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Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

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Haemozoin detection by densitometric analysis. Transmitted light images (grey scale) were taken from unstained 7 μm thick cryosections from livers of uninfected mice (A) and from mice infected with PcAS (B) or PbNK65 (C), from PcAS-infected spleens (D), PbNK65-infected lungs (E) and kidneys (F). In panel G, the relative densitometric value obtained from cryosections of gelatin blocks with different concentrations of sHz were analyzed and the formula used to calculate the relative quantity of Hz/μm2 is shown. In panel H, the relative Hz content was measured in liver sections from uninfected (Con), PcAS and PbNK65-infected mice ten days post-infection. Each dot represents the result from an individual mouse. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001
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Figure 1: Haemozoin detection by densitometric analysis. Transmitted light images (grey scale) were taken from unstained 7 μm thick cryosections from livers of uninfected mice (A) and from mice infected with PcAS (B) or PbNK65 (C), from PcAS-infected spleens (D), PbNK65-infected lungs (E) and kidneys (F). In panel G, the relative densitometric value obtained from cryosections of gelatin blocks with different concentrations of sHz were analyzed and the formula used to calculate the relative quantity of Hz/μm2 is shown. In panel H, the relative Hz content was measured in liver sections from uninfected (Con), PcAS and PbNK65-infected mice ten days post-infection. Each dot represents the result from an individual mouse. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001

Mentions: Cryosections with a thickness of 7 μm were prepared from frozen livers, spleens, lungs and kidneys and imaged by light microscopy. Transmitted light images were taken through a 20x/0.8 Plan-Apochromat objective of an Axiovert 200 M microscope equipped with an AxioCam MRm camera (Zeiss, Göttingen, Germany). For Hz quantification on liver sections, images were obtained from two rows of three consecutive fields. The densitometric analysis was performed with the AxioVision 4.6 software with a home-written script and the relative quantity of Hz/μm2 was calculated with the formula as shown in Figure 1G. The densitometric value (DV) of a pixel reflects the intensity of transmitted light at this position in the section. The densitometric background (DB) was determined in each picture by calculating the mean DV of all pixels with a DV above an empirically determined threshold. To test the linearity of the densitometric method to measure Hz on cryosections, gelatin blocks containing different concentrations of synthetic Hz (sHz) were analysed. sHz was prepared as described previously [22] and was homogenized and added in different concentrations to a 10% gelatin-PBS solution at 37°C in a 24-well plate. Upon solidification on ice (to prevent sedimentation of sHz), sHz-containing gelatin blocks were cut out of the wells, embedded in O.C.T and frozen at -80°C. For each concentration, five to six images were analysed from one section/sHz block and this was done for two blocks.


Improved methods for haemozoin quantification in tissues yield organ-and parasite-specific information in malaria-infected mice.

Deroost K, Lays N, Noppen S, Martens E, Opdenakker G, Van den Steen PE - Malar. J. (2012)

Haemozoin detection by densitometric analysis. Transmitted light images (grey scale) were taken from unstained 7 μm thick cryosections from livers of uninfected mice (A) and from mice infected with PcAS (B) or PbNK65 (C), from PcAS-infected spleens (D), PbNK65-infected lungs (E) and kidneys (F). In panel G, the relative densitometric value obtained from cryosections of gelatin blocks with different concentrations of sHz were analyzed and the formula used to calculate the relative quantity of Hz/μm2 is shown. In panel H, the relative Hz content was measured in liver sections from uninfected (Con), PcAS and PbNK65-infected mice ten days post-infection. Each dot represents the result from an individual mouse. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473299&req=5

Figure 1: Haemozoin detection by densitometric analysis. Transmitted light images (grey scale) were taken from unstained 7 μm thick cryosections from livers of uninfected mice (A) and from mice infected with PcAS (B) or PbNK65 (C), from PcAS-infected spleens (D), PbNK65-infected lungs (E) and kidneys (F). In panel G, the relative densitometric value obtained from cryosections of gelatin blocks with different concentrations of sHz were analyzed and the formula used to calculate the relative quantity of Hz/μm2 is shown. In panel H, the relative Hz content was measured in liver sections from uninfected (Con), PcAS and PbNK65-infected mice ten days post-infection. Each dot represents the result from an individual mouse. Horizontal bars represent group medians and horizontal lines with asterisks on top indicate statistical comparisons between groups. Asterisks on top of data sets indicate statistical significances compared with the uninfected control group. * p < 0.05, ** p < 0.01 and *** p < 0.001
Mentions: Cryosections with a thickness of 7 μm were prepared from frozen livers, spleens, lungs and kidneys and imaged by light microscopy. Transmitted light images were taken through a 20x/0.8 Plan-Apochromat objective of an Axiovert 200 M microscope equipped with an AxioCam MRm camera (Zeiss, Göttingen, Germany). For Hz quantification on liver sections, images were obtained from two rows of three consecutive fields. The densitometric analysis was performed with the AxioVision 4.6 software with a home-written script and the relative quantity of Hz/μm2 was calculated with the formula as shown in Figure 1G. The densitometric value (DV) of a pixel reflects the intensity of transmitted light at this position in the section. The densitometric background (DB) was determined in each picture by calculating the mean DV of all pixels with a DV above an empirically determined threshold. To test the linearity of the densitometric method to measure Hz on cryosections, gelatin blocks containing different concentrations of synthetic Hz (sHz) were analysed. sHz was prepared as described previously [22] and was homogenized and added in different concentrations to a 10% gelatin-PBS solution at 37°C in a 24-well plate. Upon solidification on ice (to prevent sedimentation of sHz), sHz-containing gelatin blocks were cut out of the wells, embedded in O.C.T and frozen at -80°C. For each concentration, five to six images were analysed from one section/sHz block and this was done for two blocks.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Immunobiology, Rega Institute, University of Leuven, Leuven, Belgium.

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus