Experimental splenectomies and malaria in mice

Purpose: To evaluate the importance of spleen in malaric infection in murino model, comparing the parasitemia and the titles of imunoglobulins in the different groups. Methods: It was used female mice non-isogenic, in inoculated with Plasmodium berghei, cepa ANKA, intraperitoneally. The parasitemia was analyzed in 23rd, 25th, 27th and 32nd day of the experiment, being the stained blood’ exam colored by Giemsa. The titles of the total serum immunoglobulins IgM and IgG were analyzed by Dot-ELISA technique, at 6th, 22nd and 32nd day, when the animals were sacrificed. Results: The parasitemia was gradual in all the inoculated groups. In the end of the experiment, the animals with partial parasitemia present superior parasitemia, but next to the non-splenectomized, while the asplenics present difference bigger than the double. The levels of total serum IgM and IgG didin ́t have significant changes with the removal partial or total splenic. Conclusion: The techniques conservatives in splenic trauma are possible and necessary. The importance of remaining spleen in the clearance of red blood cells parasitized by Plasmodium berghei showed being efficient, in order to avoid serious complications resulting of the malaria in mice.


Introduction
Trauma is a severe public health issue across the world, one that has had a major impact on human mortality attributed to external causes.Currently, trauma is the third cause of mortality in the world 1 .The epidemiological situation in Brazil is even worse: trauma it is the second leading cause of death, and the leading cause in the first four decades of life; 2 therefore, it represents a huge and growing challenge to the country in both economic and social terms.
The external causes are on the rise in Brazil.The program that addressed emergency and trauma of the Ministry of Health 3 reported, at the time, 100,000 deaths per year 3 .At the end of the 20th century, around 124,000 deaths were attributed to trauma 4 and this rose to 150,000 in 2004 2 .Hundreds of thousands of those who suffer trauma have permanent sequelae and represent a socially marginalized group.
The spleen is one of the organs most frequently injured in closed abdominal traumas, especially in children.Spleen injuries frequently result from penetrating wounds on the left side of the body, of the transition thoracic-abdominal and of the back.The most common cause of splenic injury is the closed trauma produced by an automobile accident; such injuries have predominant conduct in the lacerations the total splenic ablation 5 .Asplenic patients are more susceptible to severe infections by encapsulated bacteria 5 and malaria 6 .
Despite significant technological and scientific developments, malaria remains a major public health problem.According to the World Health Organization, it is the more concerning tropical disease because of the high rate of mortality of the illness; in addition, it is a cause of many socio-economic problems.It is estimated that 40% of the world's population live in areas at risk of malaria transmission in more than 100 countries 7 .According to the Department of Health Surveillance of the Ministry of Health (2003), in Brazil, there are 600,000 cases of malaria per year, mainly in the Amazon Basin, where 99.5% of the cases are recorded 8 .
It is remarkable that a person can survive after an important body organ like the spleen is removed.A full splenectomy generally does not involve severe complications, suggesting the presence of compensatory mechanisms for the loss of several splenic functions 9 .However, many effects of total ablation of the spleen are reported, mostly related to the functioning of the secondary lymphoid organ that is responsible for immune response to the stock antigen circulate 10 .A total splenectomy was reported to reduce the levels of immunoglobulins in serum, especially the IgM 11 .The removal of the spleen had effects on other organs, particularly the liver 9 .It appeared that after the total ablation of spleen, the patients die in greater numbers and earlier than the expected age of the general population, not only as a result of severe infections, but also due to pulmonary embolism, acute myocardial infarction 12 and atherosclerotic phenomena 13 .
Partial splenectomies have been done since the beginning of the nineteenth century, but only after the studies by Campos Christo 14 was the surgical procedure done based on segmental and terminal vascularization.The preservation of the upper pole's spleen, after ligation of the vascular pedicle was proposed by Petroianu 17 .This has been studied both clinically and experimentally in several works, and has been done successfully since 1984, because it provides the spleen with twice the area of irrigation 11 .
In the present study, we assessed the effects of partial and full splenectomies in mice infected by Plasmodium berghei.

Methods
This was an experimental prospective study with a longitudinal cohort design in mice (Mus domesticus domesticus), a Swiss house mouse, to evaluate the action of Plasmodium berghei in splenectomized (full and partial) and non-splenectomized animals.The study was conducted according to the Ethical Principles of the Brazilian College of Animal Experiments -COBEA and was approved by the Ethics Committee on the Use of Animals (CEUA) of the School of Medicine of Federal University of Amazonas, on 16 April 2008.
Twenty female mice were used; the mice were nonisogenic, age four to five weeks, and weighed between 15 to 20 grams.The animals were provided by the vivarium of Physiological Sciences Department at the Biological Sciences Institute, Federal University of Amazonas (UFAM).The animals went through an acclimation period of seven days in polypropylene cages with wood shavings, mineral water and ration Labina® autoclaved, at room temperature (25ºC) with light-dark cycles of 12 hours.
The animals were separated into four groups of five and procedures performed with each group as described below: Group I: No intervention.
Group II: Anesthesia, laparotomy, inoculation of contaminated blood.

Surgical procedures
On the eighth day of the trial, groups II, III and IV were submitted to pre-anesthesia with atropine sulphate (0.045mg/kg to 0.025%, intramuscular) fifteen minutes before anesthesia.The animals were anesthetized with a combination of ketamine hydrochloride (60mg/kg to 5%, intramuscular) and xylazine hydrochloride (16mg/kg to 2%, intramuscular).This was done with anti-sepsis and sterilized, median laparotomy and spleen exposure.At the end of surgery, the abdominal wall was closed.
In Group III, after exposure of the spleen, the ligation of corresponding vessels was done for the segment to be resected; the spleen tissue was sutured with U-shaped stitches and 50% of the spleen was removed.In Group IV, the ligation of the main pedicle and of the main spleen-gastric vessel was held and organ was subject to total ablation.
At the end of surgery, the mice received subcutaneous hydration with 2mL of sodium chloride 0.9% in the areas of the dorsolateral neck.The animals were then placed on Mayo's tables covered with doubles fields and incandescent surgical lamps to raise the temperature to around 30ºC during post-anesthetic recovery.The animals were evaluated twice at the end of surgery, after 30 and 60 minutes.

Infection by plasmodium berghei
On the 20th day of the experiment, 10 6 parasitized red blood cells were inoculated with Plasmodium berghei, the ANKA strain.The inoculation was done intraperitoneally in the left lower quadrant for the animals in groups II, III and IV.

Tracking the parasitemia level
The evolution of the parasitemia level in the mice was done using a hemoscopic survey (blood smear) on the 23rd, 25th, 27th, 29th and 32nd day.After taking the smear, it was fixed with 100% methanol and stained using the Giemsa method.The hemoscopic examination was performed using a microscope optical, with 1000x increases and analysis of ten fields.The level of parasitemia was determined using the ratio of red blood parasitized cells to the total number of red blood cells counted.

Dosage of immunoglobulins
On the 6th, 22nd and 32nd day, blood samples of 200µL were collected from the venous plexus retro-eye of all the mice in the study.The samples were centrifuged on low and serum was stored at a temperature of -20ºC.The titles of total serum immunoglobulin class M and G were titulated using the Dot-ELISA technique described by Towbin and Gordon 15 .The technique consists in two micro-liters of each serial dilution of sera (1:16 until 1:262144) applied at membranes of nitrocellulose 0,45µm.After drying at room temperature, the membranes were immersed in blocking solution containing 5% of skimmed milk dissolved in buffer Tris-Saline (TBS) pH 7.5 for two hours at room temperature.After the blocking, the membranes were placed in the presence of conjugated: anti-IgG Fc mouse region marked with peroxidasis or an anti-IgM chain of µ marked with peroxidasis, diluted 1:1000 in TBS pH 7,5 for one hour, at room temperature.Soon after, the membranes were washed four times with saline phosphate buffer (PBS) pH 7.2 and the antibodyconjugated reactions revealed with five milligrams of the substance chromogen 3'3' Diaminobenzidine diluted in 30 mL of TBS of pH 7,5, in the presence of the peroxidasis substrate, the hydrogen peroxide PA, to 0.015%.The reaction of FIGURE 1 -Parasitemia's curve of the different groups during the experiment P.S.: Student's t-test, paired by day: Group 2 X Group 3: p= 0,0183; Group 2 X Group 4: p= 0,0155; Group 3 X Group 4: p= 0,0170 coloring by precipitation of DAB was interrupted by adding distilled water and the membranes were then placed between two sheets of filter paper for drying.The immunoglobulin titles present in the sera of mice were obtained by the visual reading of end point reactions.For statistical analysis, the titles of immunoglobulins were presented using a neperian logarithm.

