Investigations on the neurotoxic effects of the anti-malaria components artemether and artemether-lumefantrine in dogs

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Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie;

Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.

1. Auflage 2009

ISBN 978-3-941703-07-0

Verlag: DVG Service GmbH Friedrichstraße 17

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www.dvg.net

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Tierärztliche Hochschule Hannover

Investigations on the neurotoxic effects of the anti-malaria components artemether and

artemether-lumefantrine in dogs

INAUGURAL - DISSERTATION

zur Erlangung des Grades eines Doktors der Veterinärmedizin -Doctor medicinae veterinariae-

(Dr. med. vet.)

vorgelegt von

Mohamed A. E. Elhensheri EL-Orban, Libyen

Hannover 2009

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Wissenschaftliche Betreuung: Univ.-Prof. Dr. Wolfgang Baumgärtner, Ph.D.

Institut für Pathologie der Tierärztlichen Hochschule Hannover

1. Gutachter: Univ.-Prof. Dr. Wolfgang Baumgärtner, Ph.D.

2. Gutachter: Univ.-Prof. Dr. Manfred Kietzmann, Institut für Pharmakologie, Toxikologie und Pharmazie der Tierärztlichen Hochschule Hannover

Tag der mündlichen Prüfung: 04. Mai 2009

Die Anfertigung dieser Arbeit wurde durch ein Doktorandenstipendium des "Ministry of Higher Education", Tripolis, Libyen gefördert.

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To my parents

إ

ﻰﻟ

ا

و ﻰﻣ

ا

ﻰﺑ

ا

ﻢهﺮﻤﻋ ﷲا لﺎﻃ

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CONTENTS

1 INTRODUCTION... 1

2 LITERATURE REVIEW ... 3

2.1 Malaria... 3

2.1.1 Overview ... 3

2.1.2 Geographic distribution of malaria and its incidence ... 3

2.1.3 Risk factors ... 4

2.1.4 Clinical disease and complications... 5

2.1.5 Life cycle and transmission of the Plasmodium parasite... 5

2.1.6 Treatment and drug resistance ... 6

2.2 Artemisinin and its derivatives... 8

2.2.1 Overview ... 8

2.2.2 Candidate partner drugs in artemisinin-based-combination therapy (ACT) ... 10

2.2.3 Use of artemisinin-based-combination therapy ... 11

2.2.4 Neurotoxicity of artemisinin and its derivatives... 12

2.3 Neuropathological alterations... 14

2.3.1 Chromatolysis ... 14

2.3.1.1 Definition and causes ... 14

2.3.1.2 Nissl’s substance... 15

2.3.1.3 Morphological classification of chromatolysis ... 15

2.3.1.4 Chromatolysis in various diseases ... 16

2.3.1.5 Cytoskeleton changes in chromatolytic neurons... 17

2.3.1.6 Neurofilaments ... 18

2.3.1.7 Axonal pathophysiology in chromatolytic neurons ... 18

2.3.1.8 Selected methods for detection of axonopathies ... 20

3 MATERIALS AND METHODS... 23

3.1 Studied animals and experimental design... 23

3.2 Histology... 25

3.3 Histochemistry... 26

3.3.1 Luxol-Fast-Blue/Cresyl violet stain ... 26

3.3.2 Bielschowsky's silver stain ... 29

3.4 Immunohistochemistry... 30

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3.4.1 Reagents... 31

3.4.1.1 Primary reagents ... 31

3.4.1.2 Blocking serum ... 31

3.4.1.3 Secondary antibodies ... 31

3.4.1.4 Detection system ... 32

3.4.2 Protocol for immunohistochemistry (ABC method)... 32

3.4.2.1 Tissue pretreatment... 33

3.4.2.2 Glial fibrillary acidic protein ... 33

3.4.2.3 Bandeiraea simplicifolia-immunohistochemistry ... 34

3.4.2.4 TUNEL-assay ... 34

3.5 Evaluation... 36

3.5.1 Classification of investigated locations... 36

3.5.1.1 “Gray matter group no. 1” ... 36

3.5.1.1.1 Alphabetic list of abbreviations and code number of the histological locations ... 36

3.5.1.1.2 Code number of the lesions, lesions, and detection method ... 37

3.5.1.2 “Gray matter group no. 2” ... 38

3.5.1.2.2 Code number of the lesions ... 40

3.5.1.3 “White matter group no. 1”... 40

3.5.1.4 “White matter group no. 2”... 41

3.5.1.4.1 Code number of the lesions ... 42

3.5.1.5 “Remaining histomorphological locations group no. 1” ... 43

3.5.1.6 Histomorphological evaluation... 43

3.6 Statistical analysis... 44

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4 RESULTS... 47

4.1 Morphological and immunohistological findings... 47

4.1.1 Neuropathologic findings... 47

4.1.2 Experimental group no. 1 ... 61

4.2 Statistical analysis... 86

5 DISCUSSION ... 87

5.1 Dose and route of application dependent neurotoxicity... 87

5.2 Quantitative and topographic evaluation of the lesions... 89

5.3 Qualitative evaluation of lesions... 90

5.3.1 Chromatolysis ... 91

5.3.2 Axonal damage ... 93

5.3.3 Gliosis ... 95

5.3.4 Microgliosis and neuronophagia... 96

5.3.5 Apoptosis ... 98

5.4 The mechanism of neurotoxicity of artemisinin... 99

6 SUMMARY ...103

7 ZUSAMMENFASSUNG...105

8 REFERENCES...107

9 APPENDIX ...125

9.1 Abbreviations and symbols... 125

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9.2 Solutions and buffer... 126

9.3 Sources of chemicals, reagents and antibodies... 127

9.4 Sources of equipment and disposable items... 129

9.5 Statistical data... 131

9.6 P-values... 209

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1 INTRODUCTION

Falciparum malaria continues to be a disease that is difficult to control, causing high mortality rates, with an estimated mortality of 90% among African children.The drug treatments of this potentially lethal infection that have been most widely recommended and provided over the past 50, with years (i. e., chloroquine and sulfadoxine–pyrimethamine) are now regarded as ineffective in most tropical countries. Resistance to these drugs has led to a resurgence of malaria-related morbidity and mortality in many countries of Asia, South America and Africa.

Nowadays, there is considerably more funding available for malaria control in endemiccountries. The treatmentsrecommended by the World Health Organization for uncomplicated falciparum malaria are artemisinin-based combination therapies (ACT). These are combinations of an artemisinin derivative and another structurally unrelated and more slowlyeliminated anti-malaria drug.

Although the safety profiles of these drugs are thought to be excellent (ANONYMUS, 2005; GORDI and LEPIST, 2004; HIEN and WHITE, 1993; PRICE et al., 1999) concerns about their potential neurotoxicity remain, based on pathological findings in animal models. In rodents and monkeys, intramuscular injections of the lipophilic derivatives result in damage to certain areas of the brain stem (BREVER et al., 1994b; GENOVESE et al., 1999; KAMCHONWONGPAISAN et al., 1997; PETRAS et al., 1997). In rats and mice, parenterally applicated artesunate, a water-soluble derivative of dihydroartemisinin, is significantly less neurotoxic than the intramuscular administration of the lipophilic artemether and arteether (GENOVESE et al., 2000;

NONTPRASERT et al., 1998; NONTPRASERT et al., 2000). Oral artemether given once or twice a day is less neurotoxic than parenteral artemether (CLASSEN et al., 1999). However, a daily oral intake of 150 mg/kg for 28 days of oil-soluble artemether was recently shown to be more neurotoxic than a daily oral administration of water- soluble artemether at the same dose and treatment period (NONTPRASERT et al., 2000).

