cellular level.
In mammals, miRs interfere with 3’-UTR of their target mRNAs by partial
complementary, leading to a translation inhibition [8]. Mounting evidence
demonstrates that a single mRNA can be targeted by several miRs, whereas a single
miR can target several mRNAs.
In this chapter, we will describe miRs biogenesis and its role in maintaining
normal brain physiology. Primarily, we will describe miRs function in all brain cells
(neurons, astrocytes, oligodendrocytes, and microglia) under normal conditions.

1.2 microRNA Biogenesis

MiRs have a unique biogenesis pathway but also a variety of expression patterns that
enable their specific functions in various biological processes. We generally consider
two pathways for miR biogenesis: (1) the canonical pathway, with RNA polymerase
II (Pol II) transcription, leading to the generation of a long transcript (several
1.2 microRNA Biogenesis 5

Nucleus
Genomic DNA

E1 E2

Canonical pathway miRtron pathway

E1 E2

Pri-miRNA
DROSHA
5’ AAA 3’

Pre-miRNA

EXPORTIN

AGO proteins Pre-miRNA

RISC complex
DiCER GW182 proteins

5’

3’ 3’
Ribosome

5’

Fig. 1.1 Schematic representation of mature microRNA biogenesis

kilobases) with a hairpin structure, so-called the pri-miR; and (2) the miRtron
pathway, non-canonical pri-miRNAs are encoded in introns of coding genes
(miRtrons) [9–11] (Fig. 1.1).
Canonical miRNA biogenesis begins with post-transcriptional or
co-transcriptional processing of primary miRNA transcripts (pri-miRNAs) from
the genome. The canonical pri-miRNAs are transcribed by RNA Polymerase II
(POL II), possess a 50 -cap, and do not usually have a poly-A tail. Pri-miRNAs
6 1 microRNAs in Normal Brain Physiology

form a hairpin structure in the nucleus and are cleaved into premature miRNAs
(pre-miRNAs) by the RNase III enzyme DROSHA, which is in complex with
RNA-binding protein (RBP) DiGeorge syndrome chromosome region 8 (DGCR8).
The pre-miRNA is released into the cytoplasm from the nucleus by Exportin-5
(XPO-5) and further processed by the RNase III enzyme DICER, bound to trans-
activation-responsive RNA-binding protein (TRBP) [12]. TRBP binds to the
pre-miRNA and DICER, and cleaves the loop of the hairpin, resulting in a
miRNA duplex. The miRNA duplex interacts with components of the
RNA-induced silencing complex (RISC) loading complex (RLC). The Argonaute
2 (AGO2) destabilizes the miRNA duplex, which is unwinded by RNA helicases.
The miRNA loaded RISC (miRISC) binds to reverse complementary sequences
within the 30 -untranslated region (UTR) of target mRNAs. Mirtrons are introns-
encoded non-canonical microRNAs, the biogenesis of which begins with splicing.
They are not being processed by Drosha and join the canonical pathway at the
Exportin-5 level.
The critical functional outcome of miRNA activity is a decreased abundance of
translated protein from a target mRNA. The guide miRNA leads the RISC complex
to a corresponding sequence on the target mRNA, typically located on the 3’UTR,
resulting in translational inhibition and mRNA destabilization.

1.3 miRNA in the Brain

Literature reveals that 70% of miR are expressed in the brain; however, only a
shortlist of them is enriched in the brain or are brain-specific [13, 14]. In 2013,
Jovičić et al. described miR expression in four dcell types in culture, and determine
specifically enriched miRs in each [15]. We will explain in the following sections
brain cell-specific microRNAs and their functions in brain physiology.

1.3.1 miRNA in Neurons

In the brain, we generally discriminate neurons from other glial cells (astrocytes,
oligodendrocytes, and microglia). However, astrocytes and oligodendrocytes are
differentiated from the same neural stem cells (NSCs) that neurons are differentiated
[9, 16, 17]. During neurogenesis, microRNAs contribute to the cell fate decision of
astrocytes versus neural progenitor cells (NPCs) that will become adult neurons.
Similar mechanisms occur in adult neurogenesis [17]. Figure 1.2 groups some of the
miRs associated with neuronal differentiation, proliferation, axonal outgrowth, and
synaptogenesis.
miR-9 enhances differentiation of neuronal stem cells, promotes neuronal migra-
tion, and inhibits neural proliferation [18–20]. miR-124 also promotes neuronal
differentiation by stimulation of neurite branching; it also modulates synaptic activ-
ity [21–23]. Let-7 is a primary miR that acts in many levels, such as proliferation,
auto-renovation, and differentiation of NSCs [24, 25]. miR-106-25 works with
1.3 miRNA in the Brain 7

Differenaon/proliferaon Axonal/dendrite outgrowth Synaptogenesis & plascity

• Let-7 • miR-9 • miR-132

• miR-9 • miR-124 • miR-134
• miR-34 • miR-132 • miR-135
• miR-124 • miR-134 • miR-138
• miR-134 • miR-218 • miR-219
• miR-137 • miR-430 family • miR-284
• miR-184 • miR-17-92 cluster • miR-485
• miR-17-92 • miR-125b
• miR-106-25

Fig. 1.2 Major functional role miRNAs in neuronal cells

miR-25 to enhance the proliferation of NSCs and NPCs [26]. miR-134 acts during
development on differentiation and synaptogenesis, and during adulthood, it
regulates synaptic plasticity [27]. miR-137 inhibits proliferation, induce neuronal
maturation, regulates differentiation of neural stem cells [28, 29].

