ABSTRACT

Malaria continues to constitute a major public health burden, particularly in sub-Saharan Africa, where Artemisinin-based Combination Therapies (ACTs) remain the recommended first-line treatment for uncomplicated malaria. Among these therapies, the combination of artesunate (AS) and amodiaquine (AQ) has demonstrated remarkable antimalarial efficacy and has significantly contributed to reducing malaria-related morbidity and mortality. Despite its therapeutic success, emerging scientific evidence suggests that prolonged or delayed neurological consequences may occur following exposure to ACTs, particularly involving brain regions responsible for learning, memory, motor coordination, and cognitive processing. However, available findings remain inconsistent, and limited information exists regarding the delayed neurotoxic effects of acute artesunate-amodiaquine administration on the hippocampus and cerebellum.

This study investigated the delayed neurobehavioral, biochemical, hematological, histopathological, and immunohistochemical effects of acute administration of artesunate and amodiaquine, administered individually and in combination, on the hippocampus and cerebellum of adult Wistar rats. Eighty healthy adult male Wistar rats weighing approximately 120 ± 5 g were randomly assigned into four experimental groups (n = 20). Group A served as the control and received distilled water, Group B received artesunate (4 mg/kg), Group C received amodiaquine (10 mg/kg), while Group D received a combination of artesunate (4 mg/kg) and amodiaquine (10 mg/kg). Treatments were administered orally once daily for three consecutive days, after which the animals were observed for an additional fourteen-day post-treatment period to evaluate delayed neurotoxic manifestations.

Body weight was monitored throughout the experimental period, while cognitive and motor functions were assessed using the Morris Water Maze and Forelimb Grip Strength Test, respectively. Animals were sacrificed on Days 4 and 15 following treatment. Blood samples were collected for hematological evaluation, including complete blood count analysis. Excised hippocampal and cerebellar tissues were subjected to biochemical analyses of oxidative stress markers, including lipid peroxidation and nitric oxide production. Histological examination using hematoxylin and Eosin staining, together with immunohistochemical assessment of Glial Fibrillary Acidic Protein (GFAP) and Cyclooxygenase-2 (COX-2), was performed to determine structural and inflammatory alterations. Statistical analysis was conducted using one-way analysis of variance (ANOVA) with significance established at p < 0.05.

The findings demonstrated that no statistically significant alterations were observed across body weight, behavioral performance, hematological indices, oxidative stress biomarkers, or brain histomorphology immediately following the three-day treatment regimen. However, significant delayed effects became evident fourteen days after treatment cessation. Rats treated with amodiaquine exhibited marked body weight reduction and impaired spatial learning, as reflected by prolonged escape latency in the Morris Water Maze. Both artesunate- and amodiaquine-treated groups demonstrated significant reductions in forelimb grip strength, indicating compromised motor coordination. Hematological analysis further revealed elevated red blood cell count and packed cell volume in the Amodiaquine-treated animals.

Biochemical investigations revealed significant increases in lipid peroxidation and nitric oxide concentrations within the hippocampus and cerebellum of treated animals, suggesting enhanced oxidative stress and free radical generation. Histopathological examination showed significant degeneration and reduced populations of Purkinje cells within the cerebellum, alongside neuronal loss within the Cornu Ammonis 1 (CA1) region of the hippocampus, characterized by cellular pyknosis and neurodegenerative changes. Immunohistochemical analysis demonstrated marked overexpression of GFAP, indicating reactive astrogliosis, while increased COX-2 immunoreactivity within the hippocampus suggested activation of neuroinflammatory pathways.

Overall, the study establishes that although short-term administration of artesunate and amodiaquine does not produce immediate neurological abnormalities, delayed neurotoxic effects become evident following treatment withdrawal. These alterations are characterized by cognitive impairment, motor dysfunction, oxidative stress, neuronal degeneration, astrocyte activation, and neuroinflammation. The findings provide important experimental evidence supporting the possibility of delayed central nervous system toxicity following acute exposure to artesunate and amodiaquine and underscore the need for further mechanistic investigations and long-term neurotoxicity surveillance of artemisinin-based combination therapies.