The aim of the present study was to characterize the neuropathological changes in dogs exposed to artemether or a combination of artemether-lumefantrine given at different doses and in various modes of administration.

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2 LITERATURE REVIEW

2.1 Malaria

2.1.1 Overview

Malaria is an acute flu-like parasitic illness that affects humans at any age. The disease is caused by one of the four species of the genus Plasmodium (P.), P.

falciparum, P. vivax, P. ovale, or P. malariae, respectively. The protozoon is transmitted by infected female mosquitoes of the genus Anopheles when it settles on a person to take a blood meal. The disease is characterized by recurrent periods of chills and fever. Infection with P. falciparum can be fatal, whereas infection with P.

vivax and P. ovale does not result in death; however, these strains have the ability to remain dormant in the liver for many months and can delay the occurrence of clinical signs of malaria for several months after the initial infection. Relapses of malaria infection may also occur with these strains. The incidence of P. malariae infection is patchy. This variant causes malaria with paroxysms recurring every fourth day, so called quartan malaria, due to schizogony and invasion of new red blood cells by parasites (ANONYMUS, 2005; McADAM and SHARPE, 2005).

At first, it was thought that malaria originated in marshes and swamps and was transmitted through the air. According to the Italian expression "mal" and "aria" - meaning "bad air" - the term “malaria” was applied to this disease. The real cause was discovered on 6^th November 1880, when Charles Louis Alphonse Laveran, a French army surgeon stationed in Constantine, Algeria, noticed parasites in the blood of a patient suffering from malaria. Subsequently it was discovered that malaria is caused by a single cell parasite, which was called “Plasmodium” (RAMHARTER and WINKLER, 2005).

2.1.2 Geographic distribution of malaria and its incidence

Malaria is endemic in most tropical and sub-tropical countries of sub-Saharan Africa, in large areas of the Middle East, South Asia, South East Asia, Oceania, Haiti, Central and South America and in parts of Mexico, North Africa and the Dominican Republic (Fig. 2.1). It represents one of the largest scourges in third world countries, affecting approximately 300 - 500 million people, and killing three million people

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annually. In endemic areas, the number of malaria cases increases from time to time dramatically to an epidemic level (ANONYMUS, 2003; McADAM and SHARPE, 2005;

RAMHARTER and WINKLER, 2005). The population of Africa suffers the most from malaria, and it is reported that 90% of malaria cases are diagnosed in Africa, mainly among young children and pregnant women. It is reported that malaria causes the death of an African child every 30 seconds (ANONYMUS, 2003).

Fig. 2.1 Geographic distribution of malaria (CENTERS FOR DISEASE CONTROL AND PREVENTION http://www.dpd.cdc.gov)

2.1.3 Risk factors

All human beings are at risk to acquire malaria. However, this tropical infection is more severe particularly in the following individuals:

Children up to an age of 5 years

Adults over 65 years old

Pregnant women

People treated with steroids or those receiving chemotherapy

Patients with acquired HIV infection (Aids patients)

Splenectomized people

People suffering from porphyry, epilepsy or chronic illness (BECK et al., 1994).

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2.1.4 Clinical disease and complications

Infected people may show one or more of the following clinical signs:

1. Periods of chills and sweats 2. High fever

3. Anorexia

4. Orthostatic hypotension characterized by low blood pressure causing dizziness when moving from a lying or sitting to a standing position

5. Muscle aches 6. Headache 7. Abdominal pain

8. Diarrhea, nausea, and vomiting

In untreated cases of P. falciparum infection additional signs may develop including:

Infection of the brain (cerebral malaria) with or without seizures and confusion resulting in coma and death

Renal failure

Abnormal and compromised liver function

Anemia

Pulmonary edema

Leukopenia

Hypoglycemia

Lactic acidosis

Hyponatremia

Hematuria indicating hemolysis due to parasitic destruction (so called Black water fever) (ANONYMUS, 2005).

Correct and rapid diagnosis of malaria infection is required, and treatment can be successful with a variety of drugs.

2.1.5 Life cycle and transmission of the Plasmodium parasite

When a mosquito bites a person infected with malaria, it ingests male and female individuals of the parasite (gametocytes). The gametocytes unite in the stomach of the mosquito forming an oocyst. The oocyst takes about a week to mature followed by sporogony in the midgut. Subsequently the sporocyst ruptures releasing thousands of sporozoites, which migrate through the hemocoel to invade the salivary

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glands of the mosquito. When this mosquito bites a human, sporozoites are injected into the bloodstream. The sporozoites reach rapidly the liver and infect hepatocytes.

They develop within 6 – 8 days into schizonts, which rupture and release merozoites into the blood stream. In some cases of P. vivax or P. ovale infection, the merozoites can remain inactive in the liver for extended periods of time. Later, reactivation of the parasite's life cycle causes a relapse.

Upon maturation, merozoites in the blood invade erythrocytes, followed by development of trophozoites, which can infect erythrocytes again or generate gametocytes. The latter can be ingested by a mosquito and perpetuates the developmental cycle.

With the burst of one infected erythrocyte, plenty of parasites are released ready for infection of numerous red blood cells. With each wave of hemolysis - about every 48 to 72 hours (Fig. 2.2), depending on the Plasmodium species - the person suffers from periods of chill, fever and sweating. Typically, signs start between 10 - 28 days after the initial mosquito bite, although they can appear as early as eight days or as late as one year after infection (BLOLAND and WILLIAMS, 2003).

In many cases, treatment or the immune response eliminates the parasite.

Particularly in children, who have yet to acquire immunity against the parasite, complications of the infection may lead to death. In addition, P. falciparum is capable of invading a much greater number of red blood cells than the other species of Plasmodium, and the infection can be fatal within a few hours after initial hemolysis (CENTERS for DISEASE CONTROL and PREVENTION http://www.dpd.cdc.gov/DPDx/). In pregnant women, the infection may be transmitted transplacentarly to the fetus. In addition, malaria has been transmitted via blood transfusion using infected donor blood samples (DUFFY and DESOWITZ, 2001).

2.1.6 Treatment and drug resistance

A malaria infection, particularly with P. falciparum, requires prompt diagnosis and treatment. In most cases, malaria can be effectively treated with one or more of the following drugs:

Chloroquine

Quinine sulfate

Hydroxychloroquine

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Fig. 2.2 Schema of the Life Cycle of Malaria

The malaria parasite life cycle involves two hosts. During a blood meal, a malaria-infected female Anopheles mosquito inoculates sporozoites into the human host . Sporozoites infect liver cells and mature into schizonts , which rupture and release merozoites . (Of note, in P. vivax and P.

ovale a dormant stage [hypnozoites] can persist in the liver and cause relapses by invading the bloodstream weeks, or even years later.) After this initial replication in the liver (exo-erythrocytic schizogony ), the parasites undergo asexual multiplication in the erythrocytes (erythrocytic schizogony ). Merozoites infect red blood cells . The ring stage trophozoites mature into schizonts, which rupture releasing merozoites . Some parasites differentiate into sexual erythrocytic stages (gametocytes) . Blood stage parasites are responsible for the clinical manifestations of the disease.