1.3.2 miRNA in Oligodendrocytes

Oligodendrocytes (OLs) represent 20–40% of brain cells. OLs are myelin-producing

cells that surround neuronal axons for effective saltatory conduction of electrophysi-
ological signals. miRNAs play critical roles in normal cellular physiology and
development of OL lineage cells. During development, OLs pass through specific
stages of proliferation and differentiation. Recent evidence demonstrates that these
stages are fine-tuned and regulated by miRs [30, 31]. During the first stage, NSCs
differentiate into oligodendrocytes progenitor cells (OPCs) by inhibition of neurons
differentiation promoters via miR-219, miR-145, miR-7a, and miR-19b
[32, 33]. OPCs are highly proliferative cells due to their sensitivity to the platelet-
derived growth factor (PDGF). Data demonstrated that PDGFRα promotes OPCs
proliferation and inhibition of their differentiation. In a specific time window of
development, OPCs overexpress miR-219, miR-138, miR-297c-5p, and miR-338
that will target PDGFRα, Sox6, Hes5, and other proteins to inhibit proliferation
[32, 34–36]. At the same time, a decreased expression of miR-7a and miR-9 allows
SRF, PMP22, CNPase, and Sp1 expression, pre-myelinating OLs (pre-OLs) differ-
entiation promoters [33, 37, 38]. The final step of OLs differentiation is myelin
production. Pre-OLs increase the expression of miR-138 and miR-23a to inhibit
UHRF1 and LaminB1 [39–41]. The overexpression of miR-23a, miR-125-3p, and
miR146a activate signaling pathways leading to myelin proteins production [36, 42–
45]. In normal brain physiology, miRs also modulate metabolism and gene expres-
sion in OLs, and more specifically, lipid metabolism via the expression of miR-219
and miR-32 [44, 46] (Fig. 1.3).
8 1 microRNAs in Normal Brain Physiology

• miR-9 • miR-23a • miR-17-92

Differentiation/maturation

• miR-27a • miR-138
• miR-138 • miR-219

OPCs expansion
Myelinaon
• miR-145 • miR-146a
• miR-205 • miR-125-3p
• miR-214
• miR-338
• miR-715
• miR-125-3p
• miR-297c-5p

Fig. 1.3 miRNAs involved in oligodendrocyte differentiation, maturation and myelination

1.3.3 miRNA in Astrocytes

Astrocytes represent the largest population of glial cells in the CNS. They are
traditionally viewed as structural cells supporting the brain structure in terms of
homeostasis by regulation of ion, pH, neurotransmitter, and blood flow. Astrocytes
are also essential for proper neuronal functioning during development via their roles
in synaptogenesis, and in the adult brain for synaptic transmission and information
processing [47]. Moreover, due to the presence of damage-associated molecular
pattern (DAMP) and pathogen-associated molecular pattern molecule (PAMP)
receptors, astrocytes also contribute to neuroinflammation in the case of infection
via the expression of specific markers [48].
As described before, miRs contribute to the cell specification of neurons. Inter-
estingly, miRs that are enriched in neurons are diminished in astrocytes,
demonstrating that the regulation of differentiation from one cell type to another at
the stem cell level is miRs dependent (Table 1.1).

1.3.4 miRNA in Microglia

The last major component of the brain cell population is microglia. Microglia are
CNS-resident macrophages that have essential physiological roles in the brain in
addition to their inflammatory activity [62]. They are derived from erythromyeloid
precursors from the yolk sac that migrate to the neuroepithelium during early
embryonic development [63]. As macrophages, in healthy conditions, they continu-
ally scan their environment and participate in synaptic plasticity. However, during
stress conditions such as bacterial and/or infection, trauma or ischemia, microglia
will activate and adopt a pro-inflammatory phenotype [62, 64, 65]. This phenotype is
1.4 Conclusion 9

Table 1.1 Astrocyte specific microRNAs

miRNAs Targets References
miR-124a GLT-1 [49, 50]
miR-125b Fyn, Nrg1, Smad4, RhoA [51]
miR-143 HK2, ADAR1, PUMA, PDGFRA, PRKCE, MAPK7, [52]
DSSP, DMP-1, KRAS, and BCL-2
miR-145 GFAP, AQP-4 [53]
miR-146a IRAK1, TRAF6 [54, 55]
miR-181a MeCP2 [56]
miR-21 PTEN, PDCD4 [57, 58]
miR-210 AKT [59]
miR-221 CX43, ICAM1 [60]
miR-223 SDF-1 [61]

harmful to the brain, especially during ischemia, and will further be described in
Chap. 4. All of these processes and phenotypes have been recently linked to miRs.
Some miRs are enriched in microglia and inhibit neuronal phenotypes. Interest-
ingly, miR-124 that is particularly enriched in the brain and has a major function in
neurons and astrocytes, is also present in microglia; and seems to be a microglia-miR
in comparison to periphery macrophages [65]. Let-7a reduces neurite production and
limits pro-inflammatory cytokines production; it also increases the production of
neuroprotective molecules [66]. Data suggest that miR-204 and miR-424 may
regulate microglial proliferation and apoptosis [67, 68]. Microglia specific
miR-155 has also been linked to angiogenesis by targeting CCN1 [69]. Many
miRs such as miR-146a/b, miR-155, miR-124, miR-689 have been correlated with
the microglia inflammatory polarization [65, 70].

