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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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Quantification of haemozoin in tissues. C57BL/6 J mice were infected with 104PbANKA, PbNK65 or PcAS parasites or were left uninfected (Con). At the indicated time intervals after infection, mice were dissected after heart puncture and perfusion. Extracted Hz from 30 – 60 mg tissue of livers (A), spleens (B), lungs (C) and kidneys (D), from half brains (E) and from whole hearts (F) were quantified by haem-enhanced luminescence and expressed as nmol haematin/organ (liver, spleen, lungs and kidneys) or pmol haematin/organ (brain and heart). Each group consisted of 15 to 20 mice, with each dot indicating individual data points. Horizontal dashed lines were used to denote the accuracy limit of the assay for each organ separately. 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 3: Quantification of haemozoin in tissues. C57BL/6 J mice were infected with 104PbANKA, PbNK65 or PcAS parasites or were left uninfected (Con). At the indicated time intervals after infection, mice were dissected after heart puncture and perfusion. Extracted Hz from 30 – 60 mg tissue of livers (A), spleens (B), lungs (C) and kidneys (D), from half brains (E) and from whole hearts (F) were quantified by haem-enhanced luminescence and expressed as nmol haematin/organ (liver, spleen, lungs and kidneys) or pmol haematin/organ (brain and heart). Each group consisted of 15 to 20 mice, with each dot indicating individual data points. Horizontal dashed lines were used to denote the accuracy limit of the assay for each organ separately. 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: The optimized haem-enhanced luminescence technique was used to study differences in the amount of Hz between various organs and between the same organs of mice infected with parasites of different pathogenicity (PbANKA, PbNK65 or PcAS). Mice were sacrificed at the indicated times post-infection and perfused systemically. Even though no difference was found in the amount of Hz before and after perfusion ( Additional file 1), it seemed more reasonable to apply perfusion on all samples tested. In this way, there could be no doubt that the detected Hz represented organ-trapped Hz and not Hz present in the circulation. Quantification of the total amount of Hz per organ revealed that most Hz was present in livers followed by spleens (Figure 3A–B). Far less Hz was detected in lungs and kidneys (Figure 3C–D), whereas subnanomolar levels of Hz were found in brains and hearts, irrespectively of the parasite species used (Figure 3E–F). Livers, lungs, kidneys and hearts from PbNK65-infected mice nine to ten days post-infection contained significantly more Hz compared with the same organs from PbANKA-infected mice seven to eight days post-infection or PcAS-infected mice ten days post-infection, even though livers of PcAS-infected mice were significantly larger (p < 0.0001 for liver weights between PcAS d10 and PbNK65 d9-10 and between PcAS d10 and PbANKA d7–8). In addition, lungs and hearts from PbANKA-infected mice seven to eight days post-infection had significantly more Hz compared with the same organs from PcAS-infected mice after ten days of infection. The total amount of Hz was similar in spleens of PcAS and PbNK65-infected mice ten days post-infection (Figure 3B), although the amount of Hz/mg spleen tissue was six-fold lower in mice infected with PcAS compared to PbNK65 (median value was 1164.4 pmol haematin/mg spleen for PbNK65 and 199.9 pmol haematin/mg spleen for PcAS; p < 0.0001). This was compensated by the three- to four-fold larger spleen size in PcAS-infected mice (p < 0.0001). Furthermore, similar amounts of Hz were observed in brains from PbNK65 and PbANKA-infected mice, whereas less Hz was found in brains of PcAS-infected mice.


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)

Quantification of haemozoin in tissues. C57BL/6 J mice were infected with 104PbANKA, PbNK65 or PcAS parasites or were left uninfected (Con). At the indicated time intervals after infection, mice were dissected after heart puncture and perfusion. Extracted Hz from 30 – 60 mg tissue of livers (A), spleens (B), lungs (C) and kidneys (D), from half brains (E) and from whole hearts (F) were quantified by haem-enhanced luminescence and expressed as nmol haematin/organ (liver, spleen, lungs and kidneys) or pmol haematin/organ (brain and heart). Each group consisted of 15 to 20 mice, with each dot indicating individual data points. Horizontal dashed lines were used to denote the accuracy limit of the assay for each organ separately. 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 3: Quantification of haemozoin in tissues. C57BL/6 J mice were infected with 104PbANKA, PbNK65 or PcAS parasites or were left uninfected (Con). At the indicated time intervals after infection, mice were dissected after heart puncture and perfusion. Extracted Hz from 30 – 60 mg tissue of livers (A), spleens (B), lungs (C) and kidneys (D), from half brains (E) and from whole hearts (F) were quantified by haem-enhanced luminescence and expressed as nmol haematin/organ (liver, spleen, lungs and kidneys) or pmol haematin/organ (brain and heart). Each group consisted of 15 to 20 mice, with each dot indicating individual data points. Horizontal dashed lines were used to denote the accuracy limit of the assay for each organ separately. 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: The optimized haem-enhanced luminescence technique was used to study differences in the amount of Hz between various organs and between the same organs of mice infected with parasites of different pathogenicity (PbANKA, PbNK65 or PcAS). Mice were sacrificed at the indicated times post-infection and perfused systemically. Even though no difference was found in the amount of Hz before and after perfusion ( Additional file 1), it seemed more reasonable to apply perfusion on all samples tested. In this way, there could be no doubt that the detected Hz represented organ-trapped Hz and not Hz present in the circulation. Quantification of the total amount of Hz per organ revealed that most Hz was present in livers followed by spleens (Figure 3A–B). Far less Hz was detected in lungs and kidneys (Figure 3C–D), whereas subnanomolar levels of Hz were found in brains and hearts, irrespectively of the parasite species used (Figure 3E–F). Livers, lungs, kidneys and hearts from PbNK65-infected mice nine to ten days post-infection contained significantly more Hz compared with the same organs from PbANKA-infected mice seven to eight days post-infection or PcAS-infected mice ten days post-infection, even though livers of PcAS-infected mice were significantly larger (p < 0.0001 for liver weights between PcAS d10 and PbNK65 d9-10 and between PcAS d10 and PbANKA d7–8). In addition, lungs and hearts from PbANKA-infected mice seven to eight days post-infection had significantly more Hz compared with the same organs from PcAS-infected mice after ten days of infection. The total amount of Hz was similar in spleens of PcAS and PbNK65-infected mice ten days post-infection (Figure 3B), although the amount of Hz/mg spleen tissue was six-fold lower in mice infected with PcAS compared to PbNK65 (median value was 1164.4 pmol haematin/mg spleen for PbNK65 and 199.9 pmol haematin/mg spleen for PcAS; p < 0.0001). This was compensated by the three- to four-fold larger spleen size in PcAS-infected mice (p < 0.0001). Furthermore, similar amounts of Hz were observed in brains from PbNK65 and PbANKA-infected mice, whereas less Hz was found in brains of PcAS-infected mice.

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