Male, Sprague-Dawley rats (180-200 gm.)
SIGNALMENT:
Male, Sprague-Dawley rats (180-200 gm.)us)
HISTORY:
Domoic acid (1-4mg/kg.b.w.) or kainic acids (12-25 mg/kg.b.w.) were administered i.p. as a single dose. These excitatory compounds induce neurological disturbances including salivation, wet dog shakes, waddling gait, convulsion and death.
GROSS PATHOLOGY:
All animals were placed under deep anesthesia and perfused through an intra cardiac puncture with 10% buffered formalin or 4% para formaldehyde as previously described (Tryphonas et al, 1990; Truelove et al, 1996). There were no obvious changes upon gross examination. Immunohistochemical studies of archived and recently collected specimens were carried out on paraffin sections microwaved in citric acid for antigen retrieval (Gill et al, 1998; Pulido et al, 1995).
LABORATORY RESULTS:
N/A
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Fig. 1. Sections of the hippocampus of a rat treated with 4 mg/kg.b.w/ i.p. of domoic acid showing a cell drop out and neurons with vascular cytoplasm, particularly within the CA3/CA4 regions. H&E x5. |
Fig. 7. Hippocampus of a rat treated with kainic acid (25 mg/kg.b.w./i.p.) stained with MAP2 (microtubule associated protein) immunohistochemistry shows fragmentation of the microtubules and a decrease in the intensity of the stain when compared to the control. |
Fig. 9 Hippocampus of a rat treated wtih kainic acid (25 mg/kg.b.w./i.p) stained with a glutamate receptor (GluR2/3) immunohistochemistry, showing a marked decrease in the intensity of the stain as compared to the control. |
 Fig. 10 Hippocampus of a control rat showing intensely staining with GluR2/3 immunohistochemistry and a preferential affinity for the neuronal cell bodies. |
MORPHOLOGIC DIAGNOSI(E)S AND ETIOLOGY:
Severe necrosis of the pyramidal neurons with marked cells drop out in the CA3/CA4 regions of the hippocampus. The remaining neurons show marked vacuolization of the cytoplasm. The most affected region is the CA3/CA4, followed by the CA1>CA2 and dentate. The drop out of neurons is best visualized with the Chen/Bodian silver stain. Sections of the hippocampus stained with the microtubule associated protein (MAP2) immunohistochemistry, showed a marked fragmentation of microtubules and decreased immunolabelling in the treated animals. The immunolabelling of the AMPA and kainic subtypes of glutamate receptors is markedly reduced in the treated animals. This is particularly evident within the pyramidal neurons of the CA3/CA4 regions.
Dx: Acute excitatory injury of the hippocampus induced by domoic and kainic acid
CONTRIBUTOR'S COMMENTS:
Domoic and kainic acid are powerful excitatory neurotoxins that induce brain injury. They are both glutamate analogs. Domoic acid was responsible for the seafood poisoning incident that occurred in Canada in 1987. It caused acute neurological disturbances and chronic memory impairment in some victims, with the highest incidence of mortality and neuropsychological disturbances in the elderly. Recently, we conducted a study on aged vs. young rats, using kainic acid as the excitatory toxin (manuscript in preparation). We included samples of the injury induced by kainic acid to compare with domoic acid.
The photographs are characteristic of the acute excitotoxicity of domoic and kainic acid. The difference between them is their potency. Domoic acid is the most potent. Neurologic abnormalities appeared earlier in aged animals treated with kainic acid than in the young. The cellular injury is not limited to the neurons of the hippocampus. Astrocytes are also affected. The other target regions included the nucleus accumbens, the area postrema and the retina. Evidence indicates that the excitotoxicity of these compounds is mediated by the ionotropic glutamate receptors (GluR). The marked decrease of the intensity of the stain, observed with GluR immunohistochemistry, supports this view. The MAP2 antibody preferentially binds to the microtubules of the dendritic tree. The marked decrease of the intensity of the stain and fragmentation of the microtubules, support the view that the dendrites are a preferential target structure for these neurotoxins. Animals that survived the acute toxic events and were alive for a longer period of time showed increase glial fibrillary acid protein (GFAP) immunolabelling. These changes are not evident in the acute lesions.
In additional studies we have demonstrated the presence of glutamate receptors in heart and other peripheral tissues, suggesting a common mechanism for excitotoxicity and wider range of potential target sites for excitatory neurotoxins. Further investigations are required to elucidate the role of these receptors in peripheral tissues. (Gill et al)
All images were captured on a ZEISS Axiophot microscope with a Roche Progress digital high resolution camera.
REFERENCES:
- Iverson,F., Truelove, J., Nera, E. et al. Domoic Acid Poisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Fd. Chem. Toxic. 27:377,1989.
- Tryphonas, L., and Iverson, F. Neuropathology of Excitatory Neurotoxins: The domoic acid model. Tox. Path. 18: 165, 1990.
- Tryphonas, L., Truelove, J., Nera, E. and Iverson F. Acute neurotoxicity of domoic acid in the rat. Tox. Path.