1.4 Conclusion

In many regards, the brain is a privileged organ, with a fine-tune regulation of all
processes during the lifetime. One of the actors of this regulation are nc-RNAs, and
more specifically miRs, that are post-transcriptionally modulators of gene expres-
sion. We have described the four major cell types of the brains and how miRs
participate in brain physiology. In an interesting manner, many miRs are present in
different cell types, and their functions are cell-context specific during time and
place. As an example, miR-146 has been associated with both microglia and
oligodendrocytes, respectively, for the regulation of neuroinflammation and myelin
production. In light of these observations, we should consider the use of miRs as
therapeutic candidates due to their ability to target several pathways in different cell
types to protect the brain from internal and external aggressions.
10 1 microRNAs in Normal Brain Physiology

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Ischemic Stroke: An Imperative Need
for Effective Therapy 2

Abstract

While it is currently estimated that stroke causes eight million annual deaths
worldwide, the death rate only gives a first account of the overall burden of
disease. Ischemic stroke is caused by the blockade or occlusion of the cerebral
artery compromising blood supply and energy flow to the brain. The likelihood of
restoration of blood flow depends on the extent of thrombosis within the cerebral
artery. Thrombolysis is currently coupled with endovascular approaches for the
removal and dissolution of occlusive thrombus. This chapter provides an over-
view of the current thrombolytic and endovascular treatment approaches for post-
stroke care along with the emerging neuroprotective agents that are lost in
translation.

Keywords
Ischemic stroke · Thrombolysis · Endovascular recanalization · Neuroprotection

2.1 Introduction

Therapeutic skepticism and inadequate treatment opportunities make ischemic

stroke the third most common cause of death and disability worldwide [1, 2]. Ische-
mic stroke induces a broad spectrum of sensory, motor, and cognitive impairments
resulting in a massive personal and financial burden on society [2]. The amplitude of
brain damage and the outcome depends on the tolerance threshold of the brain region
and neuronal population exposed to ischemic insult (Box 2.1). Though thrombolytic
therapy with recombinant tissue-plasminogen activator (rt-PA) remains the first and
the only Food and Drug Administration (FDA) approved treatment [3], intravenous
(IV) rt-PA thrombolysis is no longer the only panacea for acute ischemic stroke.
Currently, thrombolytic treatment is coupled with endovascular approaches for the
removal and dissolution of occlusive thrombus. Besides, preclinical research

# Springer Nature Singapore Pte Ltd. 2020 15

R. G. K. et al., IschemiRs: MicroRNAs in Ischemic Stroke,
https://doi.org/10.1007/978-981-15-4798-0_2
16 2 Ischemic Stroke: An Imperative Need for Effective Therapy

continues to discover and improve appropriate neuroprotective agents with little

progress in clinical trials, primarily due to the overestimation of therapeutic efficacy
in rodent models. This chapter describes the current landscape surrounding the
thrombolytic and endovascular methods used for ischemic stroke treatment, the
role of emerging neuroprotectants, their successes and failures, and
recommendations essential for the improvement of the quality of research at both
preclinical and translational levels.

Box 2.1: Stroke Incidence and Prevalence

• Stroke incidence refers to the number of individuals experiencing stroke for
the first time.
• Stroke incidence varies around the world depending on age, sex, suscepti-
bility to vascular diseases and other risk factors in a population.
• Between 2009 and 2016, ischemic stroke incidence is reserved at approxi-
mately 795,000 victims in the USA
• Stroke incidence is lower in France compared to East European countries
• In the developing nations such as China and India, stroke rate is highly
precarious with more than one billion population, diverse socio-economic
and cultural backgrounds, and varying vascular risk factors.
• Stroke prevalence is the total number of individuals who have had a stroke
at any time within a population.
• 1.5–2 people per 1000 population experience stroke each year.
• Overall prevalence rate is estimated to be about 10 per 1000 population,
which corresponds to 50 per 1000 for those aged above 65 years.

2.2 Current Treatment Choices for Ischemic Stroke Victims

This section enumerates currently accepted and utilized treatments for ischemic
stroke that can be broadly categorized into (1) IV pharmacological thrombolysis
and (2) endovascular procedures (thrombectomy or embolectomy) with or without
the administration of thrombolytic agents (Table 2.1).

2.2.1 IV Pharmacological Thrombolysis

The first generation thrombolytic agents involve streptokinase, a protein secreted by

streptococci, and urokinase, a human serine protease [4]. Their use in stroke throm-
bolysis has been hampered by the high risk of intracerebral hemorrhage (ICH),
non-specificity to fibrin clots, low clinical efficiency, and allergic reactions, particu-
larly with streptokinase [5]. The second-generation recombinant tissue-plasminogen
activator thrombolytic therapy is based on the adsorption of endogenous tissue-
plasminogen activator (t-PA, a serine protease enzyme in the plasma) to the surface
Another random document with
no related content on Scribd:
will make this one look like the last rose of summer. Leave the
hideous thing alone. Why, the Dromans will think that you are dippy.
Fact is, I believe that they are beginning to think so already."
"Let 'em!" said Rhodes with true philosophic indifference. "People
thought that Galileo was crazy, and Newton and Darwin; Columbus
was non compos mentis,[13] Fulton was dippy, and Edison was
looney. Yes, at one time the great inventor bore the beautiful
sobriquet of Looney Edison. Listen to me, Billy, me lad: the greatest
compliment that a scientist can ever receive is to be called a sap by
sapheads."
All that, I admitted, was very true and truly cogent in its place; but
this was not its place, and the Dromans certainly were neither
sapheads nor saps. To my relief, and, indeed to my surprise, I
dissuaded him from taking the thing as a specimen, and on we went
once more.
At length we left the stream, which went plunging into a most
fearsome place, into which no man could even dream of following it.
Soon after that, the descent became very steep. The going,
however, was good, and we went down at a rapid rate. This lasted
for two or three hours, and we had descended many hundreds of
feet. The slope then suddenly became gentle, and we were making
our way through a perfect maze of tortuous galleries and passages,
which at times opened out into halls and chambers.
The light was no longer in masses but in streams—streams that
crawled and shivered and shook, as though in it spirit-things were
immersed and were struggling to break from it. The fungal growths
were everywhere now. There were mushrooms with pilei bigger than
umbrellas. Shapes as grotesque as if seen through the eyes of
madness. There were growths, too, that one could almost think
beautiful, and masses hideous and slimy as so many octopi. A
strong and most unpleasant odor filled the place. And here and
there, almost everywhere in the strange fungoid growth, were things
creeping, crawling—things for which I can find no name, and for
some of them I am glad that I can not.
It was a weird scene, an indescribable scene, one horrible,
mysterious and yet strangely wonderful too. A place gloomy and
weird as any ever conceived by Dante or Doré. And through it
human forms were moving, and its stillness was broken by human
voices raised in song; and, moving with those human beings, those
inhabitants of a world as alien as that of Venus or of Mars, were
Rhodes and I, we two modern men from the great modern world
above—the wonderful, the awful world of the sun.
Of a sudden an exclamation rang out—an exclamation that stilled
the song on the instant, brought the party to an abrupt halt and the
bow of Ondonarkus and that of Zenvothunbro from the cases.
The exclamation had broken from Rhodes; he was pointing into the
gloom off to our right, a tense, expectant look on his face.
I peered with straining eyes, but I could see nothing there. A few
moments passed, and still nothing was seen. I then turned to
Rhodes to ask him what it was; but the words I was about to speak
were never uttered. Instead, I gave something like a cry and whirled
round. For a sound had come from out the fungoid growth and the
darkness behind us, a sound as if of a slimy thing moving, slipping.
Nothing, however, was to be seen there, and silence, utter silence,
had fallen upon the spot—silence abruptly broken by another
exclamation from Milton Rhodes.
"Great Heaven!" I cried as I whirled back to the direction in which he
was pointing. "They are all around us!"
"Look, Bill—look at that!"
I saw nothing for a second or two. And then, off in the darkness
beyond the reach of our lights, the darkness itself was moving—yes,
the very darkness itself.
"See that, Bill?"
I saw it. And the next instant I saw two great eyes, eyes that were
watching us. And those eyes were moving.
 Chapter 31
"GOGRUGRON!"
They were visible for a few seconds only—those great eyes burning
with a greenish fire.
"Where did they go?" exclaimed Rhodes.
"And," said I, "what can it be? An ape-bat?"
"That is no ape-bat."
He turned to Ondonarkus.
"Loopmuke?" he queried.
No; it was not a loopmuke. But what it was neither Ondonarkus's
pantomime nor Drorathusa's could tell us.
"I don't think it's as bad as that. But the Dromans all know what it is
themselves."
"There!" I cried, whirling round. "Did you hear that, Milton? There's
that other thing again—that thing behind us!"
"I heard nothing."
"I heard something, I tell you. That mystery with the eyes is not the
only thing that is watching us, watching us and waiting."
Some moments passed, perhaps minutes, in expectant watching,
our glances incessantly darting about the cavern, through which the
light-mist was moving in troubled, writhing streams, the nebulous,
spectral glow of it seeming to enhance the fearsome gloom of that
dreadful place.
"I see nothing," Rhodes said at last, "and the cavern is as silent as a
tomb."
"But we are seen. And, if we don't get out of this, it may be our
tomb."
"I don't think it's as bad as that. But the Dromans are signing to us to
come on—let us hope to a place more pleasant than this one."
