**Meet the editor**

Maurizio Balestrino was born in Genoa, Italy, the birthplace of Christopher Columbus and of the blue jeans. He received there the Degree in Medicine and the Diploma of Specialist in Neurology. In 1983-1986 he was Research Associate in the Department of Physiology at Duke University, where he started an interest in ischemic brain damage. Back in Italy he has always combined clinical

responsibilities as a neurologist and experimental research in brain anoxia or ischemia. He is currently Senior Researcher in the Stroke Unit of the University of Genoa, where he also directs the Laboratory of Experimental Neurophysiology. He has been partner or coordinator in national and international research projects aiming at bridging the gap between experimental research and clinical therapy.

Contents

**Preface IX** 

Kazuo Yamagata

Chapter 3 **A Master Key to Assess Stroke** 

**Consequences Across Species: The Adhesive Removal Test 47**  Valentine Bouet and Thomas Freret

**Part 1 Animal Models and Techniques 1** 

Chapter 1 **Ischemic Neurodegeneration in Stroke-Prone** 

**Spontaneously Hypertensive Rats and Its** 

Chapter 2 **Frameless Stereotaxy in Sheep – Neurosurgical and** 

Antje Dreyer, Albrecht Stroh, Claudia Pösel, Matthias Findeisen, Teresa von Geymüller,

Chapter 4 **Variations in Origin of Arteries Supplying the Brain** 

David Mazensky, Jan Danko, Emil Pilipcinec,

Chapter 5 **Cerebral Ischemia Induced Proteomic Alterations:** 

Marie-Soleil Giguere and Jacqueline Slinn

Chapter 6 **Delayed Neuronal Death in Ischemic Stroke: Molecular Pathways 117** 

**Part 2 Pathophysiology of Ischemic or Anoxic Damage 83** 

**Consequences for the Synapse and Organelles 85**  Willard J. Costain, Arsalan S. Haqqani, Ingrid Rasquinha,

Victor Li, Xiaoying Bi, Paul Szelemej and Jiming Kong

Eva Petrovova and Lenka Luptakova

Donald Lobsien, Björn Nitzsche and Johannes Boltze

**Prevention with Antioxidants Such as Polyphenols 3** 

**Imaging Techniques for Translational Stroke Research 21** 

**in Rabbit and Their Impact on Total Cerebral Ischemia 65** 

### Contents

### **Preface** XIII

	- **Part 2 Pathophysiology of Ischemic or Anoxic Damage 83**

X Contents


Contents VII

Chapter 17 **Could Mannitol-Induced Delay of Anoxic** 

Chapter 18 **Fasudil (a Rho Kinase Inhibitor)** 

Chapter 20 **Time-Window of Progesterone** 

Chapter 21 **The Na<sup>+</sup>**

Chapter 19 **Endogenous Agents That Contribute** 

Ornella Piazza and Giuliana Scarpati

**Neuroprotection After Stroke and** 

**Depolarization be Relevant in Stroke Patients? 399** Maurizio Balestrino, Enrico Adriano and Patrizia Garbati

**Specifically Increases Cerebral Blood Flow in Area of Vasospasm After Subarachnoid Hemorrhage 409** 

Masato Shibuya, Kenko Meda and Akira Ikeda

**to Generate or Prevent Ischemic Damage 419**

**Its Underlying Molecular Mechanisms 479**  Weiyan Cai, Masahiro Sokabe and Ling Chen

**/H+ Exchanger-1 as a New Molecular Target in Stroke Interventions 497** Vishal Chanana, Dandan Sun and Peter Ferrazzano

**to the Management of Acute Ischemic Stroke 511** Elisa Benetti, Nimesh Patel and Massimo Collino

Chapter 22 **PPAR Agonism as New Pharmacological Approach** 

Joseph T. McCabe, Michael W. Bentley and Joseph C. O'Sullivan


VI Contents

Chapter 7 **The Matrix Metalloproteinases and Cerebral Ischemia 145**

**in the Hippocampus After Transient Cerebral Ischemia 155**

Chapter 8 **Folate Deficiency Enhances Delayed Neuronal Death** 

**Shock Proteins in Cerebral Ischemia 177** Vivianne L. Tawfik, Robin E. White and Rona Giffard

**Complex in the Migration of Adenine** 

Chapter 11 **Diabetes-Mediated Exacerbation of Neuronal Damage** 

Shinya Kamiuchi, Fumiko Suzuki, Hiroshi Iizuka, Hirokazu Matsuzaki and Yasuhide Hibino