18: 1,1990.
- Truelove, J., Mueller, R., Pulido, O. and Iverson, F. Sub chronic Toxicity Study of Domoic Acid in the Rat. Fd. Chem. Toxic. 34: 525,1996.
- Gill, S., Pulido, O., Mueller, R. et al. Molecular and immunochemical characterization of the ionotropic glutamate receptors in the rat heart. Brain Res. Bulletin 40: 429, 1998.
- Gill, S., Pulido, O., Mueller, R. and McGuire, P. Immunochemical localization of the metabotropic glutamate receptors in the rat heart. Brain Res. Bulletin 48: 143, 1999.
- Gill, S., Mueller, R., McGuire, P. And Pulido, O. Potential target sites in peripheral tissues for excitatory neurotransmission and excitotoxicity. Tox. Path. 28: 277, 2000.
- Pulido, O., Mueller, R., Smyth, P., Clausen, J. and Gill, S. Heart and Brain Glutamate Receptors: A Common Mechanism for Excitotoxicity? Brain Pathology 10: 681, 2000.
- Gill, S. and Pulido, O. Glutamate Receptors in Peripheral Tissues. Tox. Path. (Accepted)
Case Summary Report (R064) - by Moderator Dr. Fred Hines
"The incident that first revealed domoic acid's toxicity occurred on Prince Edward Island, Canada, in 1987. Shellfish contaminated with domoic acid were consumed, yielding 4 deaths and over 100 people suffering various toxic symptoms. The effects of domoic acid poisoning vary from slight nausea and vomiting, to muscle weakness, disorientation, and … complete short-term memory loss [and this is why domoic acid poisoning was referred to as ASP or amnesiac shellfish poisoning]."1
This case's contributor is Toxicology Research Division, Bureau of Chemical Safety, Food Directorate, Health Products & Food Branch, Health Canada, Ottawa, Ontario, Canada KIY OL2 (please note the two citations from this laboratory in the early work on domoic acid; Iverson et al 1989 and Tryphonas and Iverson 1990).
You have an opportunity to download a PowerPoint presentation the contributor created. Please take advantage of that opportunity - and also copy the legends for the slides. The contributor gives an overview of the lesions - comparing controls to treated animals (H&E and special stains). It will make an excellent addition to your personal library of educational and reference material.
The U.S. FDA is also doing research on domoic acid toxicity. An outbreak of domoic acid toxicity affecting large numbers of California sea lions occurred in the United States in 1998. The main culprits were razor clams containing Pseudonitzschia sp which were producing high levels of domoic acid.2
If I may say a few words about my organization (the Center for Food Safety and Applied Nutrition of the Food and Drug Administration):
The mission of the U.S. FDA is to promote and protect the public health.3
A major goal of the last administration was to reduce the incidence of foodborne illness in the United States by creating the Food Safety Initiative:
"In his January 25, 1997 radio address, President Clinton announced he would request [money in the 1998 budget] to fund a nationwide early-warning system for foodborne illness, increase seafood safety inspections, and expand food-safety research, training, and education….
The goal of this initiative is to further reduce the incidence of foodborne illness to the greatest extent feasible. The recommendations presented in this report are based on the public-health principles that the public and private sectors should identify and take preventive measures to reduce risk of illness, should focus our efforts on hazards that present the greatest risk, and should make the best use of public and private resources. The initiative also seeks to further collaboration between public and private organizations and to improve coordination within the government as we work toward our common goal of improving the safety of the nation's food supply."4
According to the President's Council on Food this past January,
"there has been a 20% decrease in foodborne illness due to the major bacterial food pathogens across the United States [comparison of 1997 levels to 2000 levels]….The real benefits of this accomplishment are being reaped by consumers across the nation: this 20% decline in illness suggests that about a million foodborne illnesses have been prevented."5
Everyone I know at CFSAN/FDA is quite proud of that accomplishment and job satisfaction is high, but I have a sore arm from patting myself on my back, so I will continue now with the mission of this conference.
Most participants mentioned that they would have difficulty diagnosing neuronal necrosis, or at least differentiating the lesion from artifact. The comparison of images with the controls, however, convinced all that neuronal necrosis was the correct diagnosis. Also, note that in the PowerPoint presentation provided by the contributor, they mentioned that the animals were perfused which would also make less likely the occurrence of artifact. It is hypothesized that domoic acid may induce neurotoxicity because of the failure of astrocytes to remove extracellular glutamate.6
REFERENCES:
- http://www.transy.edu/homepages/chem4014/mathis/domoicacid/history.htm
- http://www.wa.gov/wdfw/fish/shelfish/razorclm/domacid.htm
- http://www.fda.gov/opacom/morechoices/mission.html
- http://vm.cfsan.fda.gov/~dms/fsreport.html
- http://www.foodsafety.gov/~fsg/cstrpl-4.html
- Ross IA, Johnson, W, Sapienza PP, Kim CS (2000). Effects of the seafood toxin domoic acid on glutamate uptake by rat astrocytes. Food Chem Toxicol 38(11): 1005 - 1111.