The gametocytes, male (microgametocytes) and female (macrogametocytes), are ingested by an Anopheles mosquito during a blood meal . The parasites’ multiplication in the mosquito is known as the sporogonic cycle . While in the mosquito's stomach, the microgametes penetrate the macrogametes generating zygotes . The zygotes in turn become motile and elongated (ookinetes)

which invade the midgut wall of the mosquito where they develop into oocysts . The oocysts grow, rupture, and release sporozoites , which make their way to the mosquito's salivary glands.

Inoculation of the sporozoites into a new human host perpetuates the malaria life cycle . (CENTERS FOR DISEASE CONTROL AND PREVENTION http://www.dpd.cdc.gov)

Combination of sulfadoxine and pyrimethamine

Mefloquine

Combination of atovaquone and proguanil

Doxycycline

= Infective Stage

= Diagnostic Stage

MosquitoStages Ruptured Oocyst

Oocyst Release of

sporozoites

Sporogeniccycle

Ookinete

Macrogametocyte

Microgamete entering macrogamete

P. Falceparum

P. vivax P. ovale P. malariae

Mosquitotakes a blood meal (injects Sporozoites)

Mosquito takes a blood meal (injects gametocytes)

Ruptured schizont

schizont

Gametocytes Mature trophozoite

Exflagellated microgametocyte

Immaturetrophozoite (ring stage) Human BloodStages

Erythrocytic Cycle

Schizont Infectedliver

cell Human Liver Stages

Liver cell

Exo-erythrocytic Cycle Ruptured schizont

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Because of the relentless increase in resistance of malaria parasites to conventional drugs, new therapeutic approaches have been developed, among them the artemisinin-based-combination therapy (ACT) is most important (SULLIVAN and KRISHNA, 2005).

2.2 Artemisinin and its derivatives

2.2.1 Overview

The artemisinin compounds are new, highly-effective anti-malaria drugs, discovered in China. Their chemical structure is depicted in Fig. 2.3. A crude extract of the wormwood plant Artemisia annua (qinghao) was first used as an antipyretic substance 2000 years ago. Its specific effect on the fever of malaria was already reported in the 16^th century (KLAYMAN, 1985; DAVIS et al., 2005a). The active constituent of the extract was identified and purified in the 1970s, and named qinghaosu or artemisinin (C15H22O5). Artemisinin has been developed pharmacologically into different derivatives with various properties and antimalarial potency (KLAYMAN, 1985; DAVIS et al., 2005a; HIEN and WHITE, 1993). Several million patients have been treated successfully with these compounds during the past three decades (HIEN and WHITE, 1993). Each artemisinin derivative is highly active against asexual forms of the four species of Plasmodium that infect humans. The initial reduction of parasitemia by these compounds is the most rapid of all available antimalarial drugs (HIEN and WHITE, 1993; VRIES and DIEN, 1996). Their half-lives are short (Fig. 2.3) relative to the duration of their effect on parasite clearance, suggesting an equivalent to a “post-antibiotic effect”, characterized by persistent suppression of bacterial growth following limited exposure to an antimicrobial agent (DAVIS et al., 2005a).

The artemisinin derivatives are also active against the sexual form of the parasites (gametocytes) taken up by the mosquito and can therefore reduce transmission rates (CHEN et al., 1994). Their exact mechanism of action is unknown. An endoperoxide moiety, which is essential for the antimalarial activity, may generate destructive free radicals within the parasite and form covalent bonds impairing the function of key parasite proteins, including membrane transport ways (ECKSTEIN-LUDWIG et al., 2003). The artemisinin derived free radicals appear to damage specific intracellular

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Fig. 2.3 Chemical structure and pharmacology of artemisinin derivatives

*Arteether is a closely related compound with an ethylether instead of a methylether group that has been developed for intramuscular use “elimination half-life, 12–30 hours”

(DAVIS et al., 2005a)

targets, possibly via alkylation. This observation suggested that free radicals might be involved in the mechanism of action (MESHNICK, 2002). Short treatment periods (3 - 5 days) with low doses (about 50 mg/kg) are associated with high recrudescence rates (HIEN and WHITE, 1993; VRIES and DIEN, 1996). Although such recrudescence can be re-treated successfully with an artemisinin drug, this phenomenon highlights the limitations of a monotherapy. There are no documented cases of parasite resistance to artemisinin derivatives in humans (WHITE, 2004).

Artemisinin

Route of administration: oral, rectal;

Elimination half-life: 2–3 hours

Artesunate (artesunic acid) Route of administration: oral, rectal, intravenous, intramuscular;

Elimination half-life: 2–5 minutes (converted to dihydroartemisinin)

Dihydroartemisinin

Route of administration: oral, rectal;

Elimination half-life: 40–60 minutes Artemether*

Route of administration: oral, intramuscular;

Elimination half-life: 3–7 hours (converted to dihydroartemisinin)

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The therapeutic index, i. e. the relation of the therapeutic to the toxic dose of artemisinin derivatives is wide. The most common reported adverse effects include nausea, vomiting, bowel disturbance, abdominal pain, headache and dizziness.

However, these signs may also result from the malaria infection itself. Mild and reversible hematological and electrocardiographic abnormalities, such as thrombocytopenia and first-degree heart block, are observed infrequently (PRICE et al., 1999). Neurotoxicity, principally in the form of brain stem lesions, were first identified in animals (dog, rat, mouse, and monkey) receiving high doses over long periods (BREWER et al., 1994a; 1994b; GENOVESE and NEWMAN, 2007;

NONTPRASERT et al., 2002). Neurological side-effects such as ataxia, slurred speech and hearing loss have also been reported in few adult humans (MILLER and PANOSIAN, 1997; TOOVEY and JAMIESON, 2004), but are unlikely to be of clinical importance (JOHANN-LIANG and ALBRECHT, 2003; KISSINGER et al., 2000), especially as there is limited penetration of artemisinin derivatives into the cerebrospinal fluid (DAVIS et al., 2003; VRIES and DIEN, 1996). The pharmacokinetics of artemisinin derivatives such as artesunate is not influenced significantly by the severity of the infection (DAVIS et al., 2003).

Among the available derivatives, artesunate has the most favourable pharmacological profile for use in ACT treatment of uncomplicated malaria. The presence of a hemisuccinate group in the molecule confers water solubility and relatively high oral bioavailability. It is rapidly and quantitatively converted in vivo to the potent active metabolite (DAVIS et al., 2003). Dihydroartemisinin, artemisinin and artemether are all poorly water-soluble, resulting in slow and incomplete absorption.

Furthermore, the Role Back Malaria (RBM) strategy parallels multidrug treatment used successfully in patients with HIV and cancer, and combines the rapid schizontocidal effect of an artemisinin compound with a longer half-life of the drug.

The World Health Organization (WHO) has recently endorsed ACT as the “policy standard” for all malaria infections in areas where P. falciparum is the predominant infecting species (ANONYMUS “RBM”, 2004-2008).

2.2.2 Candidate partner drugs in artemisinin-based-combination therapy (ACT) In South-East Asian countries, artesunate-mefloquine has been used widely for many years and remained highly effective for uncomplicated cases of P. falciparum

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malaria. Under the WHO system of efficacy assessment, more than 95% of patients will remain free of malaria 28 days after such a treatment (HIEN et al., 2004).

