Ethanol-Induced Apoptotic Neurodegeneration and Fetal Alcohol Syndrome

1. Ethanol-Induced Apoptotic Neurodegeneration and Fetal Alcohol Syndrome Dawn Barclay

2. Purpose • To show that ethanol acting by a dual mechanism (NMDA receptor blockage and over activation of GABAA receptors) triggers widespread apoptotic neurodegeneration in the developing rat forebrain during the period of synaptogenesis.

3. Outline • Background • Experiment 1 (Methods and Results) • Experiment 2 (Methods and Results) • Experiment 3 (Methods and Results) • Experiment 4 (Methods and Results) • Experiment 5 (Methods and Results) • Experiment 6 (Methods and Results) • Conclusions • Closing Thoughts

4. Background • What is fetal alcohol syndrome (FAS)? • What are N-methyl-D-aspartate (NMDA) receptors and GABAA receptors? • Why test NMDA receptor antagonists and GABAA receptor activating agents in conjunction with FAS? • Why the rat as a model system? • How does the rat compare with the human developmentally?

5. What is FAS? • Intrauterine exposure of the human fetus to ethanol causes a neurotoxic syndrome. • Features of FAS are neurobehavioral disturbances ranging from hyperactivity to learning disabilities to depression and psychosis.

6. What are N-methyl-D-aspartate (NMDA) glutamate receptors and GABAA receptors? • NMDA glutamate receptors are found in the central nervous system and respond to excitatory amino acids--neurotransmitters such as glutamate.* • GABAA receptors are found in the central nervous system and respond to inhibitory amino acid—neurotransmitters such as GABA (gamma-aminobutyric acid).* *(Vander, 1998)

7. Why test NMDA receptor antagonists and GABAA receptor activating agents in conjunction with FAS? • A previous study (Ikonomidou, 1999) found that blockage of NMDA glutamate receptors during the period of synaptogenesis causes widespread apoptotic neurodegeneration in the developing rat brain. • After completing Experiment 1, the use of GABA activating compounds will be discussed.

8. Why the rat as a model system? • The rat is a mammal. • FAS has been studied extensively on this model system. • The rat is commonly used as a model system for human development.

9. How does the rat compare with the human developmentally? • Days 1-9 in the rat correspond to the 1st trimester of human development.* • Days 10-21 in the rat correspond to the 2nd trimester of human development.* • Postnatal period of days 1-10 corresponds to the 3rd trimester of human development.* *(West, 1987)

10. Experiment 1 • Does ethanol trigger apoptotic neurodegeneration in the developing rat brain? • If so, does it act by the NMDA glutamate receptor antagonist mechanism?

11. Methods-Experiment 1 • A 20% solution of ethanol in normal saline was administered to 7-day-old Sprague Dawley rats in two separate treatments. • Each treatment delivered 2.5g/kg subcutaneously (sc). • Control rats were given normal saline. • The treatments were separated by 2 hours. • Twenty-four hours following the first treatment the rats were anesthetized and perfused with aldehyde fixative.

12. Methods-Exp. 1 cont. • Three histological methods were used to study the brains DeOlmos silver impregnation, TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) and electron microscopy.

13. Figure 1

14. Results-Experiment 1 • In the brains of saline-treated rat, both silver and TUNEL staining techniques revealed a very light pattern of neurodegeneration. • This neurodegeneration was attributed to normal physiological cell death (PCD) which occurs during brain development.

15. Results-Exp. 1 cont. • In the brains of ethanol-treated rats, both silver and TUNEL staining techniques revealed a very dense and widely distributed pattern of neurodegeneration. • The pattern overlapped but was much more extensive than that induced by NMDA* antagonist alone. • Electron microscopy confirmed apoptosis. *Samples were obtained from previous study. Methods: Seven-day-old rats were injected intraperitoneally (ip) with vehicle or dizocilpine [(+)MK801] (0.5 mg per kilogram of bodyweight) at 0, 8, and 16 hours and the brains examined at 24 hoursby TUNEL and silver staining to detect degeneratingcells (Ikonomidou,1999).

16. Experiment 2 • What possible mechanisms could explain such results? • Could agents that act as either agonists or antagonists at dopamine receptors, block kainic acid or muscarinic cholinergic receptors, or block voltage-gated ion channels explain results? • Are benzodiazepines and barbituates, “GABAergic” agents, a plausible explanation?

17. Methods-Experiment 2 • Diazepam (10-30 mg/kg) intraperitoneally (ip) at 0 hours, n=6. • Clonazepam (0.5 to 4 mg/kg) ip at 0 hours,n=6. • Pentobarbital (10- mg/kg) ip at 0 and 4 hours, n=6. • Phenobarbital (50 to 75 mg/kg) ip at 0 hours, n=6. Note: Histological studies were performed using the same methodology as Experiment 1.

18. Figure 1

19. Results-Experiment 2 • All non “GABAergic” agents tested showed no significant findings. • “GABAergic” agents triggered widespread cell death in the rat brains. • These findings were in a dose-dependent trend. • Pattern of degeneration was similar for all agents utilized (“GABAergic”).

20. Results-Exp. 2 cont. • Pattern differed siginificantly from NMDA antagonists. • Superimposing both NMDA antagonist and “GABAergic” results created a composite pattern closely resembling the ethanol sample. • Vulnerability was assessed to be the period of synaptogenesis. • Electron microscopy confirmed apoptosis.

21. Experiment 3 • Quantitative evaluation of apoptotic neurodegeneration in 15 brain regions using samples obtained from Experiment 1, Experiment 2 and NMDA antagonist receptor study.

22. Methods-Experiment 3 • Stereological dissector method to quantify the numerical density (neurons/mm3) of normal neurons in 70 micrometer Nissl-stained sections or of degenerating neurons in 70 micrometer DeOlmos silver method. • 8-10 samples were used per brain region with dimensions (0.05 mm by 0.05 mm, dissector height 0.07 mm). • Counts were performed in a blind manner.

23. Methods-Exp. 3 cont. • Absolute number of degenerating neurons was established using Nissl-stained sections and an image analysis system, NIH Image 1.54. • Boundaries of individual brain region were identified. • Volume determination for each brain regions were made.

24. Methods-Exp. 3 cont. • Multiplication of the volume (mm3) of a given brain region by the numerical density (neurons/mm3) of the degenerating neurons in that region provides an estimate of total numbers of neurons deleted from that region by ethanol treatment.

25. Table 1 • See handout.

26. Results-Experiment 3 • Density of degenerating neurons in saline-treated rats varied from 0.13-1.55% of the total neuron density. • This corresponds to the expected neurodegeneration in postnatal day 8 (P8) rat brains and is attributed to PCD. • Density of degenerating neurons in ethanol-treated rat brains ranged from 5-30% of total neuron density in the same brain regions.

27. Experiment 4 • What dosing regimen triggers the most severe apoptotic neurodegeneration in P7 rats?

28. Methods-Experiment 4 • Ethanol was administered to P7 rats by four dosing regimes. • 2.5 g/kg x 1 sc. • 1.5 g/kg x 2 sc. • 1 g/kg x 5 sc. • 2.5 g/kg x 2 sc. • Normal saline was given to control group. • Injections were given 2 hours apart.

29. Methods-Exp. 4 cont. • Blood ethanol levels were assessed for hours 1-15 following injections. • Severity of neurodegeneration associated with the blood ethanol levels were concluded using the quantitative assessment outlined in Experiment 3. • Thirteen brain regions were evaluated.

30. Figure 2

31. Results-Experiment 4 • Severity of apoptotic degeneration does not correlate with total dose, but rather with the rate at which the dose is given and the length of time the blood ethanol level remains elevated above a toxic threshold. • This toxic threshold was found to be 180-200 mg/dl.

32. Results-Exp. 4 cont. • Dosing regimes that elevated blood ethanol levels above 200 mg/dl for 4 hours significantly increased the rate of apoptotic neurodegeneration. • Dosing regimes that elevated blood ethanol levels above 200 mg/dl for greater than 4 hours showed a degenerative response that became progressively more severe in proportion to exposure time.

33. Experiment 5 • What is the age dependency of ethanol-induced apoptosis in developing rat brains?

34. Methods-Experiment 5 • Immature rats at 7 developmental ages (E17-P21) were used. • E17, E19, P0, P3, P7, P14, and P21 were ages used. • 2.5 g/kg x 2 • Injections were given 2 hours apart. • Injections were given subcutaneously. • Twenty four hours following initial injection the specimens were sacrificed.

35. Methods-Exp. 5 cont. • The quantitative evaluation outlined in Experiment 3 was used. • To determine the number of neurons undergoing apoptotic neurodegeneration due to ethanol, the number of neurons counted in the saline-treated samples (n=6) in a given brain region was subtracted from those counted in the ethanol group (n=6) in the same region.

36. Figure 3

37. Results-Experiment 5 • A window of time corresponding to E19-P14 was found to show sensitivity to ethanol-induced neurodegeneration. • This time period also corresponds with the period of synaptogenesis. • Different neuronal populations showed sensitivity at varying times. • Three trends were noted.

38. Results-Exp. 5 cont. • Neuronal populations showing the early-stage profile included the ventromedial hypothalamus, mediodorsal and ventral thalamus. • Early-stage profile began to display a significant response to ethanol at day E19, peaked at day P0 and declined rapidly thereafter.

39. Results-Exp. 5 cont. • Neuronal populations showing the middle-stage profile included the subiculum, hippocampus, caudate, and laterodorsal and anteroventral thalamus. • Middle-stage profile began to display a significant response to ethanol at day E19, peaked at day P3, and declined to zero by P14.

40. Results-Exp. 5 cont. • Neuronal populations showing the late-stage profile included frontal, parietal, temporal, cingulate, and retrosplenial cortices. • Late-stage profile began to display a significant response to ethanol at day P3, peaked at P7, and decreased significantly thereafter.

41. Experiment 6 • Is the apoptotic response to ethanol associated with loss of brain mass?

42. Methods-Experiment 6 • Day P7 rats were used, n=6. • Control rats were injected with normal saline, n=6. • 2.5 g/kg x 2 • Injections were 2 hours apart. • Injections were given subcutaneously. • Specimens were sacrificed on day P12.

43. Methods-Exp. 6 cont. • Weights were taken for the whole brain. • The brains were then dissected at the level of the pons into two portions. • One included the forebrain (FB) and midbrain (MB). • The second included the cerebellum (CB) and brainstem (BS). • Both were weighed. • All weights were conducted in a blind manner.

44. Figure 4 * P<0.05 ** P<0.01

45. Results-Experiment 6 • Brain weights whether weighed as a whole or in parts were significantly lower in the ethanol-treated group versus the saline-treated group.

46. Conclusions • Exposure of the developing rat brain to ethanol for extended periods of time (hours) during the period of synaptogenesis induces an apoptotic neurodegenerative response that deletes large numbers of neurons. • Ethanol acts by many mechanisms within the brain, it appears that two mechanisms—NMDA receptor antagonist and GABAA potentiation is at least partly responsible for the proapoptotic effects.

47. Conclusions cont. • The developmental period during which the developing brain is vulnerable is the same for ethanol, NMDA antagonists, and “GABAergic” agents. • It was found to be during the period of synaptogenesis.

48. Closing Remarks • Period of synaptogenesis occurs prenatally in humans during the third trimester or last three months of gestation. • Therefore, ingestion of ethanol or drugs of abuse (NMDA antagonists/GABAA agonists) by the pregnant mother puts her child at risk.

49. Closing Remarks cont. • The use of NMDA antagonists and GABAA agonists are frequently used as sedatives, tranquilizers, anticonvulsants, or anesthetics in pediatrics and/or obstetric medicine. • Since the human brain’s growth spurt occurs not only prenatally but also several years after birth, the use of these agents needs to be thoroughly explored to ensure their safety.

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