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| TITLE: | Alterations in astrocyte functioning and regulation of cytokine mRNAs during development following perinatal exposure to environmental agents (EMF) | ||
| Principal Investigator |
Jean Harry, Ph.D. | Laboratory of Biochemical Risk Analysis, NIEHS | |
| Health Relevance |
Neurobehavioral Dysfunction | ||
| Research Categories |
Whole Animals Toxicology | Reproductive Teratology | Neural Development |
| FY95 Funds | DIR-4 $ 10,000 | Start Date 9/93 | End Date 9/95 |
| Rationale and Summary |
This developmental period is highly sensitive to environmental insults. This project addresses the
possible effects of electromagnetic fields (EMF) during the neonatal period. Studies in our
laboratory have demonstrated that exposure to low levels of a known neurotoxicant, lead acetate,
during the developmental period result in a shift in the developmental pattern for mRNA
associated with the outgrowth of the neuronal axon and the maturation of the astrocyte. During
development, the axon of the neuron elongates to form connections (synapses) with various target
sites. This process is guided by the axonal growth cone at the leading edge of the axon. During
development and regeneration, the developmentally regulated protein, growth associated protein-
43 (GAP-43) is expressed maximally and located prominently along axons as they are elongating.
This protein is enriched at the tip of the growing axon at the cytosolic surface of the growth cone
membrane. Alterations in this protein during critical periods of development could result in
altered axonal elongation and subsequent connectivity of the neural network.
During the formation of the brain, there is an over abundance in axonal outgrowth in order to insure contact with the proper target site. Once this contact has been made, the excess material is removed by a process of pruning. This process may be the function of immune-like phagocytic cells of the brain, the microglia. When activated, the microglia up-regulate expression of pro- inflammatory cytokines. We have identified a developmental profile for the mRNA for Interleukin 1, Interleukin-6, and Tumor Necrosis Factor alpha. This profile appears to be altered by developmental lead exposure suggesting a link between the abortive axonal elongation and the decrease in the material needed to phagocytize and the induction of pro-inflammatory cytokines in the developing brain. Given the significance of the growth of the axon, the formation of appropriate synapses on target sites, and the process of "pruning" in the final formation of the neural network of the brain, we will measure mRNA for GAP-43 and proinflammatory cytokines early in the postnatal development of EMF exposed animals in an attempt to detect any alterations which could suggest a subtle perturbation to the developing nervous system. |
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| Experimental Design and Exposure Conditions |
Pregnant rats were exposed to EMF for 18.5 hours per day or intermittently (1hr on 1hr off for
18.5 hours) during their entire pregnancies and during the postnatal period using the continuous
breeding protocol at IIT Research Institute. The exposure conditions were control, 20 mG, 2G,
and 10G continuous exposure, 10G intermittent exposure. Brain tissue was collected from
postnatal day 2, 3, and 5, coded, and shipped on dry ice to NIEHS. Additional brain samples were
collected at the end of exposure when the offspring were adults.
RNA was isolated from the cerebellum of brains at each time point, subjected to Northern blot analysis and probed with radiolabelled cDNA for GAP-43 and Actin. The amount of radioactivity in each band representing mRNA for GAP-43 or Actin was determined. Following Northern hybridization, the RNA samples from postnatal days 2, 3, and 5 will be analyzed for the level of mRNA for interleukin 1, interleukin 6, and tumor necrosis factor alpha by RT-PCR. Forebrain tissue is in the process of analysis for PND 2. Adult tissue will be evaluated for a glia specific protein - GFAP by ELISA. |
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| Quality Assurance Measures |
The identity of the exposure conditions has remained unknown throughout these experiments. For each time point, 5 samples per exposure condition were isolated together, run on the same gel, and hybridized together to minimize variability. Exposure QA measures are associated with the ITI Research Institute. | ||
| Results and Discussion |
At both postnatal day 2 and 5 there was no apparent difference between exposure groups with
regard to levels of GAP-43 and Actin mRNA. At postnatal day 3 the control and low dose groups
showed a low level of GAP-43 mRNA, the 10G continuous and intermittent groups were 2x
higher, while the 2G continuous group was dramatically higher than all other groups (5x higher
than 0 and 2mG, 2.5x higher than groups 10G continuous and intermittent).
The developmental onset of GAP-43 mRNA expression occurs at a time of terminal cell division and accompanies the neuronal decision of cell fate while the continued elevation is associated with the developmental progression of the axon. This protein is enriched in the leading edge of the axon, the growth cone which provides crucial guidance in response to appropriate targets and other environmental cues. The increase in mRNA level seen at postnatal day 3 suggests that exposure to EMF produces subtle alterations in the neural network pattern of the brain. The increase could be representative of a stimulation in axonal growth as the result of a decrease in target sites or other factors involved in regulating the stability of neural connections. This observation suggests the need for further studies to examine the developmental pattern of expression of this gene as well as other associated genes that are developmentally regulated. The results from this pilot project do not appear to support further studies. However, the data are being reviewed by the NIEHS Molecular Oncology faculty to determine whether additional studies are warranted. |
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| Recent Publications |
None. | ||