This study assessed the consequences of a single binge exposure to ethanol on postnatal day (PN) 4 in the Purkinje cell population of the rat cerebellar vermis. The acute temporal pattern of apoptosis and the long-term permanent deficit in Purkinje cells across the entire vermis and on a lobular basis was investigated. It also investigated the lobular based Purkinje cell vulnerability to ethanol-induced apoptosis.
On PN4 Sprague-Dawley rat pups from timed matings were randomly assigned to either an alcohol exposed (AE; 4.5g/kg, 10.2% (v/v) as 2 doses 2 hours apart) via intragastric intubation, or sham intubated control (IC) group. Immunolabelling methods were used to detect active capase-3 in cerebellar vermal Purkinje cells hourly from 1 to 12 hours and 14, 16, 20, and 24 hours, following the first intubation. Semi-quantitative methods were used to determine the ratio of active caspase-3, a marker of apoptosis, labelled Purkinje cells per total Purkinje cells within the entire cerebellar vermis and on a lobular basis. The onset of caspase-3 activation occurred 3-4 hours following the initial ethanol exposure and peaked 10 hours post-ethanol, the magnitude of increase in active caspase-3 varied across lobules. To determine the long-term consequences a second cohort of pups was treated as per above and the total number of Purkinje cells in the adult (PN60) cerebellar vermis, as well as in lobules I/II, VII and IX, was determined using the optical disector method combined with the Cavalieri technique. A significant deficit was found in the entire vermis (21%), as well as lobules I/II (48%), VII (25%) and IX (38%).
A third cohort of suckle control (SC) pups was used to investigate the lobular based vulnerability to apoptosis in normal cerebellar vermal Purkinje cells on PN4. The levels of Bcl-2 and Bax mRNA expression were investigated using laser capture microdissection (LCM) to isolate the Purkinje cell lobular based populations combined with quantitative RTPCR (qPCR) technique. This method enabled the determination of comparative levels of mRNA expression attributable to the Purkinje cell population in identified lobules. Results indicated that Bcl-2 is expressed in cerebellar Purkinje cells and does not vary across the lobules. However, Bax expression could not be confirmed.
This thesis demonstrates the effects of a moderate ethanol binge on cerebellar vermal Purkinje cells on PN4. Findings demonstrate that apoptosis is initiated rapidly and results in the permanent loss of Purkinje cells, the degree of which is lobular dependent and greater than seen in the vermis as a whole. Thus significant cell loss can occur in discrete regions which may have specific discrete impairments in cerebellar function. Importantly for the human population, this study indicates that a single moderate alcohol binge is sufficient to cause significant permanent neuronal loss in the developing fetus, the onset of which occurs rapidly, and may result in specific brain dysfunctions.
| Identifer | oai:union.ndltd.org:ADTP/197605 |
| Date | January 1900 |
| Creators | Fowler, Anna-Kate, n/a |
| Publisher | University of Otago. Department of Anatomy & Structural Biology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://policy01.otago.ac.nz/policies/FMPro?-db=policies.fm&-format=viewpolicy.html&-lay=viewpolicy&-sortfield=Title&Type=Academic&-recid=33025&-find), Copyright Anna-Kate Fowler |
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