Drug administration commenced on day 8 after inoculation. Two Camosunate tablets® (100mg of Artesunate + 300mg of Amodiaquine) and one P-Alaxin tablet® (40mg of Dihydroartemisinin + 320mg of Piperaquine) were powdered separately in a mortar, mixed with 20ml of distilled water and administered in mg/kg body weight as recommended by the World Health Organization (2015b).
Drugs were administered as aqueous suspensions with oral gavage using the formula;
Weight of animal(kg) X Dosage (mg/kg) Concentration of drug (mg/ml)
The drug suspension was continuously agitated in order to deliver the drugs homogeneously to the animals. This experiment was to mimic the dose and duration of administration of the drugs in human as recommended by the World Health Organization (WHO, 2015b). Drug administration started at day 8 after the establishment of parasitemia, as follows:
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Table1: Experimental design for drug administration in the Plasmodium parasitized mice
GROUPS TREATMENT DOSAGE (mg/kg).
Group A Normal saline Infected control Group B Artesunate+Amodiaquine 4/10mg/kg for 3 days
Group C Dihydroartemisinin+Piperaquine 4/18mg/kg for 3days
Group D Artesunate+Amodiaquine 4/10mg/kg for 3days, allowed
to recover for 28 days Group E Dihydroartemisinin+Piperaquine 4/18mg/kg for 3days, allowed
to recover for 28 days Group F Normal Saline Uninfected control
Animals in groups D and E were used to study recrudescence and reversibility of antimalaria drug-induced toxicity in P. berghei infected mice.
3. 8. Data Collection 3. 8.1. Weight Assessment
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As an index of the physical status of the animals, the weight of each animal was monitored over the period of the study. The animals were weighed before and after drug administration using Electronic weighing balance (Labtech; model; BL7501;
Range: 750g - 0.1g)
3. 8.2 Collection of blood samples
After 24 hours, blood samples were collected from all the experimental groups After 28 days post - recovery, blood samples were also collected from animals in groups, D and E for haematological investigation. Blood samples were collected through retro – orbital sinus with a capillary tube into ethylenediaminetetraacetic acid (EDTA) tubes.
3. 9. Estimation of haematological parameters:
Blood was collected from the animals in all the groups and assayed for White blood cell count, White blood cell differential count, Red blood cell count and Haemoglobin concentration using standard methods.
3. 9.1 White blood cell count
White blood cell count was estimated with haematocytometer using the principle of a calibrated capillary tube for blood sampling described by Baker et al (1998).
Briefly, 950μl of diluting fluid was dispensed into a small clean dry test tube using micropipette, then, 50μl of well mixed EDTA anticoagulated blood was added to the diluting fluid in the test tube. The diluted sample was then mixed and loaded
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into the improve Neubauer counting chamber. The white cells present in the 4 corners of the chamber were counted and recorded.
3. 9.2 White blood cell differential count
A drop of blood was taken from the tail of the infected mice and placed at a distance of about an inch from one end of the microscopic slide and dispersed along the length of the slide width with another slide, the thin film was air-dried and fixed with methanol for 3 seconds, air-dried and then stained with field stain A and B for 6 seconds. The slides were washed, drained and air-dried. Lymphocytes, neutrophils, eosinophils, basophils and monocytes were identified and counted under the 100x objective lens.
3. 9.3 Estimation of Packed Cell Volume (PCV)
Packed cell volume was estimated using centrifugation method of Dacie and Lewis (1984). A small volume of blood was collected from the tip of the animal tail (tail tip amputation) into a heparinized capillary tube, about three-quarters of the tube. The capillary tubes were sealed with sealant and placed in the numbered slots of the haematocrit centrifuged with the sealed end against the rim, then, spun at 10,000rpm for 5 minutes to separate the blood into plasma and packed cells.
Immediately after centrifuging, the packed cell volume was read using the microhaematocrit reader.
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3. 9.4 Red Blood Cell count
Red blood cell count was estimated with haematocytometer using the principle of a calibrated capillary tube for blood sampling described by Baker et al (1998).
Briefly, 4ml of red blood cell diluting fluid was drawn with a Pasteur pipette and then transferred into a small, clean and dried test tube. Then, 20μl of blood was drawn with a micropipette and then added to the tube, the diluted blood was mixed gently, 10μl was withdrawn from the diluted blood and then loaded into the counting chamber. When the cells have settled out of suspension, the number of red blood cells lying in the 5 of the 0.04 mm2 areas were counted and recorded.
3. 9.5 Estimation of Haemoglobin concentration
Haemoglobin concentration was estimated by Sahli‟s haemoglobinometric method.
Hydrogen chloride was placed in diluting tube up to the lowest mark; 0.02ml of well mixed whole blood was dispensed with micropipette into diluting tube and mixed. The diluted haemoglobin was allowed to sit at room temperature for 10 minutes. After 10 minutes, distilled water was added in drops and mixed until it has exactly the same colour with comparism standards.