I had turned to quit the spot, my look, however, lingering in that
direction whence had come those low, mysterious sounds, a
direction right opposite to that in which the moving eyes had shone.
And scarcely had I taken a step forward when I fetched up, cried out
and pointed.
"See that! See it moving?"
A large fungus-tree, its form one indescribably grotesque, was
quivering. It began to shake violently. Some heavy body, hidden from
our eyes, was moving there, and it was moving toward us.
Of a sudden the tree was thrust far over, there was a squishy,
sickening sound, then down it came, the spot where it fell involved in
a cloud of phosphorescence, which thinned and faded in the air like
dust or mist as it settles.
"Shades of the Gorgons," I cried, "what is in there?"
A sound from Milton Rhodes turned me round on the instant.
"The eyes again!" he cried. "There they are. See them? Have we at
last got into Dante's Inferno itself?"
I was beginning to think that we had got into something worse.
Yes, there the eyes were, and they were nearer this time. But that
was all we saw, eyes and nothing more. The thing itself was hidden
in the fungoid growth and the shadows.
Rhodes raised his revolver, rested it on his left arm, took careful aim
and fired.
The report seemed to bellow like thunder through the cavern. There
was a scream from the Dromans, none of whom, save Drorathusa,
had ever heard a firearm before; and I doubt that even Drorathusa
knew what had killed her demon. On the instant, whilst the report of
the weapon and the cry of the Dromans were ringing in our ears,
came another sound; it was a shriek, high, piercing, unearthly, one
that seemed to arrest and curdle the very blood in our hearts.
It sank, ceased. But almost instantaneously it came again; it rose
until the air seemed to quiver to the sound.
The effect upon the Dromans was most sudden and pronounced.
A nameless fear, and something worse, seized upon me as I saw it.
They started from the spot as if in a panic, signing to us with frantic
gestures to follow.
I started; but Rhodes, for some inexplicable reason, stood there, his
look fixed on the spot whence came those demoniacal shrieks. The
eyes had disappeared, but in almost that very instant that I turned,
they shone again. I gazed at them as though in fascinated horror,
forgetting for the moment that there was something behind me.
Up the eyes rose. A black thing was visible there in the darkness, but
its shape was amorphous, mysterious. Up the eyes rose, seeming to
dilate, and the fire in them grew brighter and brighter, became so
unearthly that I began to wonder if I were going insane. The eyes
swayed, swayed back and forth for some moments, then gave a
sudden lurch into darkness. The shrieks broke, then came again,
more horrible, if that were possible, than before.
"Come on!" I cried, starting. "For Heaven's sake, Milton, let's get out
of this, or I'll go mad!"
"What in the world," said Rhodes, reluctantly turning to follow, "can
that thing be?"
"Let's get out of this hellish place. Let's do it before it's too late.
Remember, there is something behind us. Maybe monsters in other
directions too."
"Well," said Rhodes complacently as he followed along in my wake,
"we have our revolvers."
"Revolvers? Just see what your revolver has done. A revolver is only
a revolver, while that thing—who knows what that monster is?"
"The Dromans know, or at any rate, they think that they do."
"And look at the Dromans. Fear has them. Did you ever see fear like
that before? See how they are signing to us to come on. Even
Drorathusa is shaken to the very soul."
"After all, 'tis no wonder, Bill, that she is. Those shrieks! How can it
continue to shriek and shriek like that?"
Ere long we had come up with the Dromans, who at once quickened
their pace. On we went, casting apprehensive glances into the gloom
about us. Those frightful sounds sank as we moved onward. They
became faint, fainter still, and at last, to my profound thankfulness,
they were no longer to be heard, even when we paused to listen.
"If that," said I during one of those pauses, "is a good sample of what
we are to have here in Drome, then I wish that, instead of coming
down here, I had stepped into a den of cobras or something."
Drorathusa's eyes were upon me. As I ceased speaking, she raised
a hand and pointed in the direction whence we had come.
"Gogrugron!" she said.
And I saw fear and horror unutterable well up in her eyes as she said
it.
 Chapter 32
"LEPRAYLYA!"
Steadily we made our way along and downward. The light-streams
were increasing in volume, the luminosity becoming stronger and
stronger, the vegetation more abundant, the weird shapes larger and
more unearthly than ever. The silence was broken by the drone of
insects—creatures meet inhabitants, forsooth, for a place so
indescribably strange and dreadful.
The cavern we were following was very tortuous, our route even
more so, what with the twists and turns which we had to make in
order to get through that phantasmagoria of fungal things. I do not
mean to say that all of those growths were horrible, but most of them
were, and some were as repulsive to the touch as they were to the
sight.
As we toiled our way through them, my heart was replete with dire
apprehension. I could not banish the vision of those great burning
eyes, the horror of those shrieks, which perhaps were still ringing
out. What if we were suddenly to find ourselves face to face with one
of those monsters (or more than one) here in this nightmare-forest?
Gogrugron! Gogrugron! What in the world was that monstrosity
known to the Dromans as a gogrugron? Well, most certainly, I was
not desirous of securing firsthand knowledge, upon that interesting
item, for the great science of natural history.
At length the light no longer lay in streams and rifts in the darkness,