However, the neuropsychiatric effects of mefloquine as well as its relative cost are problematic. No coformulation is available, but in Cambodia there is a blister pack available containing tablets of each constituent drug grouped by the doses to be administered over 3 days. It is therefore possible for patients to identify and take the artesunate and avoiding the less tolerated mefloquine (SHWE et al., 1998), which results in a reduction of therapeutic efficacy.

Artemether-lumefantrine has been given priority as the first-line ACT combination for uncomplicated malaria (ANONYMUS “RBM”, 2004-2008). In comparison to halofantrine and quinine as partner drugs of ACT lumefantrine exhibits no significant cardiotoxicity (TYLOR and WHITE, 2004; KARUNAJEEWA et al., 2003), but initial studies with a four doses artemether–lumefantrine regimen showed a relatively high rate of late treatment failure (OMARI et al., 2004).

The most promising form of ACT seems to be dihydroartemisinin-piperaquine.

Piperaquine is a bisaminoquinoline frequently used in China in the 1970s and 1980s, when chloroquine resistance increased in the south of the country (DAVIS et al., 2005b). It was rediscovered in the 1990s as a candidate for ACT, and is currently combined with dihydroartemisinin. There have been two recent Indo-Chinese studies that have demonstrated its effectiveness, with 28-day cure rates greater than 95%

(DENIS et al., 2002; HUNG et al., 2003). The 2-day, 4-dose recommended treatment regimen is well tolerated and there are no significant side-effects (KARUNAJEEWA et al., 2003). Although the long half-life of piperaquine may theoretically allow resistant parasites to be selected by the subtherapeutic concentrations present after treatment (HUNG et al., 2003), this has not been of concern in areas of Thailand where the transmission rate is low and where mefloquine (a drug with a similarly long elimination phase) has been used extensively with artesunate (NOSTEN et al., 2000).

2.2.3 Use of artemisinin-based-combination therapy

Malaria can be particularly severe in pregnancy causing low birth weight, anemia and an increased risk of a fatal outcome. Because of the potential benefits of the artemisinin derivatives in pregnancy, a WHO review of available safety data states

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(ANONYMUS, 2002; 2007a) that use of artemisinin derivatives in 731 pregnancies was not associated with human fetal toxicity. In contrast, animal studies have shown significant adverse effects. Due to these data, the WHO currently advises against the use of artemisinin drugs in the first trimester, unless in a life threatening situation where no other drugs are suitable, while, in later pregnancy, alternative therapies should be used if available.

Antimalarial treatment per se was not associated with an increased risk of abortion.

Recent studies show, that the risks of abortion due to malaria infection far outweighed any risks of abortion due to the use of antimalarial medicines including artemisinin derivatives. The evidence regarding first trimester exposures to artemisinins was reassuring but still inadequate and warrants a change in the current WHO recommendations for the treatment of malaria in the first trimester of pregnancy (ANONYMUS, 2007a; DUFFY and DESOWITZ, 2001). ACT has been used safely in children up to 5 years of age in Africa, south-east Asia and Europe and shows a high cure rate and tolerability. The combination should be taken as a 6-dose regimen. The safety of ACT has not yet been established in children of less than 5 kg body weight, and its use in this group is not recommended until further safety data are available (ANONYMUS, 2005).

2.2.4 Neurotoxicity of artemisinin and its derivatives

The use of artemisinins has risen substantially in the last 5 years and large increases in their deployment are impending as countries systematically introduce and adopt ACT to replace existing therapies, which have fading effectiveness as a result of drug resistance (ANONYMUS, 2007b; LALLOO et al., 2007). While generally believed to be well tolerated, the safety of artemisinins has been the subject of some debate (GORDI and LEPIST, 2004; TOOVEY, 2006).

Numerous studies with artemisinins have demonstrated that, under certain conditions, their administration can result in a relatively specific brain stem lesion in laboratory animals. Furthermore, the relationship between neuropathology and the application form, treatment duration, dose and type of artemisinin is not completely understood. The mode of action in neurotoxicity and the mode of action in antimalarial efficacy of artemisinins are also not yet defined (GOLENSER et al., 2006). Despite the documented occurrence of artemisinin induced brain stem

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neurotoxicity in laboratory animals 14 years ago (BREWER et al., 1994a; 1994b), there is a distinct paucity of recent information on the subject, even as the number and amount of artemisinins in use grows at an accelerating pace as does the situations in which their use is recommended.

Neurotoxicity of artemisinins has been observed in mice (NONTPRASERT et al., 2002), rats (GENOVESE et al., 1998; 1999; 2000; 2001), and rhesus monkeys (PETRAS et al., 1997). The occurrence and severity of lesions are dose dependent and ranged from minimal neuronal abnormalities (BREWER et al., 1994a; 1994b;

CLASSEN et al., 1999; GENOVESE et al., 1999) to extensive necrosis associated with morbidity (BREWER et al., 1994a; KAMCHONWONGPAISAN et al., 1997). The lesions occur in a specific pattern affecting predominantly selected brain stem nuclei of the medulla oblongata, pons, and mesencephalon. Affected areas in the telencephalon and diencephalon are conspicuously spared (KAMCHONWONGPAISAN et al., 1997). Within the pons and medulla oblongata, nuclei associated with auditory and vestibular functions are particularly vulnerable.

The auditory pathway includes Corti’s organ, ganglion spirale, cochlear nerve, nucleus cochlearis, corpus trapezoideum, nucleus olivaris anterior (nucleus trapezoidium dorsalis), leminiscus lateralis and its nuclei, colliculus superior, corpus geniculatum medialis, capsula interna and auditory cortex at the sulcus ectosylvius (Fig. 2.4). In addition, precerebellar nuclei including lateral reticular and reticulotegmental nuclei and nuclei in the large medial reticular core (e. g., nucleus pontis caudalis and nucleus gigantocellularis) were also affected. Alterations in the mesencephalon including the red nucleus were notably observed. Deep cerebellar nuclei, such as the fastigial nucleus, are also damaged. The cause of the topographical selectivity of the lesions is not known. The neuronal damage is characterized by swollen cell bodies, dissolution of Nissl’s substance (chromatolysis), cytoplasmic vacuolization, nuclear eccentricity, nuclear shrinkage, nucleolar swelling, karyopyknosis, satellitosis, axonal degeneration, necrosis and neuronophagia.

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Sulcus ectosylvius Auditory

cortex

Corpus geniculatum

medialis Colliculus superior

Leminiscus lateralis Nuclei

cochleares

Nucleiolivares anterior

Nervus cochlearis

Corpus trapezoieum

2.3 Neuropathological alterations

2.3.1 Chromatolysis

2.3.1.1 Definition and causes

The term chromatolysis describes a disintegration and dispersal appearance of chromatin granules of the rough endoplasmic reticulum in a nerve cell body (MAXIE and YOUSSEF, 2007). Chromatolysis may occur after overwork exhaustion of the cell or as a response to damage of its peripheral processes (ANDREW, 1936). It also occurs after injuries to neurons due to various toxins including acrylamide, actinomycin, alcohol, 6-aminonicotinamide, capsaicin, triethyltin, trimethyltin, and lectins (LEVINE et al., 2004). Other changes considered as part of chromatolytic changes include swelling of the perikaryon and shifting of the nucleus from its central position to the periphery.