Animal euthanization
The animals were euthanized by applying three times the dosage of anesthesia through intramuscular injections.

Statistical analysis
The correlation of the data and the statistical analysis were performed (or done) by taking the averages of the groups and adding or subtracting the standard deviation of the average number of experiments.The results were analyzed by the statistical method considered by ANOVA and Student's t-test, using the program GraphPad Prism® 3.0.Differences were considered significant when the probability was less than 5% (p <0.05).

Results
The parasitemia of animals was progressive in all groups inoculated by Plasmodium berghei (Figure 1).At the end of the experiment (on day 32nd), the parasitemia of the group of non-splenectomized animals (Group II) was 31.04%; the group of There was no difference among the titles of immunoglobulin of the total serum of classes M and G (p>0.05) among groups in the study during the days upon which blood was collected (Figures 2 and 3).

Discussion
The spleen performed a fundamental role in the clearance of Plasmodium berghei 12 , through the removal of red blood cells infected with the parasite 14 .The maintenance of the portion of spleen tissue was satisfactory in controlling parasitemia, because the mice subjected to partial splenectomy presented a level of parasites similar to that of non-splenectomized mice, while the animals subjected to full splenectomy had twice the amount of circulating parasites (Figure 1).This corroborated previous studies indicating that the asplenia hampers control of parasitemia 11 .
The animals without spleens also present deficiencies in the production of IgM, due to sharp decrease of B residents lymphocytes, especially those in the area surrounding the spleen 16 .In the samples collected on the 6th, 22nd and 32nd days of the trial, no significant changes (p>0.05) were observed for the titles of IgM in any of the four groups.The titles of IgM detected on the last day of the trial (day 32nd) show that the mice inoculated and animals treated with partial splenectomy (Group III), 35.01%, and 63.22% (p<0.05) for the group of completely splenectomized animals (Group IV).The average parasitemia among the groups was statistically significant (G3xG4: p = 0.0183; G3xG5: p = 0.0155 and G4xG5: p = 0.0170, paired by day Student's t-test ).not subjected to splenectomy (Group II) displayed a decrease of title of IgM when compared to the individuals of control group (Group I), but this difference was not significant (p>0.05).In mice inoculated and subjected to partial splenectomy (Group III), despite the fact that the level of parasites had been similar to that of Group II, the levels of immunoglobulins were lower on the 32nd day (p>0.05),probably due to the decrease of B lymphocytes as described by WARDEMANN et al. 16 .However, on the 6th day the titles of IgM were higher in the animals of Group III (p>0.05).In the mice inoculated and subjected to full splenectomy (Group IV), the parasitemia was exacerbated, indicating the inability to fully eliminate parasites.However, although the difference is not significant (p>0.05), a small increase of the titles IgM was observed on the 32nd day for Group III, and, on the same day, the titles of IgG were increased in relation the other groups.It is worth noting that the immunoglobulins present in the plasma of Group IV animals were not specific to Plasmodium berghei, because they were not effective in neutralizing the circulating parasites.The results show that levels of total serum IgM and IgG did not change with partial or total removal of the spleen.These levels may be reached by secondary lymphoid organs or even by the liver which would again perform the immune function, as it does in the fetal stage.

Conclusion
The use of conservative techniques aimed at partial or full preservation of the spleen should always be sought given the importance of the spleen in clearing red blood cells parasitized by Plasmodium berghei.This could prove key to preventing serious infections arising from malaria.