but the darkness, instead, lay in streams through the light. The
Dromans quickened their already hurried pace, and there were
exclamations of "Drome! Drome!"
"Drome!" echoed Milton Rhodes. "I wonder what we are going to
find."
"Something wonderful," said I, "or something worse, perhaps, than
anything that we have seen."
Rhodes laughed, and I saw Drorathusa (Ondonarkus was leading
the way) turn and send a curious glance in our direction.
"Well," I added, "anything to get out of this horrible forest of fungi and
things."
Some minutes passed, perhaps a half-hour, perhaps only fifteen. Of
a sudden the great tunnel, now as light as a place on a sunless day,
gave a sharp turn to the right; a glad cry broke from the Hypogeans.
"Drome! Drome!" they cried.
We all hurried forward.
"Look!" I said as we reached the turn. "The mouth, the mouth! The
tunnel ends!"
There, but two hundred feet or so away, was the great yawning
mouth of it; nothing was visible through that opening, however, but
light, pearly, opalescent, mystic, beautiful.
"Drome!" cried Nandradelphis, clapping her hands.
A few moments, and we were standing at the entrance and gazing
out over the weird and beautiful scene.
"Drome!"
I turned at the sound and saw Drorathusa, her figure and mien
ineffably Sibylline and majestic, pointing out over the strange
landscape, her eyes on the face of Milton Rhodes.
"Drome," she said again.
"Drome," echoed Milton.
"I wonder, Bill, what this Drome really is. And I have an idea that this
is only the outskirts that we see. Can we at last be near our journey's
end, or is that end still far away?"
"Who can tell? This place seems to be a wilderness."
"Yes; a forest primeval."
"What," said I, "are we destined to find down there?"
"Things stranger, Bill, in all likelihood, than any explorer ever found
anywhere in that strange world above us."
"No gogrugrons, I hope."
Rhodes laughed.
"Gogrugron!" said Drorathusa.
And I saw that horror and fear again in her eyes.
The cavern had come out high up on a broken, jagged wall, which
went beetling up for hundreds of feet, up to the roof, which arched
away over the landscape before us. We were fully half a thousand
feet above the floor, which was a mass of luxuriant forest. Glimpses
were caught of a stream down to the left, possibly the one which we
had followed for so long. I judged the place to be over a mile wide;
Rhodes, however, thought that it was perhaps not quite a mile in the
widest part. Down this enormous cavern, the eye could range for
three or four miles, at which distance the misty light drew its veil over
the forest, the dark walls, and the roof arching across.
At times the light quivered and shook and there were strange
flickerings, and dartings of opalescent streaks through it—streaks
ineffably beautiful and yet, strangely enough, terrible too, terrible as
the blades of plunging swords in hands savage and murderous.
Once more Drorathusa raised a hand, and this time she pointed into
the misty distance.
"Le-prayl-ya!" she said.
Again her eyes were on Milton Rhodes, and, as she spoke that
name, I saw in those wondrous orbs of hers one of the strangest
looks that I have ever seen. I wondered if Rhodes too saw it. I found
his eyes upon Drorathusa, but there was in them so abstracted an
expression that I believed his thoughts were far away and that he
had not noticed. When I turned to Drorathusa again, it was to find
that that strange look was gone.
What a fascinating and mysterious creature this woman was!
"Lepraylya," she said again.
"Lepraylya," Milton nodded.
He looked at me.
"I wonder who or what this Lepraylya can be, Bill."
"King maybe."
"Queen, I hope," said Milton Rhodes.
He drew forth his notebook and pencil and handed them to
Drorathusa, pronouncing as she took them that mysterious name:
"Lepraylya?"
A few strokes with the pencil, and Drorathusa had given us the
answer.
"You see, Bill?" said Rhodes, smiling. "A woman. In all likelihood,
too, the queen."
Drorathusa's Sibylline look was upon him once more—and she did
not smile.
 Chapter 33
FACE TO FACE
We found the wall even more broken and difficult than it had
appeared from the entrance. It was almost destitute of vegetation, a
circumstance that contributed not a little to the slowness of the
descent. Indeed, making our way down over those pitching naked
rocks was a ticklish, an unpleasant business, I want to tell you; at
times it was really precarious.
We had halted to rest above one of these difficult spots, and every
one was either seated or leaning against the rock, when of a sudden
Milton, who was nearest the edge, arose and pointed, pointed down
and off to the right.
"Hello!" said he. "What's that?"
All of us arose, moved over and looked.
"Where?" I asked.
"Down there by that strange clump of cycadaceous trees, that clump
near those tall trees that look so much like Douglas firs. But 'tis gone
now."
"What did you see?"
"I haven't the faintest idea, Bill. But there certainly was something
there, and it was moving. And, if I were imaginative, I would probably
say that it was watching us, that, at the moment I arose and pointed,
it glided back to the concealment of the trees."
"Well, did it?"
"It certainly seemed to do so, Bill."
I peered down there again, but I could not see anything moving.
There was silence for some moments. The Dromans stood watching,
waiting; stood expectant, puzzled.
"Oh, well," Rhodes said, turning a quizzical look in my direction and
then to the face of Drorathusa, "we must expect to find live things in
that forest."
I saw Drorathusa's eyes fixed upon his face, then, a few moments
after he ceased speaking, return to the clump of cycads.
"Live things?" said I. "There may be things in this place of mystery
more terrible than any live thing."