Fig. 2.4 A simplified diagram of the auditory pathway;

Image created by Diana Weedman Molavi, PhD, at the Washington University School of Medicine.

http://thalamus.wustl.edu/course/

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The morphologic changes of chromatolysis following injury to Nissl’s bodies, cytoskeleton and axon were first described by Nissl in 1892 (MACKEY et al., 1964).

The studies were designed to characterize a localized axonal injury but also generalized changes in the organism due to anoxia, toxemia, hyperpyrexia, exhaustion, neurotropic virus infections, and hibernation in other lower vertebrates (GERSH and BODIAN, 1943).

Chromatolysis is the primary response of the nerve cell to injury resulting from morphological, electrical, and biochemical alterations and represents the most relevant morphological feature at the light microscopic level (MACKEY et al., 1964).

2.3.1.2 Nissl’s substance

The Nissl’s substance are chromophilic granules consisting of endoplasmic reticulum and ribosomes located in neuronal perikarya and dendrites, but they are absent from axons. They consist principally of the ribose type of nucleic acid and the nucleoprotein and stain strongly with basic aniline dyes. They are concerned with protein synthesis and metabolism at variable physiological and pathological conditions (ANDREW, 1936).

The amount of Nissl’s substance is variable and depends on the physiological state of the cell. Electron microscopic studies showed that, the Nissl’s substance is composed of electron dense particles that occur either free among the membrane structures or are bound to the endoplasmic reticulum (ribosomes) (PALAY and PALADE, 1955). Chromatolysis, a fading, disorganization, or entire loss of the basophilic substance in the cytoplasm, was shown by DOLLEY (1911) to accompany fatigue in dogs resulting from excessive activity in a treadmill. Working with Purkinje cells, he traced carefully the steps in chromatolysis and showed that, the nucleus is able to elaborate chromatin which passes into the cytoplasm. Overwork may destroy this capability resulting finally in cell death (DOLLEY, 1911).

2.3.1.3 Morphological classification of chromatolysis

Chromatolysis may occur in various forms and can be subclassified as central, peripheral or total chromatolysis according to its location within the cell body, severity and stage of injury (BAUMGÄRTNER, 2007; SUMMERS et al., 1995).

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Central chromatolysis

Neurons with central chromatolysis are characterized by a swollen, rounded rather than angulated shape. The nucleus typically takes an eccentric position, and Nissl’s granules breakdown (lysis) and clear from the central region of cell body (MAXIE and YOUSSEF, 2007).

Peripheral chromatolysis

Peripheral chromatolysis represents the initial response to neuronal exhaustion (CAMMERMEYER, 1963), and may be regarded as an early stage on the way to necrosis. Nissl’s bodies become lose in structure and small in size at the periphery, while persisting around the nucleus. Subsequently the cell body shows slight shrinkage rather than swelling (MAXIE and YOUSSEF, 2007).

Total chromatolysis

In total chromatolysis, Nissl’s granules are completely cleared and the nucleus takes an eccentric position. In addition, the cell body is swollen and cellular organelles are dislocated (SUMMERS et al., 1995).

2.3.1.4 Chromatolysis in various diseases

Chromatolysis has been observed in different spontaneous and experimental disorders of animals and humans. It represents a distinctive lesion in certain diseases of animals, such as equine (Grass Sickness) or feline (Key Gaskell-Syndrom) dysautonomia, equine motor neuron disease and swayback of lambs and kids caused by copper deficiency (SUMMERS et al., 1995). Furthermore, chromatolysis has been described in some neurotropic virus infections including poliovirus infection of mice (BLEDSOE et al., 2000), in certain hereditary diseases, such as “hereditary canine spinal muscular atrophy (SMA)”, and in human “motor neuron disease”

(CORK et al., 1982). In chicken, fish, rats, and cynomolgus monkeys chromatolysis can be induced by administration of certain substances, e. g. lithium compounds or triphenyl-phosphate (FIORONI et al., 1995; GARRUTO et al., 1989; GUARNIERI et al., 1994; LEVINE et al., 2004). Therefore chromatolysis represents a specific neurodegenerative effect. Chromatolysis has been also interpreted as a response to axonal injury (GRAHAM et al., 2002; McILWAIN and HOKE, 2005).

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2.3.1.5 Cytoskeleton changes in chromatolytic neurons

The morphology of each type of nerve cell is characterized by its distinctive shape and cytoplasmic organization of the intracellular components. The ultrastructure of the cytoplasm in the perinuclear soma, dendrites and axons displays typical morphological features. The most relevant morphological structure is a finely meshed and regular network of microfilaments (4 - 7 nm in diameter). A small annular region around the nucleus contains fine microfilaments radially disposed to the outer nuclear membrane. These filaments seem to anchor the nucleus in the cytoplasmic network.

Concentrically to this small fibrillar layer a wide perinuclear zone of the same meshwork surrounds numerous membrane-bounded organelles such as endoplasmic reticulum and mitochondria, orientated predominantly circular around the nucleus. All membrane-bounded organelles including the Golgi apparatus are anchored with microfilaments. In this perinuclear zone the presence of microtubules is scarce.

Neurons contain three major classes of cytoskeletal elements: neurofilaments about 10 nm, microfilaments about 5 nm, and microtubules about 20 nm in diameter (LEVITAN and KACZMAREK, 1991). They are aggregated in bundles and can be observed most frequently in the basal portion of the principal dendrite. Cross-linkers between neurofilaments, microtubules and membrane-bounded organelles are found.

It is generally accepted that the cytoskeleton is primarily responsible for the form of cell soma, dendrites and axons (MELLER, 1987a; METUZALS et al., 1983).

The physiological changes, which occur in early stages of chromatolysis, are enlargement of the nucleolus, swelling, and increased size of mitochondria as well as cisternae of the endoplasmic reticulum. These changes suggest an enhanced metabolic activity with protein synthesis during chromatolysis representing probably an attempt of cell repair (MACKEY et al., 1964).

The biochemical processes are an uniform reaction pattern of a neuron to injury.

They depend, however, on certain functional parameters, such as site of the lesion and its distance from the cell soma, stage of maturation of the cell, and species- specific factors. Not all neurons develop these reactions at the same time and to the same degree of intensity. It is assumed that these mechanisms are related to repair of the neurons in order to compensate the loss of axonal material and to establish a new functional relation to the target (MELLER, 1989). The nucleus and all organelles are embedded into a meshwork of filamentous components belonging to the cytoskeleton (MELLER, 1987a). Early alterations of the axon can be recognized in a

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remodelling process of the cytoskeletal elements, especially microtubules, neurofilaments and their cross-linking structures (MELLER, 1987b). The early chromatolytic reaction in the cell soma is a response of the cytoskeletal structures to noxious stimuli similar to that occurring in the injured axon (MELLER, 1989).

2.3.1.6 Neurofilaments

Neurofilaments (NF) are the major intermediate (10 nm) filaments in many types of mature neurons. Assembled from three polypeptide subunits, NF-L (68 kDa), NF-M (95 kDa) and NF-H (115 kDa), neurofilaments are most abundant in large myelinated axons (LEE and CLEVELAND, 1994). Phosphorylation and dephosphorylation of neurofilaments are complex processes. However, all three subunits may be phosphorylated, but NF-M and NF-H have unusually high phosphorylation levels.

Cell-cell contacts and communication between cells and axons are not well understood, but often elicit intracellular responses via changes in phosphorylation levels for specific substrates. If cell-axon contacts modulate phosphorylation- dephosphorylation cycles within axons, then a disruption of these interactions might alter not only NF phosphorylation, but also a variety of axonal parameters including axon caliber, axonal transport, and membrane organization (DE WAEGH et al., 1992). Approximately 80% of axonal NF are phosphorylated, representig a “static pool”. The remaining 20% were less phosphorylated NF forming the “dynamic pool”

(NIXON, 1993).

2.3.1.7 Axonal pathophysiology in chromatolytic neurons

The axon is the elongated fiber that extends from the cell body to the terminal endings and transmits the neural signal. The axons of the brain are extending across different layers, for example from the cerebral cortex to the subcortical white region.

These different layers of the brain have different densities, and are located at varying distances from the center of a given rotation and sliding across each other, which can put unnatural stress on the axons, which extends across these layers (GRAHAM et al., 2002). The primary role of the axon is information transfer, both from one end of the individual neuron to the other, and from one cell to another. Transfer of information within a cell is termed intracellular signalling and between cells, intercellular signalling. The intracellular signal starts at the cell body, runs down the

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axon to the synaptic terminal end. The intercellular transfer is across the synapses, where the signal jumps from one cell to another.

There are two specialized regions in the axon: immediately beneath the axolemma lies the membrane or cortical cytoskeleton, composed of a meshwork of actin filaments, fodrin-brain spectrin polymers and associated proteins, and more internally in the axoplasm, neurofilaments and microtubules form a cytoskeletal core of two overlapping networks cross-linked by numerous side-arms (GRAHAM et al., 2002).

Axons are most exposed to injury in a shear mechanism and are particularly vulnerable to injury of the long thin protrusion which can extend substantial distances across different layers of different density within the brain. The effect of this sliding is that the axon is rapidly stressed beyond its tolerance, which may result in tearing or stretching of the axon. Even if the axon is not entirely altered as a result of such force, it may be significantly damaged. The axon is protected by an insulation called myelin sheath. When the axons insulation is disrupted, the speed of information processing within the brain can be profoundly affected (GRAHAM et al., 2002;

KINNEY and ARMSTRONG, 2002; LEVITAN and KACZMAREK, 1991).

Degeneration of axons is followed rapidly by breakdown of its myelin sheaths. The usual cause of this type of injury is trauma and these changes are seen distal to the site of damage, which is called “Wallerian degeneration” (SUMMERS et al., 1995).

Proximal to the damage the nerve cell body undergoes transient swelling and breakdown of the endoplasmic reticulum (chromatolysis). In some diseases there is a tendency that injury to axons occurs first and most severely at the distal ends. Axonal degeneration then proceeds backwards toward the neuron, called “dying-back”.

Dying back neuropathies or distal axonopathies represent typical signs of a human disease called “glove and stocking anaesthesia” (MACSWEEN and WHALEY, 1992).

Finally, axonal degeneration can also be secondary to degeneration of the cell body, as in anterior poliomyelitis or motor neuron disease (MACSWEEN and WHALEY, 1992).

These are various events that have been triggered by the primary injury and include neurobiological processes involving cellular dysfunction such as free radical formation, receptor mediated mechanisms and calcium as well as inflammation mediated damage (GRAHAM et al., 2002). Axonal damage may be caused by various noxes including traumatic, ischemic, toxic, metabolic, or other injuries, resulting in:

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Â Cell miscommunication: A series of electrical and chemical changes developing over a period of hours result in changes of blood flow, metabolism and ionic balance in the brain. Brain cells communicate with each other both electronically and chemically. Trauma can disrupt the electrical and chemical balance resulting in set off wrong signals, which can damage or destroy the cell.

Â Production of neurotoxins: Distal sensorimotor polyneuropathy is probably the most common clinical manifestation of neurotoxicant exposure in man. A variety of toxicants, including hexane, methyle n-butyl ketone (2-hexanone), carbon disulfide (CS2), acrylamide and organophosphorus esters, result in degeneration of axons in the PNS and CNS which are characterized by swellings of axonal neurofilaments.

Trauma can also result in the release of chemicals which are toxic to brain cells.

Â Stimulation of micro-inflammation: Other damage is related to inflammatory CNS diseases, including poliovirus infection (DESTOMBES et al., 1979). Chromatolytic changes such as neuronal swelling can cause a pressure increase at the cellular level, which results in secondary brain damage and inflammation (GRAHAM et al., 2002).

2.3.1.8 Selected methods for detection of axonopathies Silver impregnation

Silver staining techniques, including modified Bielschowsky´s, Bodian´s, and Campbell-Switzer method, represent a major advance in neuropathological studies to detect particular pathological changes in the CNS and PNS as reviewed recently (UCHIHARA, 2007). Silver staining has been steadily improved and the most frequently used technique is a modified Bielschowsky method which is employed to detect and assess neurofibrils, dendrites and axons as well as their morphological changes (SWITZER, 2000), Furthermore, Bielschowsky´s method detects amyloid deposition in human brains with Alzheimer's disease (YAMAMOTO and HIRANO, 1986). Bodian´s method is used for the detection of the axonal structure, nerve endings and their morphological changes in the PNS, and for the demonstration of particular lesions including Alzheimer’s neurofibrillary tangles. The Campbell-Switzer method is used to detect neuritic plaques and neurons with neurofibrillary tangles, which are the hallmark in human Alzheimer's disease (CHAN and LOWE, 2002;

UCHIHARA, 2007), as well as amyloid plaques in brains of old dogs (CZASCH,

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2006). Axonal degeneration in canine brain is visualized using the amino-cupric-silver stain of de Olmos (SIEGEL et al., 1999).

Amyloid Precursor Protein (APP)

APP is a transmembrane glycoprotein that is synthetized in neurons and normally flows along the axon. APP is thought to have functions related to cellular adhesion, growth and response to injury (FERREIRA et al., 1993; MASLIAH et al., 1998;

LEBLANC et al. 1992). APP has a short half-life and is rapidly metabolized in two ways. The α-degradation pathway involves two consecutive divisions, the so-called α- and γ-secretase process. The β-degradation is also divided in two steps, the so- called β- and γ-secretase process. Both finally lead to production of β-amyloid, which eventually accumulates in Alzheimer's disease due to mutations of the APP gene or in the enzyme structure (HARDY, 1996). APP probably accumulates after disruption of the axonal cytoskeleton causing interruption of axoplasmic flow. Since the early 1980s APP immunocytochemistry has been used as a marker of diffuse axonal injury (GENNARELLI et al., 1982; GENNARELLI, 1996; GEDDES, 1997), and can be detected 35 minutes after injury in contrast to more traditional silver impregnation techniques, which only identify axonal swellings with certainty about 15 hours after injury, and there is a highlevel of background staining with silver (HORTOBÁGYI and AL-SARRAJ, 2008). APP-positive spheroids have been described after traumatic brain injury, around infarcts, abscesses, tumour metastases, neurodegenerative disorders (SHERRIFF et al., 1994), and in some neuronal viral infection such as canine distemper virus (SEEHUSEN, 2006). Thus APP-positive axonal bulbs may occur in the absence of head injury, cerebral hypoxia, especially in those cases associated with neurotoxicity due to treatment.

