The Role of Endothelin in the Pathogenesis of Cerebral Malaria in a Mouse Model

Herbert Tanowitz, M.D.

Albert Einstein College of Medicine

Funded in June, 2004: $100000 for 2 years
LAY SUMMARY . ABSTRACT . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Using Imaging to Understand How Malaria Infection Causes Brain Dysfunction

Investigators will study animals infected with malaria to try to learn how this infectious disease sometimes produces damage,called "cerebral malaria,"  to the brain The investigators hypothesize that in some people malaria infection causes their blood vessels to constrict, resulting in a reduced blood flow to the brain, which then damages brain cells.

People with cerebral malaria develop seizures, unsteadiness, and confusion; occasionally they slip into a coma.  The investigators’ prior studies of cerebral malaria in a mouse model that they developed showed increased production of a protein called “endothelin” by cells that line blood vessels in the brain, and also by certain immune cells that participate in the immune inflammatory response.  Increased endothelin is known to cause blood vessels to constrict, which reduces blood flow to brain and damages brain cells.

Now the investigators will inhibit endothelin production in the cerebral malaria mouse model, and use MRI and MRS to image the animals’ brains.  This will enable the investigators to see whether the decreased endothelin levels correlate with functional and metabolic improvements in the animals’ brains.

Significance:  The research could lead to human studies designed to identify malaria-infected people who are at risk of developing brain damage.  The findings also may lead to development of preventative treatments to inhibit endothelin production to prevent cerebral malaria from occurring.

ABSTRACT

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The Role of Endothelin in the Pathogenesis of Cerebral Malaria in a Mouse Model

Cerebral malaria is an important cause of morbidity and mortality in many parts of the world, and children and pregnant women are particularly at risk. Preliminary observations from our laboratory using MRI indicate that in a murine model of cerebral malaria there is reduced cerebral blood flow and evidence of neuronal dysfunction. Endothelin-1is a potent vasoconstrictor, and our preliminary data indicate that components of the endothelin pathway are upregulated in the brain of a mouse with cerebral malaria and could provide an important mechanism for cerebral dysfunction. Quantitative magnetic resonance imaging and spectroscopy (MRI/MRS) are powerful tools to examine the functional and metabolic state of the brain in cerebral malaria, and thus we have employed this technology in our investigations of murine cerebral malaria.

We plan to directly evaluate the role of endothelin-1 in the mouse model by utilizing inhibitors of endothelin converting enzyme, endothelin receptor blockers, and mice in which the endothelin gene has been selectively deleted from endothelial cells. These findings will be correlated with functional /metabolic studies utilizing MRI of the brain. These studies will include detailed measurements of cerebral blood flow, water compartmentation, oxygen consumption and neuronal function.

The MR methods used in this proposal can be applied in clinical scanners operating at 1.5T or higher, which are common throughout the world. Furthermore, by obtaining a better understanding of the underlying physiological changes induced by cerebral malaria, these studies can direct optimal use of other blood flow and oxygenation sensitive methodologies such as transcranial Doppler or near infrared spectroscopy that can be used at the bedside. Detection of these physiological changes should help identify those individuals at risk for adverse neurological outcome and identifying a target population for additional treatment with endothelin inhibitors aimed at salvaging neural tissue.

HYPOTHESIS

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Hypothesis: 
Cerebral malaria is an important cause of morbidity and mortality in many parts of the world and children and pregnant women are particularly at risk. It has been suggested that cerebral malaria is associated with reduced central nervous system perfusion due to the blockage of vessel by parasitized erythrocytes. However, there has not been a direct correlation between the sequestration of parasitized red blood cells and cerebral malaria. Preliminary observations from our laboratory indicate that in a murine model of cerebral malaria there is reduced cerebral blood flow and evidence of neuronal dysfunction. Endothelin-1 is a potent vasoconstrictor. On the basis of our preliminary investigations, we hypothesize that the vasoconstrictor endothelin-1 significantly contributes to the pathogenesis of cerebral malaria and that functional MRI will be useful in determining the extent of cerebral dysfunction in this disease.

Goals:
1. To evaluate the role of endothelin-1 in the mouse model by utilizing inhibitors of endothelin converting enzyme, endothelin receptor blockers, and mice in which the endothelin gene has been selectively deleted from endothelial cells.

2. To correlate our findings with functional /metabolic studies utilizing MRI of the brain. These studies will include detailed measurements of cerebral blood flow, water compartmentation, oxygen consumption and neuronal function.

Methods: 
Molecular techniques and functional magnetic resonance imaging (MRI) of the mouse brain in mice infected with a strain of malaria that causes cerebral malaria.

Follow-on Funding:
Dr. Mahalia Desruisseaux, a research fellow in the laboratories of Dr. Herb Tanowitz, professor of pathology and of medicine, and Dr. David Spray, professor of neuroscience and of medicine, is the recipient of a five-year Burroughs-Wellcome Fund Career Award for Medical Scientists. The award will support Dr. Desruisseaux's investigations of a mouse model of cerebral malaria.  

FINDINGS

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We found that infected mice had a significant impairment in visual memory at day 7 (p < 0.05) that worsened by day 11 of infection.  At day 11, the infected mice also seemed to have a decrease in the time spent exploring new objects.  These impairments were observed in the absence of confounding effects of infection, as the infected mice showed no alteration in locomotor activity, arousal, reflexes, or motor coordination on a functional observation battery.  The cognitive dysfunction correlated with immunostaining evidence for prominent microglial activity throughout the brain of the infected mice at days 8 and 11, with hypertrophy of the microglia and retraction and thickening of the processes.  This was absent in the control mice where the microglia retained their delicate morphology.  Furthermore, astrocytes in the infected mice appeared to display increased affinity for the perivascular region, and double-labeling revealed that the normal polarized distribution of Aquaporin-4 at the astrocytic end-feet was disrupted, being redistributed throughout the cell processes.   With this model, we establish that malaria infection is associated with cognitive impairment that is associated with a loss of integrity in brain microglial and astrocytic morphology and activity.  Thus, this model of cerebral malaria demonstrates that the disease may be considered to some extent a gliopathy, in addition to its effects on vasculature.  Further studies using this mouse model may suggest testing strategies that could potentially identify subjects at risk for adverse cognitive outcome as well as elucidate underlying pathogenesis in cerebral malaria.

 

SELECTED PUBLICATIONS

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Kennan R.P., Machado F.S., Lee S.C., Desruisseaux M., Wittner M., Tsuji M., and Tanowitz H.B. Reduced cerebral blood flow and N-acetyl aspartate in a murine model of cerebral malaria.  Parasitol Res. 2005 Jul;96(5):302-7

Machado F.S., Desruisseaux M.S., Nagajyothi F., Kennan R.P., Hetherington H.P., Wittner M., Weiss L.M., Lee S.C., Scherer P.E., Tsuji M., and Tanowitz H.B. Endothelin in a murine model of cerebral malaria. Exp Biol Med (Maywood). 2006 Jun;231(6):1176-81.