"Come, Bill, come. It can't be so bad as you imagine it, or our
Dromans wouldn't be here.
"I wish," he added, "I knew what that thing is that I saw."
"Hello!" I cried the next moment, my look raised up to the vaulted
roof. "What does that mean? Good Heaven, what next?"
The light, which was brightest up along the roof—in fact, it seemed
pressed up against the rock-canopy like glowing, diaphanous mist—
was changing, fading. The wonderful opalescence of it was
disappearing before our eyes.
Of a sudden the spot where we stood was involved in a gloom
indescribably strange. Up above, the light-mist was quivering and
flickering, pale and dreadful.
"What in the world is it?" I cried.
"Queer place, this!" said Milton Rhodes.
"What can it mean?" I asked.
He did not answer. He sent a questioning look toward Drorathusa
and her companions. Mine followed. The faces of the Dromans
seemed to glimmer ghostlike in the thickening, awful darkness. Upon
those pale features, however, was no discoverable sign of alarm, of
uneasiness even.
The gloom deepened about us. Pitchy darkness came down with a
rush. Far away, and up along the roof, there were pale flickerings
and flashes. Then the light burst out, so sudden and so strong that
pain shot through the eyes.
Came a cry, and I turned to see Drorathusa pointing, pointing down
toward those cycads.
"There it is, Bill!" exclaimed Milton. "There it is again! See it
moving?"
I saw it, but it was for a fleeting moment only. And, I thought, I saw
something else.
"A little nearer this time," Rhodes told me. "There can be no doubt
that it is watching us."
"Evidently," I said, "it is moving over to lie in wait for us. And, unless
I'm very much deceived, it isn't alone."
"Hum," said Rhodes. "Queer place, Bill, to go into. Even our
Hypogeans, it seems, don't know what to make of that apparition."
They were conversing in low tones, casting searching, apprehensive
looks along the ragged margin of the forest.
The gloom was falling again. Denser and denser it grew about us.
Fainter, more and more dreadful became those distant flickerings
and flashes along the great vaulted roof. Darkness, blackness was
involving everything. Dimmer still became the flickerings. The
stillness was utter, portentous. There was not the gentlest movement
of air. The light gave a last faint, angry gleam and went out
altogether.
Abruptly, from out of the darkness, a voice came sounding, and,
though I knew that the voice was Drorathusa's, I started and almost
gave a cry. I pressed the button, and the rays of the lamp flashed
out, lighting up the spot and showing the tall figure of Drorathusa
with arms extended upward in some mystic invocation. The others
were kneeling, and the words that Drorathusa spoke were echoed,
as it were, in their low responsive voices. It was a strange scene,
truly—the dark, savage masses of rock, the tall Sibylline figure of the
woman, the kneeling forms of the others and we two men from the
sunlit world looking on in wonder and in awe.
Minutes passed. The wondrous, eerie voice of Drorathusa never
ceased, though there were moments when those echoing voices
were silent.
Look! Far away, there was a faint, ghostly flicker. Another and
another. Brighter they became and brighter still, at last opalescent;
soon rocks and forest, soon the whole weird landscape was again
bathed in the mystic pearly light.
"What, in the name of wonder," I said, "was it?"
"An eclipse," smiled Rhodes. "Queer place, this."
"Queer place? Can't you hit another tune? You don't have to keep
telling me that this is a queer place. I am not at all likely to forget that
fact. And I wonder if these 'eclipses' are a frequent phenomenon.
Certainly I hope that they are not."
"I wish that I could tell you, Bill, tell you that and a few other things."
"And," I added, "that forest, when the light goes, must be a queer
place truly—gosh, I'm catching it from you! But I'll tell you what: I
wouldn't like to find myself, in the depths of those woods, face to
face with a loopmuke or a gogrugron or something and in that instant
have the darkness come down."
"It would be rather unpleasant, I fancy. But unfortunately our likes or
our dislikes are not likely to alter in any way the scheme of things."
The Dromans, all standing now, were singing a low and sweet song
of thanksgiving and gladness. Yes, so sweet were the tones that
they seemed to linger in the air, for some moments, even after the
song had ceased.
We cast our looks along the margin of the forest, but not a single
glimpse was caught of that mysterious object, or objects, that we had
seen moving down there.
It was patent that the Dromans knew no more what to make of that
apparition than we did ourselves and that they looked forward with
no little apprehension to our entry into that wood.
The descent was resumed. Were eyes, somewhere below, watching
our every movement? I feared that it was indeed so, and, as I well
knew, every other member of our little band feared it, too. There was
nothing, however, that we could do except descend and face the
issue. To turn aside would be futile, for the watcher, or the watchers,
would turn aside also to meet us.
Ere long we reached the talus, and our troubles were then over; that
is, as regards the descent. But Heaven only knew what troubles
were awaiting us somewhere in that forest, to which we were now
drawing so very near.
As we made our way down over the rock-fragments, amidst which
shrubs and stunted trees were growing, more than once did we
pause and send keen, searching looks and glances into the silent
recess of that mysterious wood.
Some of those sylvan depths were enshadowed, gloomy; others
were pervaded with the strong, transparent light-mist, the objects
involved in which cast no shadows.
At the foot of the talus, almost beneath the branches of great palm-
