Combination Therapy Antimalarial Drugs Mefloquine And Artequin Induce Reactive Astrocytes Formation In The Hippocampus Of Rats

The normal adult vertebrate nervous system is a relative quiescent tissue in terms of cell proliferation. However, astrocytes in many region of the central nervous system (CNS) like hippocampus retain the capacity to undergo cell division. Understanding the growth of this glial cell is a key to repairing neurons in response to insult. We studied reaction of astrocytes in the hippocampus after a three day administration of antimalarial agent mefloquine and Artequin to forty-two Wistar rats. The rats received 1.07mg/kg, 2.14mg/kg, 4.28mg/kg of mefloquine and 0.86/1.07mg/kg, 171/2.14mg/kg and 3.24/4.28mg/kg of Artequin. Hippocampal sections of treated rats stained by Hortega’s lithium carbonate method revealed astrocytes stained black as in the control, but hippocampal sections of rats treated with 2.14mg/kg, 4.28mg/kg of mefloquine, and 1.71/2.14mg/kg and 3.24/4.28mg/kg of Artequin showed large, numerous and some few paired astrocytes. We conclude that mefloquine and Artequin induced dose dependant reactive astrocytes formation in the hippocampus. This may impair uptake of neurotransmitters and alter neuronal environment, thus altering hippocampal functions like memory and learning.


INTRODUCTION
In most parts of Nigeria including Calabar, malaria has become a familiar sickness. Health centres are not often patronized rather home/self treatment is the first-line of treatment as antimalarial drugs are purchased at the open markets like other commodities. In addition, sales persons have little or no formal education on the various antimalarial therapies in use, knowledge of side effects of the drug or contradictions and appropriate dosages. The risk of toxicity this way is high mainly from over dose and misuse; this indiscriminate misuse of drug could endanger the life of the vast population 1 .
Antimalarial agents are drugs used in the treatment of malaria infections caused by the Plasmodium species; falciparum, vivax, ovale and malariae. Some of this antimalarial monotherapies include; chloroquine, amodiaquine, mefloquine and artesunate. Mefloquine is a 4quinoline methanol derivative that has been the drug of choice in most regions of the world. Like chloroquine, it is a blood schizonticide, though has long acting half life than any known antimalarial 1 . Over the years, there has been global decline in the use of mefloquine regime due to its documented neurological adverse effects such as neuropsychiatric disorders, forgetfulness and seizure 2 . In 1980 mefloquine was withdrawn from the pharmaceutical market 1 .
The recent trend of malaria therapy with the artemisinin based combination therapy (ACT) such as artemether or artesunate combined with a more slowly eliminated drug has again re-introduced mefloquine combined with artesunate known as Artequin 3  Astrocytes make up half of the cell population in the CNS apart from neurons, they are known to provide neurons with rich nutritional environment for survival. Understanding this glial activity is a clue to neuronal responses. Mefloquine presence in Artequin may indicate a possibility of danger because of the adverse events documented against mefloquine on the hippocampal functions like memory, learning and emotion 6 .
Since self medication is especially common in developing countries. In Africa, this has resulted in indiscriminate use of drug and sometimes at dosage above the therapeutic dose. It becomes important to checkmate drug administration for human safety; this study determines the effect of these drugs on astrocytes in the hippocampus.

ANIMAL CARE
Forty-two Wistar rats weighing 200g were purchased from the animal house of the Department of Anatomy, College of Medical Sciences, University of Calabar, Nigeria. The animals were maintained at temperatures of 30-36 o C and were fed with growers mash obtained from Pfizer Nigeria Limited, twice daily and allowed water ad-libitum. They were cared for in compliance with applicable guidelines for animal research study. The approval of animal study and experiment was obtained from the Ethics Committee of the University of Calabar, Calabar, Nigeria.

DRUG ADMINISTRATION
The mefloquine and Artequin used in this research study produced by Mepha Pharmaceutical Limited, Asch-basal, Switzerland was purchased from the University of Calabar Pharmacy Shop, Calabar, Nigeria. The mefloquine and Artequin were dissolved in 100ml distilled water separately. The drug suspensions was administered to animals based on body weight, the therapeutic dosages for the rats were determined against the therapeutic doses for humans, which is 750mg/kg of mefloquine and 600/750mg/kg of Artequin. These suspensions were administered orally with the aid of orogastric tubes. The rats were divided into groups as shown in Table 1 below.
The animals in all the groups were sacrificed on day four. The skulls was dissected and the whole brains were removed and preserved in neutral formal saline for seven days, thereafter the hippocampus were dissected by first removing the cerebellum and brainstem, then scrapping the inferior aspects of the dorsum of cerebrum on either side revealing the entire extent of the hippocampus. They were removed, processed and stained using the Hortega's lithium carbonate method for reactive astrocytes. Photomicrographs were taken using the research microscope.