**Neuronal Death and Ischemic Tolerance 241** Jan Lehotsky, Martina Pavlikova, Stanislav Straka, Maria Kovalska, Peter Kaplan and Zuzana Tatarkova

Tatyana I. Gudz and Sergei A. Novgorodov

**Models of Global Cerebral Ischemia 305**  Miguel Cervantes, Ignacio González-Burgos,

Graciela Letechipía-Vallejo, María Esther Olvera-Cortés

**Therapeutic Alternative in Cerebral Ischemia 347** 

Joseph T. McCabe, Michael W. Bentley and Joseph C. O'Sullivan

**Part 3 Novel Approaches to Neuroprotection 303** 

Carlos Silva-Islas, Ricardo A. Santana,

Chapter 16 **Preconditioning and Postconditioning 379** 

Ana L. Colín-González and Perla D. Maldonado

Elena Erlykina and Tatiana Sergeeva

**Nucleotides in Mitochondrial Dysfunction 193** 

**and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water-Soluble Extract**

**from Culture Medium of** *Ganoderma lucidum* **Mycelia 215** Naohiro Iwata, Mari Okazaki, Rika Nakano, Chisato Kasahara,

Wan Yang and Guangqin Li

Chapter 9 **Glial Cells, Inflammation and Heat** 

Chapter 10 **Role of Creatine Kinase – Hexokinase**

Chapter 12 **Mechanisms of Ischemic Induced** 

Chapter 14 **Neuroprotection in Animal** 

and Gabriela Moralí

Chapter 15 **Nrf2 Activation, an Innovative** 

Chapter 13 **Mitochondrial Ceramide in Stroke 269**

Jun Hyun Yoo


Preface

predicted.

"neuroprotective" compound in stroke.

In the last part of the 20th century scientists discovered drugs that made the brain more resistant to ischemia, to such an extent that cerebral tissue treated with them was only little damaged, or was not damaged at all, by an ischemic insult that badly damaged control, untreated tissue. It was the beginning of a very exciting era in neuroscience research, a period when academic and industrial scientists all pursued the research of a "neuroprotectant" that could defend the ischemic brain from irreversible damage. As most people know, the search turned out to be mostly unsuccessful because the drugs that in the animal models were effective were not so effective in the clinics. Frustratingly enough, one compound after another failed in clinical trials of stroke patients. The easiest and most common explanation given was that something was wrong with animal studies, and this is still the leading belief of mainstream neurologists. So, skepticism grew among clinicians, and nowadays it is very rare to find a clinician that gets excited by the idea of trying or studying a

However, I think that such a dismissal is plain wrong. It is unconceivable that hundreds of scientists throughout the world have for 30 years all carried out flawed or even fraudulent research demonstrating that several compounds improve the resistance of the brain to ischemic damage. Granted, that may have happened sometimes, but hundreds of laboratories around the world cannot have been run during 30 years by incompetent or criminal scientists. At least, all successful animal research in neuroprotection must be seen as having provided a "proof-of-concept" demonstrating that it is possible to use drugs to protect the brain from ischemic damage. And in fact, that neuroprotection is indeed possible is demonstrated beyond doubt by the neglected survivor of that host of neuroprotectant agents: hypothermia. Hypothermia was demonstrated to be effective in a score of animal experiments, and it has now become recommended intervention in out-of-hospital cardiac arrest. Hypothermia is not a drug, but it demonstrates that neuroprotection is a reality, not a myth. Besides, it obviously shows that animal experiments were right, humans treated with hypothermia fare better than untreated ones, just like animal studies had

Thus, the fact that hypothermia is now successful in clinical practice, at least in out-ofhospital cardiac arrest, tells us one simple truth: neuroprotection is possible. Another

