Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN.
Cultures of dorsal root ganglia (DRG) can achieve neuronal maturation with axons, making them useful for neurobiological studies. They have not, however, previously been used to investigate subcellular events that occur following exposure to neuropathy-inducing organophosphorus (OP) esters. Recent studies in other systems demonstrated alterations of ATP concentrations and changes in mitochondrial transmembrane potential (DYm) following exposure to neuropathy-inducing OP compounds, suggesting that mitochondrial dysfunction occurs. The present dissertation proposed an investigation using chick embryo DRG cultures to explore early mechanisms associated with exposure to these toxicants. This approach uses an in vitro neuronal system from the species that provides the animal model for OP-induced delayed neuropathy (OPIDN). DRG were obtained from 9-10 day old chick embryos, and grown for 14 days in minimal essential media (MEM) supplemented with bovine and human placental sera and growth factors. Cultures were then treated with 1 mM OP compounds, or the DMSO vehicle control. OP compounds used were phenylsaligenin phosphate (PSP) and mipafox, which readily elicit OPIDN in hens, and paraoxon, which does not cause OPIDN. Confocal microscopic evaluation of neuronal populations treated with PSP and mipafox showed opening of mitochondrial permeability transition (MPT) pores, and significantly lower mitochondrial tetramethylrhodamine fluorescence, suggesting alteration of mitochondrial structure and function. This supports our conclusion that mitochondria are a target for neuropathy-inducing OP compounds by inducing mitochondrial permeability transition. For further evaluation of mitochondrial function, mitochondrial respiratory chain reactions were measured. In situ evaluation of ATP production measured by bioluminescence assay showed decreased ATP concentrations in neurons treated with PSP and mipafox, but not paraoxon. This low energy state was present in several levels of the mitochondrial respiratory chain, including complexes I, III and IV, although complex I was the most severely affected. For morphological studies, the media containing the aforementioned toxicants was removed after 12 hours, and cultures maintained for 4 to 7 days post-exposure. Morphometric analysis of neurites in DRG was performed by inverted microscopy, using a system that was entirely computerized. Morphometric estimation of neurites treated with mipafox or PSP but not with paraoxon suggested that reversible axonal swelling at day 4 post-exposure had reversed by 7 days post-challenge. Ultrastructural alterations were described by electron microscopy. Damage to neurons was more severe following exposure to PSP and mipafox, with mitochondrial swelling and rarefaction of microtubules and neurofilaments observed within the cytoplasm. This study supports others that suggested mitochondria are a primary target for neuropathy-inducing OP compounds. We suggest that mitochondrial permeability transition (MPT) induce abrupt changes in mitochondrial membrane potentials, altering the proton gradient across the mitochondria membrane, decreasing ATP production within the cell. In addition, reduction in ATP production can be related to specific-complex alteration of the mitochondria respiratory chain following neuropathy-inducing OP compounds. The profound ATP depletion and the induction of MPT can induce the release of apoptotic factors and intramitochondrial ions, leading to axonal damage observed later in the course of OPIDN. This study provides evidence that chick DRG cell cultures are an excellent model to study early structural and functional features of OPIDN. It is likely that the alteration in energy lead to ultrastructural defects in these cells. These early events can contribute to alteration in neuronal ATP production previously reported in OPIDN. / Ph. D.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/30004 |
| Date | 09 December 2001 |
| Creators | Massicotte, Christiane |
| Contributors | Veterinary Medical Sciences, Ehrich, Marion F., Van der Schyf, Cornelis, Inzana, Karen D., Hoyner, Bernard, Klein, Bradley G., Bloomquist, Jeffrey R. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | application/pdf, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
| Relation | Fig4Dch6p223.pdf, introliteraturereview.pdf, PhD_discussion_c.pdf |
Page generated in 0.0029 seconds