CHAPTER ONE

INTRODUCTION

1.1 Background to the Study

Malaria remains one of the world's most devastating infectious diseases and continues to pose a major public health challenge, particularly in tropical and subtropical regions. According to the World Health Organization, hundreds of millions of malaria cases are reported annually, with sub-Saharan Africa accounting for the overwhelming majority of infections and malaria-related deaths. Nigeria bears one of the highest global malaria burdens, making effective antimalarial therapy indispensable for disease control and reduction of mortality.

The emergence and widespread distribution of drug-resistant Plasmodium falciparum strains led to the introduction of Artemisinin-based Combination Therapies (ACTs) as the recommended first-line treatment for uncomplicated malaria. Among the various ACTs, the combination of artesunate and amodiaquine has gained widespread acceptance because of its rapid parasite clearance, high therapeutic efficacy, affordability, and ability to delay the development of drug resistance. Consequently, the combination has become one of the most frequently prescribed antimalarial regimens in Nigeria and several malaria-endemic countries.

Although artesunate and amodiaquine have demonstrated remarkable effectiveness in malaria treatment, increasing experimental and clinical evidence indicates that antimalarial drugs may produce adverse effects beyond their intended therapeutic actions. In recent years, attention has shifted toward understanding the possible neurological consequences associated with repeated or delayed exposure to these medications. Since both drugs possess the ability to cross the blood-brain barrier to varying degrees, concerns have emerged regarding their influence on neuronal integrity, cognitive function, and motor coordination.

The central nervous system, particularly the hippocampus and cerebellum, is highly susceptible to oxidative stress and inflammatory injury. The hippocampus plays a fundamental role in learning, memory formation, spatial navigation, and cognitive processing, whereas the cerebellum is essential for maintaining posture, balance, muscle coordination, and motor learning. Damage to these brain regions may result in cognitive deficits, impaired motor performance, memory disturbances, and long-term neurological dysfunction.

Experimental studies have suggested that artesunate and amodiaquine may induce oxidative stress by increasing the production of reactive oxygen species, lipid peroxidation products, and inflammatory mediators. Excessive oxidative stress may overwhelm endogenous antioxidant defenses, resulting in neuronal membrane damage, mitochondrial dysfunction, DNA injury, and activation of apoptotic pathways. Furthermore, neuroinflammatory responses characterized by astrocyte activation and elevated inflammatory enzymes such as cyclooxygenase-2 (COX-2) may contribute significantly to neuronal degeneration.

Reactive astrocytes express increased levels of Glial Fibrillary Acidic Protein (GFAP), which serves as an important biomarker of neuroinflammation and central nervous system injury. Elevated GFAP expression has been associated with neuronal stress, astrocyte proliferation, and tissue remodeling following toxic or inflammatory insults. Similarly, increased COX-2 activity has been implicated in inflammatory-mediated neuronal damage and progressive neurodegeneration. Assessment of these biomarkers therefore provides valuable insight into the underlying mechanisms of drug-induced neurotoxicity.

Despite the extensive clinical use of artesunate-amodiaquine combination therapy, available literature presents conflicting reports concerning its neurological safety. While several studies have reported minimal adverse neurological effects following short-term administration, others have documented evidence of neuronal degeneration, oxidative stress, behavioral abnormalities, and structural alterations within the hippocampus and cerebellum. Moreover, most previous investigations have focused primarily on immediate or acute toxicological outcomes, with relatively little emphasis placed on delayed neurotoxic manifestations following treatment cessation.

Understanding whether neurological injury develops immediately after treatment or emerges during the recovery period has important implications for patient safety, clinical monitoring, pharmacovigilance, and future drug development. Delayed neurotoxicity may remain clinically undetected despite the apparent resolution of malaria infection, potentially contributing to subtle cognitive or behavioral impairments that become evident only after treatment has been completed.

Therefore, this study seeks to comprehensively evaluate the delayed neurobehavioral, biochemical, histopathological, hematological, and immunohistochemical effects of acute artesunate and amodiaquine administration on the hippocampus and cerebellum of Wistar rats. Findings from this investigation are expected to contribute significantly to existing knowledge regarding the neurological safety profile of artemisinin-based combination therapies and provide experimental evidence for improved pharmacological monitoring and safer antimalarial treatment strategies.

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