Biology Reference
In-Depth Information
3.3
Follow-Up
1. From day 0 to day 12, record body weight and clinical presen-
tation every day before and after treatment.
2. In order to evaluate the clinical presentation of cerebral malaria,
use the classification in clinical stages (from 0 to 4) named
BIENVENU score described below (
8
):
Stage 0: healthy mice
Stage 1: symptoms not associated with cerebral malaria
(ruffled fur)
Stage 2: ruffled fur and motor impairment
Stage 3: respiratory distress
Stage 4: convulsions and/or coma
3.4
Parasitemia
From day 4 to day 12, measure parasitemia of each mouse as
follow.
1. Collect blood in the using tail vein a 1 mL sterile syringe with
0.33 × 12 mm needle.
2. Take a drop of blood and place it on a slide.
3. Spread the drop of blood to obtain a thin film.
4. Dry the slide.
5. Fix in Diff Quick Fixative (or methanol) for 20 s.
6. Stain with Diff Quick solution I for 20 s.
7. Counterstain with Diff Quick solution II for 30 s.
8. Rinse in tap water to remove excess stain.
9. Use one drop of oil to watch the slide at immersion ×1000.
10. Calculate the number of parasitized erythrocytes per 100
erythrocytes.
11. Experiment end-point is day 12 post-infection and is defined
as the day that at least 90% of the control group mice are dead.
3.5 Statistical
Analysis
Calculate cumulative long-term survival according to the Kaplan-
Meier method. Compare groups with the log rank test. Survival
time is the dependent variable.
p
-value of <0.05 is considered
significant. From the last day of treatment (day 8 post-infection),
cumulative long-term survival analyses are performed every day.
3.6 Expected
Biological and Clinical
Presentation
Biological and clinical presentation of mice post-infection without
treatment is expected as follow (Table
1
, Fig.
2
):
Day 3: positive parasitemia.
●
Day 4: increased parasitemia not yet associated with clinical
●
signs.
Day 5: most mice with ruffle fur (first stage) related to fever.
●
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