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3 MATERIALS AND METHODS

3.1 Studied animals and experimental design

41 male Beagle dogs between 22 to 52 weeks of age were treated with test formulations in an experimental trial at Novartis Pharma, Basel. Formalin fixed tissue samples of brain, spinal cord and the pars petrosum with the inner ear were submitted for histological investigation to the department of pathology, university of veterinary medicine, Hannover. Tissues were investigated light microscopically with particular emphasis on the auditory system and its pathway. The animals were allocated in 14 experimental groups according to different modes of treatment, dose and duration of treatment, recovery period and day of euthanasia (Table 3.1). The first experimental group (dog no. 51 - 53) consisted of control animals received a vehicle control (peanut oil) intramuscularly, and the other experimental groups were treated either intramuscularly (group no. 2 - 8) or orally (experimental group no. 9 - 14) with either artemether (ARM566; experimental group no. 2 - 11) or the combined drug artemether and lumefantrine (COA566; experimental group no. 12 - 14), which was designed for the oral treatment for uncomplicated malaria of the Plasmodium falciparum type.

ARM566 was administered intramuscularly in a low dose (10 mg/kg b. w.) in experimental group no. 2 – 4, and in a high dose (40 mg/kg b. w.) in experimental group no. 5 – 8, and orally in a low dose in experimental group no. 9 – 11 (600 mg/kg b. w. on 1^st day and 300 mg/kg b. w. during the following days). Animals of experimental group no. 2, 3, 4 and 8 received the daily dose alternating in one hind leg, and animals of experimental group no. 5, 6, and 7 received half of the daily dose via injection into each hind leg.

COA566 was administered orally in a high dose (1000 mg/kg b. w.) in experimental group no. 12, 13, and 14, this dose contained 143 mg artemether and 857 mg lumefantrine.

Treatment duration differed between 3 days (experimental group no. 2, 3, 5, 6, 9, 10, 12, and 13) and 8 days (experimental group no. 4, 7, 8, 11, and 14). The recovery period was 0 (experimental group no. 2, 4, 5, 7, 8, 9, 11, 12, and 14) or 5 days after treatment duration (experimental group no. 3, 6, 10, and 13; Table 3.1). The dogs were euthanized at the end of the study.

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Table 3.1 Experimental group number, animal identification, animal number, group designation, dosage, mode and duration of treatment, dose designation and route of administration, recovery period, day of euthanasia and tissue fixation method of the experimental animals

E.G. no.: I.D. Animal no. Group designation Dose mg/kg b. w. and treatment duration Dose and administrati- on route Recovery period Day of euthanasia Fixation method

01 51-53 3 v.control 00 Control animals 00 8 PF 02 54-56 3 ARM566 10 for 3 days Low dose i. m. 00 3 PF 03 57-59 3 ARM566 10 for 3 days Low dose i. m. 05 8 PF 04 60-62 3 ARM566 10 for 8 days Low dose i. m. 00 8 PF 05 63-65 3 ARM566 40 for 3 days High dose i. m. 00 3 PF 06 66-68 3 ARM566 40 for 3 days High dose i. m. 05 8 PF 07 69-71 3 ARM566 40 for 8 days High dose i. m. 00 8 PF 08 349-350 2 ARM566 40 for 8 days High dose i. m. 00 8 PF/IM 09 151-153 3 ARM566 600/300§ 3 days Low dose orally 00 3 PF 10 154-156 3 ARM566 600/300§ 3 days Low dose orally 05 8 PF 11 157-159 3 ARM566 600/300§.8 days Low dose orally 00 8 PF 12 160-162 3 COA566 1000 for 3 days High dose orally 00 3 PF 13 163-165 3 COA566 1000 for 3 days High dose orally 05 8 PF 14 166-168 3 COA566 1000 for 8 days High dose orally 00 8 PF

§ The dose was reduced from 600 to 300 mg/kg after 1^st day for group no. 9, 10, and 11.

PF = perfusion. IM = immersion. PF/IM = PF in I.D 350, and IM in I.D 349. E.G.no. = experimental group number. I.D. = identification number. i. m. = intramuscular. v. control = vehicle control

Tissues of all dogs were fixed by perfusion with 10% neutral buffered formalin under deep anaesthesia except tissues of dog no. 349 were fixed by immersion in 10%

neutral buffered formalin.

All tissues were fixed for 20 – 29 days in 10% neutral buffered formalin except tissues of dog no. 349 (40 days). Coronal sections of the brains were cut and embedded in paraffin wax according to standard laboratory procedures. Coronal sections of the brains were obtained by slicing the tissue into 3 mm thick sections. A total of 37 tissue blocks (block no. 1-37) were prepared from most brains and spinal cords (block no.18 and 21 were not used in this study) and two additional tissue blocks from each ear. The bone of the pars petrosum was decalcified in 10% formalin

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in EDTA-disodium for 20 days at room temperature under continuous stirring of the decalcification solution. One sagittal cut was performed through each cochlea (2 blocks total). Tissue processing (dehydration, clearing and paraffin infiltration) was done automatically (shandon pathcentre^® Tissue Processor, Thermo, USA). Finally, the tissues were infiltrated with and embedded in paraffin wax (Tissue-Tek^®, Sakura, Netherlands) according to standard laboratory procedures.

3.2 Histology Histology processing

Tissue sections were cut at 5 µm thickness with a microtome (LEICA RM2035^®, Leica, Nussloch, Germany) and placed on a glass slide (SuperFrost^® Plus Objektträger, Menzel, Braunschweig, Germany). The glass slides were incubated for 20 - 30 min. at 57^ºC to enhance the adhesion of the tissue section on the slide, and were then kept at room temperature until used.

One section of each paraffin block was stained automatically with hematoxylin-eosin (H&E) according to a standard laboratory procedure (37 blocks for each animal).

From 10 selected paraffin blocks of the brain stem (block no. 2-11) 13 serial sections were cut. The first 5 sections of each series (see table 3.2) were stained with H&E, Luxol-Fast-Blue/Cresyl violet (LFB-Nissl; BÖCK, 1989; one section), modified silver stain according to Bielschowsky (BSS; BÖCK, 1989; one section), immunohistochemistry for demonstration of glial fibrillary acidic protein (GFAP; two sections including one control), lectin immunohistochemistry using Bandeiraea simplicifolia (BS-1) for identification of microglia/macrophages (two sections including one control), and the TUNEL-method for identification of apoptotic cells (two sections including one control). According to the locations of lesions in the H&E stained slides from three selected paraffin blocks (block no. 6, 8, 11) four additional serial sections were cut and stained with immunohistochemistry for demonstration of non- phosphorylated neurofilaments (n-NF) to identify axonal damage and normal neurons, phosphorylated neurofilaments (p-NF) to identify chromatolytic neurons, and beta-amyloid precursor protein (β-APP) accumulation to identify axonal damage (spheroids). One section was used as negative control. The number of sections and types of stains applied to each localization are listed in Table 3.2, 3.3, and 3.4.