### Preface

In the last part of the 20th century scientists discovered drugs that made the brain more resistant to ischemia, to such an extent that cerebral tissue treated with them was only little damaged, or was not damaged at all, by an ischemic insult that badly damaged control, untreated tissue. It was the beginning of a very exciting era in neuroscience research, a period when academic and industrial scientists all pursued the research of a "neuroprotectant" that could defend the ischemic brain from irreversible damage. As most people know, the search turned out to be mostly unsuccessful because the drugs that in the animal models were effective were not so effective in the clinics. Frustratingly enough, one compound after another failed in clinical trials of stroke patients. The easiest and most common explanation given was that something was wrong with animal studies, and this is still the leading belief of mainstream neurologists. So, skepticism grew among clinicians, and nowadays it is very rare to find a clinician that gets excited by the idea of trying or studying a "neuroprotective" compound in stroke.

However, I think that such a dismissal is plain wrong. It is unconceivable that hundreds of scientists throughout the world have for 30 years all carried out flawed or even fraudulent research demonstrating that several compounds improve the resistance of the brain to ischemic damage. Granted, that may have happened sometimes, but hundreds of laboratories around the world cannot have been run during 30 years by incompetent or criminal scientists. At least, all successful animal research in neuroprotection must be seen as having provided a "proof-of-concept" demonstrating that it is possible to use drugs to protect the brain from ischemic damage. And in fact, that neuroprotection is indeed possible is demonstrated beyond doubt by the neglected survivor of that host of neuroprotectant agents: hypothermia. Hypothermia was demonstrated to be effective in a score of animal experiments, and it has now become recommended intervention in out-of-hospital cardiac arrest. Hypothermia is not a drug, but it demonstrates that neuroprotection is a reality, not a myth. Besides, it obviously shows that animal experiments were right, humans treated with hypothermia fare better than untreated ones, just like animal studies had predicted.

Thus, the fact that hypothermia is now successful in clinical practice, at least in out-ofhospital cardiac arrest, tells us one simple truth: neuroprotection is possible. Another

#### XIV Preface

demonstration that neuroprotection is possible may be edaravone, a neuroprotective compound that has been used for years in Japan and China, and that has been declared reasonably effective in ischemic stroke, at least pending larger trials, by a recent Cochrane Review (S. Feng, et al. Edaravone for acute ischaemic stroke. *Cochrane Database Syst. Rev.* 12:CD007230, 2011).

Preface XI

improve their work, but clinicians should finally understand how to properly exploit

Second, it should not be forgot that several neuroprotectant have failed not because they lacked efficacy, but because they revealed unexpected side effects. Many NMDAreceptor antagonists were discarded because in clinical trials they showed psychedelic unwanted effects. Tirilazad, an antioxidant belonging to the "lazaroid" class of antioxidants, unexpectedly worsened outcome of ischemic stroke, a fact very likely explained by some unexpected toxic action(s) that offset its neuroprotective ability.

So, the 21st century will hopefully favor the harmonization of basic research and of clinical neurology, two realities that were too distant from each other in the second half of the 20th century. Hopefully, preclinical scientists will learn how to carry out better experiments, **and** clinicians will learn how to best apply them to their patients. To do so, preclinical studies of stroke must be continued and improved. The Authors of this book have provided their expertise and experience in reporting ways how to do

The first section of this book collects studies of animal models of stroke. Advances in this area are needed because animal experiments, carried out with proper analgesia and respect for animal lives, will still be necessary for a long time. Although the 3 "R"s (reduction, refinement, replacement) have greatly decreased the need for in vivo animal experiments, the latter ones are still needed (cf. for example "Recommendations for Standards Regarding Preclinical Neuroprotective and

The second section of the book collects studies on pathophysiology of ischemic damage. This is an area where our knowledge has greatly advanced in the past decade, mainly due to the study of novel pathways of damage and of novel techniques to investigate them. Better knowledge of how brain tissue becomes damaged in stroke will hopefully lay the foundation for better therapies, be them recanalization (like thrombolysis) or neuroprotection (like hypothermia). Novel techniques like proteomics have greatly improved our capability to study and understand the

