The online version of this article (doi:10.1186/1475-2875-11-166) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
KD participated in the design of the study, performed the animal experiments, optimized the luminescence technique, analysed and interpreted the data, performed the statistical analysis and drafted the manuscript. NL participated in Hz analysis. SN operated the microscope and designed the script for the densitometric analysis of Hz on pictures from organ cryosections. EM participated in optimizing the luminescence technique. GO participated in study design, manuscript writing and provided critical support. PVDS conceived and participated in the design of the study, optimized the method for densitometric analysis of Hz in cryosections and participated in interpreting the data and drafting the manuscript. All authors read and approved the final manuscript.
Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against life-threatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intra-erythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivo immunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence.
An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6 J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann–Whitney U test and Pearsons correlation analysis.
Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas sub-nanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels.
The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.
Additional file 1: Effect of perfusion on the organ-specific haemozoin content. To investigate the effect of perfusion on the organ-specific haemozoin content, mice were infected intraperitoneally with 104Pb NK65 parasites. Ten days later, mice were sacrificed, the right lung and kidney were pinched off, and the other organs were perfused with phosphate-buffered saline to remove circulating erythrocytes. The amount of Hz/mg tissue was quantified in both perfused and non-perfused lungs and kidneys with the modified 96-well plate based haem-enhanced luminescence assay. The total Hz content in lung and kidney was calculated by multiplying the amount of Hz/mg tissue with the organ weights. As shown in panel A and B, no difference was found in the amount of Hz in lungs and kidneys with or without perfusion. Nevertheless, perfusion was applied for all organs tested as it is conceptually more rational to remove circulating infected erythrocytes as a source of haemozoin. Each dot represents data of an individual mouse. (PDF 77 kb) (PDF 77 KB)12936_2011_2231_MOESM1_ESM.pdf
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- Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice
Philippe E Van den Steen
- BioMed Central
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