RESULTS
In control sections, there were distinct astrocytes stained black. They were few in number and unpaired (Plate 1). The sections of the hippocampus of rats treated with 1.07mg/kg of mefloquine showed many astrocytes that were unpaired compared to the control (Plate 2). The sections of the hippocampus of rats treated with 2.14mg/kg of mefloquine revealed many unpaired astrocytes that were densely stained with few paired ones (Plate 3). Sections from the hippocampus of rats treated with 4.28mg/kg of mefloquine showed many paired astrocytes deeply stained (Plate 4).
The sections from the hippocampus of rats treated with 0.86/1.07mg/kg of Artequin revealed astrocytes that were numerous and unpaired (Plate 5). Sections of hippocampus of rats treated with 1.71/2.14mg/kg of Artequin revealed many paired astrocytes while the group that received 3.42/4.28mg/kg of Artequin showed highly numerous and few paired astrocytes (Plates 6-8).

Figure 2
Photomicrographs of hippocampal section of rats given mefloquine and their control  Photomicrographs of hippocampus of rats treated with Artequin and their control

DISCUSSION
Astrocytes are star shaped glial cells in the CNS. Two forms of astrocytes exist in the mammalian brain, these are the fibrous astrocytes related to the white matter having small cell bodies with long processes and protoplasmic astrocytes that have more frequent processes which are shorter and thicker than the processes seen in the fibrous astrocytes. These astrocytes contain multitude of small, round, dark glycogen granules that make astrocytes appear dark when stained 7 . They provide metabolic support for neurons in the CNS and synaptic means of communication within the brain 8 .
They regulate ionic concentration, for tight junctions, blood-brain barrier and serve as intermediary stations for converging nutrient, gases and removal of waste products between neuron 9 . Astrocytes exert strong influences on neurons during synaptic transmission through modulation of their volume, composition and concentration of neurotransmitter, glutamate and ATP receptor 8 .
Peter et al 8 9 showed that in CNS injury astrocytes acts as neuroprotective sheath, they increase in number, fill injury zone, forming glial scar to fill defects left by loss of specialized nervous tissues. They help in healing and recovery of neurons such are called reactive astrocytes. Abbas and Nelson 10 reported that the presence of reactive astrocytes indicates early sign of cell loss and serve as indicator of pathologic process. Thus the activity of mefloquine and Artequin on astrocytes might suggest possible stages of neuronal deranged activity or loss, which could also explain the neurological effects of mefloquine therapy on the hippocampus such as forgetfulness and seizure.
In response to glutamate and adenosine receptor stimulation astrocytes could form swelling leading to increased size of the astrocytes 8 . In this study findings revealed large and paired astrocytes in rats treated with 2.14mg/kg, 4.28mg/kg of MQ and 3.24/4.28mg/kg of Artequin per body weight when compared to the control indicating hyperplasia and hypertrophy. Peter et al 8 showed that in CNS injury zone, forming glial scar to fill defects left by loss of specialized nervous tissues. They help in healing and recovery of neurons such astrocytes are called reactive astrocytes. Abbas and Nelson 10 reported that the presence of reactive astrocytes indicates early sign of cell loss and serve as indicator of pathologic process. Thus the activity of mefloquine and Artequin on astrocytes might suggest possible early sign of cell loss and serve as indicator of pathologic process. Thus the activity of mefloquine and Artequin on astrocytes might suggest possible early stages of neuronal deranged activity or loss, which could also, explain the neurological effects of mefloquine therapy on the hippocampus such as forgetfulness and seizure.
In respond to glutamate and adenosine receptor stimulation astrocytes could form swellings leading to increased size of the astrocytes 8 . In this study findings revealed large and paired astrocytes in the rats treated with 2.14m/kg, 4.28mg/kg of MQ and 1.71/2.214mg/kg and 3.24/4.28mg/kg of Artequin. Growing evidence indicate that trophic action of extracellular nucleotides are involved in CNS development, injury and repairs for example ATP released upon CNS injury contributed to the formation of reactive astrocytes 11 . Fibroblast growth factor (FGF) is also increased after injury can stimulate astrocytes proliferation 10 . This showed that proliferation is one of the features of reactive astrogliosis particularly in traumatic injury. They also confirmed that hypertrophy could be due to accumulation of intercellular substances. These changes in normal astrocytes morphology and population could impact new functional state which might impair astrocytes activity like uptake and metabolism of monoamine and amino acid neurotransmitters.
Hippocampal hypoxia have also caused astrocytes to become reactive and hypertrophied as seen in most mental disorders and spatial loss of memory during seizure, it is also implicated in epileptogenesis 12 . In addition apart from injury, cell death, mechanical alteration of tissue structure and components also stimulated cell proliferation while certain inhibitory or excitatory signal from the microenvironment have been indicated 8 .

CONCLUSION
Our findings showed a dose dependent pattern of alteration and pairing of astrocytes observed in rats treated with increased dosages of mefloquine and Artequin (2.14mg/kg, 4.28mg/kg of MQ and 1.71/2.214mg/kg and 3.24/4.28mg/kg of Artequin). Altered neuronal environment may be implicated in the effects of these drugs on the hippocampus whose main functions include memory, learning, spatial navigation and emotion. The increase population of reactive astrocytes formation is meant to protect neurons by providing nutritionally rich environment for neuronal survival following possible adverse effects produced by these drugs. The effects of these drugs on astrocytes appeared to be dose dependent and similar. We therefore conclude and recommend that Artequin which is a new artemisinin combination therapy (ACT) drug may affect the function of the hippocampus and should be administered