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3.3 Histochemistry

3.3.1 Luxol-Fast-Blue/Cresyl violet stain

The Luxol-Fast-Blue stain combined with the Cresyl violet method for Nissl´s substance (LFB-Nissl; BÖCK, 1989) results in a bluish color of the myelinated white matter and a violet color of the nuclei and Nissl´s substance. This staining method is used to identify the basic neuronal structure as well as the loss of myelin and Nissl´s substance. The number of sections stained with LFB stain is listed in Table 3.2, 3.3, and 3.4.

Table 3.2 Slide, block number, and applied stain/immunohistochemistry for tissue sections in animal no. 51 - 71 and 151 - 168

Slide no.

Block no.

Stain / Immunohistochemistry

1 1 - 37 H&E

2 2 - 11 H&E

3 2 - 11 H&E

4 2 - 11 H&E

5 2 - 11 H&E

6 2 - 11 LFB

7 2 - 11 BSS

8 2 - 11 GFAP

9 2 - 11 GFAP-control

10 2 - 11 BS-1

11 2 - 11 BS-1-control

12 2 - 11 TUNEL

13 2 – 11 TUNEL-control

14 6, 8, 11 n-NF

15 6, 8, 11 p-NF

16 6, 8, 11 β-APP

17 6, 8, 11 Control

H&E = hematoxylin and eosin staining; LFB = Luxol-Fast-Blue/Cresyl violet-method; BSS = modified silver stain according to Bielschowsky; GFAP = glial fibrillary acidic protein; BS-1 = Bandeiraea simplicifolia-1; TUNEL = terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end-labeling (TUNEL)-assay; control = negative control; p-NF = phosphorylated neurofilament; n-NF = non-phosphorylated neurofilament; β-APP = beta-Amyloid Precursor Protein.

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Table 3.3 Slide, block number, and applied stain/immunohistochemistry for tissue sections of animal no. 349

Slide no. Block no. Stain /

Immunohistochemistry

Notes

1 1-30 H&E

1* 3-7,16,18,24 and 26 LFB

2 3-7,16,17,18,24 and 26 BSS 17 H&E

3 3-7,16,18,24,26 H&E

4 4-7 H&E

6 3-7,16,18,24,26,28 H&E 28 TUNEL

7 18,24,26,28 GFAP 28 TUNEL-control

8 3-7,16 GFAP

9 4-7,16,18,24,26 H&E

12 18,24,26 H&E

13 28 BSS

14 28 LFB

15 26 H&E

16 28 H&E

19 28 H&E

22 28 H&E

25 28 H&E

28 28 H&E

29 28 BS-1

30 28 BS-1-control

14 8,10,12 n-NF

15 8,10,12 p-NF

16 8,10,12 β-APP

17 8,10,12 Control

H&E = hematoxylin and eosin staining; LFB = Luxol-Fast-Blue/Cresyl violet-method; BSS = modified silver stain according to Bielschowsky; GFAP = glial fibrillary acidic protein: BS-1 = Bandeiraea simplicifolia-1, TUNEL = terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end-labeling (TUNEL)-assay; control = negative control, 1* = slide no. 1 was repeated twice; p-NF = phosphorylated neurofilament; n-NF = non-phosphorylated neurofilament; β-APP = beta-Amyloid Precursor Protein.

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Table 3.4 Slide, block number, and applied stain/immunohistochemistry for tissue sections of animal no. 350

Slide no.

Block no. Stain /

Immunohistochemistry

Notes

1 1-30 H&E

1* 1-4,6,8,11,14,20 LFB

2 1-4,6,8,11,14,20 BSS 17 was H&E 3 1-4,6,8,11,13,20 H&E

4 1-3 H&E

5 2 H&E 28 was TUNEL

6 1-4,6,8,11,13,20 H&E 28 was TUNEL-control

7 2,4,6,8,11,14,20 GFAP

8 1-3 GFAP

9 1-4,6,8,11,13,20 H&E

10 2,4 GFAP

11 2 GFAP-control

12 8,11,12,13,20 H&E

13 4,6,8,11,14,20 TUNEL

14 1,3,4,6,8,11,14,20 TUNEL-control 15 1,3,4,6,8,11,14,20 BS-1 16 6,8,11,14,20 BS-1-control

14 2,3,11 n-NF

15 2,3,11 p-NF

16 2,3,11 β-APP

17 2,3,11 Control

H&E = hematoxylin and eosin staining; LFB = Luxol-Fast-Blue/Cresyl violet-method; BSS = modified silver stain according to Bielschowsky; GFAP = glial fibrillary acidic protein; BS-1 = Bandeiraea simplicifolia-1; TUNEL = terminal deoxynucleotidyl transferase [TdT]-mediated deoxyuridinetriphosphate [dUTP] nick end-labeling (TUNEL)-assay; control = negative control; 1* = slide No. 1 was repeated twice; p-NF = phosphorylated neurofilament; n-NF = non-phosphorylated neurofilament; β-APP = beta-Amyloid Precursor Protein.

Protocol of Luxol-Fast-Blue/Cresyl violet staining

1. Deparaffination and rehydration by immersion twice for 5 min. in Rotihisto^® (Roth, Karlsruhe, Germany) and once in isopropanol for 5 min.

2. Rinsing in distilled water for 5 min.

3. Incubation in Luxol-Fast-Blue solution (Luxolechtblau^®, Schmid, Köngen, Germany) at 56^ºC overnight

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4. Rinsing off excess staining solution with 95% ethyl alcohol 5. Rinsing in distilled water for 5 min.

6. Differentiation in 0.1% lithium carbonate solution for approximately 30 sec.

7. Continuing differentiation in 70% ethyl alcohol for 20 - 30 sec. (until the gray matter will be clear)

8. Rinsing in distilled water

9. Microscopic check, if gray matter is clear and white matter sharply defined.

10. Differentiation in 0.05% lithium carbonate solution for approximately 10 - 20 sec.

11. Rinsing off excess staining solution with 70% ethyl alcohol for some sec.

12. Repeating the differentiation steps (step 10 - 11) if necessary (until clear differentiation between gray- and white matter)

13. After completion of differentiation, placing the slides in distilled water

14. Counter staining in the cresyl violet solution for approximately 6 min. at 37°C.

15. Rinsing in distilled water

16. Dehydrating twice in 96% alcohol, clearing in “acetic acid-n-butylester” (EBE^®, Roth, Karlsruhe, Germany), and finally automatic mounting (promountes^® RCM2000, Medite, Burgdorf, Germany)

3.3.2 Bielschowsky's silver stain

The modified silver stain according to Bielschowsky (BSS; BÖCK and ROMEIS, 1989) and the protocol manual for laboratory methods (Laboratory Methods in Histotechnology, Chapter 14, AE Downing, Armed Forces Institute of Pathology, Washington) were used to identify nerve fibers, physiological axonal structures as well as axonal lesions characterized by axonal loss or focal argyrophilic swellings of axons (spheroids). The number of sections stained with modified silver stain according to Bielschowsky is listed in Table 3.2, 3.3, and 3.4.

Protocol of Bielschowsky's silver staining

1. Deparaffination and rehydration by immersion twice for 5 min. in Rotihisto^® (Roth, Karlsruhe, Germany), once in isopropanol for 5 min. and a descending series of alcohols: 96%, 70%, and 50% each for 5 min.

2. Rinsing in distilled water for 5 min.