The third section deals with neuroprotection. As we have discussed above, this has become a kind of Holy Grail for stroke scientists. Contributors to this section reviewed the state of the art in this quest or reported their experience in exploring novel ways of neuroprotection. Their work will be a useful addition to the store of knowledge that

I am most grateful to the Authors of the various chapters, who have expertly written and patiently revised their very interesting work for this book. I also would like to thank the InTech publisher, who has invited me to edit this book and has provided me with the online tools and assistance that made the job possible. In particular I am most

Restorative Drug Development". *Stroke* 30 (12):2752-2758, 1999).

pathological changes that are caused by ischemia.

the modern Parsifal will exploit to finally find his Grail.

it.

so.

Why, then, have scores of drugs, previously found to be effective in animal models, failed clinical trials? The average clinician will answer this question by saying that animal experiments are useless, that they do not reflect the human situation, that they are badly designed and carried out, and so forth. Meetings and committees have even been celebrated to declare this truth, and to teach preclinical neuroscientists how to properly carry out their experiments, see for example the "STAIR" (Stroke Therapy Academic Industry Roundtable) meetings in the US.

Of course, there is some truth in this answer, and probably much more than "some". We are in the 21st century, and even animal experiments must be updated and modernized. I am perfectly convinced that preclinical scientists (among whom I proudly list myself) must learn, as they have done for a couple of centuries, new ways of designing and carrying out their experiments. "Blind" treatment and evaluation, use of older animals (more similar to stroke patients), reliance on permanent rather than on transient models of ischemia are just the simplest and most obvious improvements that should be implemented in the laboratory, at least for those experiments that are meant to build the foundation for a clinical translation of the treatment. All these modifications, and much more, will certainly improve the reliability and the usefulness of animal experiments, much in the same way as the use of statistics has greatly improved animal experimentation in the 20th century (remember those very old days when statistics were not required to publish an experiment?).

But I believe that other truths must be told, too.

First, clinicians have not been able to grasp the true conditions under which neuroprotection is possible. Clinicians (or maybe the drug industry?) have been blinded by the illusion that a simple cure to all ischemic strokes was at hand, and they simply treated all patients with stroke, irrespective, for example, of age, infarct size and comorbidity. Sometimes the neuroprotective treatment was administered one day after stroke onset, an obvious nonsense that was not justified by animal data. With these and other behaviors, clinicians lost the opportunity of neuroprotection by applying it to patients that were not apt to benefit from it. I was very happy in reading that this truth has finally been recognized even in published science: Reza et al. (Neuroprotection in acute ischemic stroke. *J Neurosurg Sci.* 55 (2):127-138, 2011) wrote that "Previous clinical studies have failed to show benefit [of neuroprotection] likely due to poor patient selection, altering time windows that had shown benefit in bench models and failure to link treatments with reperfusion". Alas, animal scientists must improve their work, but clinicians should finally understand how to properly exploit it.

X Preface

experiment?).

*Database Syst. Rev.* 12:CD007230, 2011).

Academic Industry Roundtable) meetings in the US.

But I believe that other truths must be told, too.

demonstration that neuroprotection is possible may be edaravone, a neuroprotective compound that has been used for years in Japan and China, and that has been declared reasonably effective in ischemic stroke, at least pending larger trials, by a recent Cochrane Review (S. Feng, et al. Edaravone for acute ischaemic stroke. *Cochrane* 

Why, then, have scores of drugs, previously found to be effective in animal models, failed clinical trials? The average clinician will answer this question by saying that animal experiments are useless, that they do not reflect the human situation, that they are badly designed and carried out, and so forth. Meetings and committees have even been celebrated to declare this truth, and to teach preclinical neuroscientists how to properly carry out their experiments, see for example the "STAIR" (Stroke Therapy