trees, there was a pause.
"Now for it!" said Milton Rhodes solemnly.
The Dromans were clustered together in earnest but laconic
dialogue, their eyes employed the while in a keen scrutiny of the
forest aisles and recesses, before us and on either hand.
Insects were in the air about us; one or two shadowy butterflies
flitted past; and that was all. Not a leaf stirred; the air was without the
slightest movement. No song, no call of a bird broke the silence,
which seemed to press down upon us and about us as though it
were a tangible thing. It was as if the spot, the forest itself had never
known either the voice or the movement of any sentient thing. But,
somewhere in that forest, hidden and close at hand, there was
something sentient—something, in all likelihood, watching us,
watching and waiting. Waiting for what?
Or, came the sudden thought, even now stealing toward the place
where we stood.
"This suspense," said I to myself, "is simply awful, is as terrible,
even, as that which we knew when we were crossing the bridge, that
chasm of unknown depth on either side."
Drorathusa turned to us and pointed in a rather vague direction out
into the trees.
"Narranawnzee," she said.
"Evidently," said Milton, "they plan to strike that stream."
"I pray Heaven," I told him, "that we live to see that narranawnzee."
Whereupon Rhodes laughed outright, and the effect of the sudden
sound was curious and startling, so great was the tension of our
nerves.
"One would think, Gloomy Face," said he, "that you had just issued
from the Cave of Trophonius. 'And he never smiled again.'"
"I have an idea, grinning Shaky Knees," I retorted, "that we have got
ourselves into a place more awful than any Cave of Trophonius. I
don't blink, that's all."
"Nor, Bill, do I," said Milton soberly. "You know, I'd feel more at ease
if it wasn't for the presence of the ladies. Why did they come on a
journey so hazardous and so terrible?"
How often had we wondered that! We didn't know the ladies of
Drome.
We at once got in motion. Ondonarkus and Rhodes were leading,
Drorathusa was just behind them, then came Nandradelphis and
Silvisiris, whilst Zenvothunbro and myself brought up the rear. This
disposition of our little party was as Drorathusa herself had desired
it, and she had been at some pains to impress upon Rhodes and me
(though there had been no necessity for that) the expediency of
keeping our weapons ready for action at any instant.
On we went, deeper and deeper into the wood. Strange forms of
vegetation, strange flowers, strange insects were everywhere. How
interesting we should have found the place! But there was that thing,
somewhere hidden, watching us perhaps—following.
Came a sharp exclamation, a dull sound from above; but it was only
a bird, a thing of silver and gold, launching itself from off a branch of
one of the trees which we were approaching. Away it went sailing,
lovely as a vision from fairyland, and disappeared amongst the tree-
trunks and foliage.
Five minutes or so passed. Another sound, an exclamation from
Drorathusa, and the party came to a sudden halt.
Every one had heard it—a clear, unmistakable but inexplicable
sound and from behind. We were being followed!
We stood listening for some moments, waiting; but the sound did not
come again. Save for the low melancholy drone of insects, the spot
was as silent as a tomb.
We resumed our advance, every sense on the alert. A few moments
passed, and then we heard it again. This time it was off to the right,
almost abreast of us, it seemed.
Had it thus quickened its movements in order to get in front of us?
We waited, but nothing was seen, nothing was heard.
We had advanced but twenty or thirty feet when a sudden gloom
involved the forest. The scene on the instant turned weird, unearthly.
This, however, was but a few moments only; then came the light.
The advance was at once resumed. But we had gone only a short
distance when the gloom came once more, grew so dense that we
had to come to a halt.
It lifted, just as I was on the point of switching on my electric light.
Then like a bolt came utter darkness. And, even as the darkness fell,
there was a velvety sound and a faint rustling from amongst the
foliage beside us. With frantic haste I sought and pressed the light-
switch. At the same instant Rhodes flashed on his light. A cry of
horror broke from me. There, thrust over the top of a great log and
but a few yards distant, was a long snaky head with a pair of great
blazing eyes fixed upon me.
We were face to face at last!
 Chapter 34
ANOTHER!
I jerked out my revolver, took swift aim, right between those great
blazing eyes, and fired.
From the beast came a fearful roar, which seemed to end in a
scream, and the long snaky head and neck (no more of the animal
had been visible) disappeared.
"Good work, Bill!" applauded Milton Rhodes.
He had hardly spoken when the light came again, strong and
beautiful. And the next instant came something else. A dark form,
with a gleam of something white, rose into the air and drove straight
toward us. I sprang aside and turned to fire, but I did not do so for
fear of hitting the Dromans or Rhodes. There came a piercing shriek
from Drorathusa, a sound made by cloth-rending talons. The
monster had her.
 "How like the big face of a fat man!"

I leaped toward it and emptied the revolver into its side, whilst
Ondonarkus and Zenvothunbro sent each an arrow into the body.
That of the former was driven with such force that it passed clear
through the body and went on for a distance of six or eight feet. And
down the beast fell dead, though still quivering, there in our very
midst.
I turned and hurried to Drorathusa. Rhodes was already beside her.
As I reached her, the darkness came down again upon the place,
pitchy and awful. The claws had ripped her dress, from the thigh