Of course, there is some truth in this answer, and probably much more than "some". We are in the 21st century, and even animal experiments must be updated and modernized. I am perfectly convinced that preclinical scientists (among whom I proudly list myself) must learn, as they have done for a couple of centuries, new ways of designing and carrying out their experiments. "Blind" treatment and evaluation, use of older animals (more similar to stroke patients), reliance on permanent rather than on transient models of ischemia are just the simplest and most obvious improvements that should be implemented in the laboratory, at least for those experiments that are meant to build the foundation for a clinical translation of the treatment. All these modifications, and much more, will certainly improve the reliability and the usefulness of animal experiments, much in the same way as the use of statistics has greatly improved animal experimentation in the 20th century (remember those very old days when statistics were not required to publish an

First, clinicians have not been able to grasp the true conditions under which neuroprotection is possible. Clinicians (or maybe the drug industry?) have been blinded by the illusion that a simple cure to all ischemic strokes was at hand, and they simply treated all patients with stroke, irrespective, for example, of age, infarct size and comorbidity. Sometimes the neuroprotective treatment was administered one day after stroke onset, an obvious nonsense that was not justified by animal data. With these and other behaviors, clinicians lost the opportunity of neuroprotection by applying it to patients that were not apt to benefit from it. I was very happy in reading that this truth has finally been recognized even in published science: Reza et al. (Neuroprotection in acute ischemic stroke. *J Neurosurg Sci.* 55 (2):127-138, 2011) wrote that "Previous clinical studies have failed to show benefit [of neuroprotection] likely due to poor patient selection, altering time windows that had shown benefit in bench models and failure to link treatments with reperfusion". Alas, animal scientists must Second, it should not be forgot that several neuroprotectant have failed not because they lacked efficacy, but because they revealed unexpected side effects. Many NMDAreceptor antagonists were discarded because in clinical trials they showed psychedelic unwanted effects. Tirilazad, an antioxidant belonging to the "lazaroid" class of antioxidants, unexpectedly worsened outcome of ischemic stroke, a fact very likely explained by some unexpected toxic action(s) that offset its neuroprotective ability.

So, the 21st century will hopefully favor the harmonization of basic research and of clinical neurology, two realities that were too distant from each other in the second half of the 20th century. Hopefully, preclinical scientists will learn how to carry out better experiments, **and** clinicians will learn how to best apply them to their patients. To do so, preclinical studies of stroke must be continued and improved. The Authors of this book have provided their expertise and experience in reporting ways how to do so.

The first section of this book collects studies of animal models of stroke. Advances in this area are needed because animal experiments, carried out with proper analgesia and respect for animal lives, will still be necessary for a long time. Although the 3 "R"s (reduction, refinement, replacement) have greatly decreased the need for in vivo animal experiments, the latter ones are still needed (cf. for example "Recommendations for Standards Regarding Preclinical Neuroprotective and Restorative Drug Development". *Stroke* 30 (12):2752-2758, 1999).

The second section of the book collects studies on pathophysiology of ischemic damage. This is an area where our knowledge has greatly advanced in the past decade, mainly due to the study of novel pathways of damage and of novel techniques to investigate them. Better knowledge of how brain tissue becomes damaged in stroke will hopefully lay the foundation for better therapies, be them recanalization (like thrombolysis) or neuroprotection (like hypothermia). Novel techniques like proteomics have greatly improved our capability to study and understand the pathological changes that are caused by ischemia.

The third section deals with neuroprotection. As we have discussed above, this has become a kind of Holy Grail for stroke scientists. Contributors to this section reviewed the state of the art in this quest or reported their experience in exploring novel ways of neuroprotection. Their work will be a useful addition to the store of knowledge that the modern Parsifal will exploit to finally find his Grail.

I am most grateful to the Authors of the various chapters, who have expertly written and patiently revised their very interesting work for this book. I also would like to thank the InTech publisher, who has invited me to edit this book and has provided me with the online tools and assistance that made the job possible. In particular I am most

#### XVI Preface

grateful to Ms. Ana Pantar, who was effective and determined in removing initial obstacles, thus making this project possible. And to Ms. Maja Bozicevic, without whose kind and efficient assistance this project could not have been successful. I hope readers will find our efforts useful.

> **Maurizio Balestrino, MD**  Department of Neuroscience, Ophthalmology and Genetics, University of Genova, Italy
