Title page for ETD etd-05102007-120107

Document Type Master's Dissertation
Author Smit, Eureka
Email eureke.smit@up.ac.za
URN etd-05102007-120107
Document Title Morphological changes in chick embryo neural tissue associated with hydrocortisone use during prenatal development
Degree MSc (Cell Biology))
Department Anatomy
Advisor Name Title
Dr M J Bester
Prof E Pretorius
  • associated
  • hydrocortisone
  • development
  • glucocorticoids
  • morphological changes
  • prenatal
  • neural tissue
  • chick embryo
Date 2006-11-24
Availability unrestricted

Glucocorticoids known to be such powerful agents that cell growth, differentiation and cell death are influenced in the brain of mammals throughout life. Despite this, relatively little toxicological information regarding prenatal exposure is available. The aim of this study was to determine the effect of prenatal hydrocortisone exposure on cell viability and cell morphology in chick embryonic neurons. Four different histological staining techniques namely, Hematoxylin and Eosin (H&E), Cresyl Fast Violet, Silver impregnation and a combination of Gold Chloride and Toluidine Blue were used to evaluate chick embryo neural tissue exposed to 0.137M or 0.685M hydrocortisone on day 3.75 (Carnegie stage 16) and day 5.5 (Carnegie stage 18) of development. Histological processing was optimized and neural tissue evaluated for any changes in neuron morphology and cell number. Specific ultrastructural changes to membraneous structures were evaluated by transmission electron microscopy (TEM). Fixation procedures that resulted in little to no disruption of these structures were optimized and used in studies evaluating the effect of hydrocortisone on neuron morphology. Primary chick embryonic neuronal cultures were prepared and increasing concentrations of hydrocortisone (26.3nM, 0.16M, 0.63M, 3.8M, and 22.8M) added. Fluorescence microscopy was applied to the in vitro hydrocortisone exposed primary neuronal cultures. A combination of fluorescein diacetate (FDA) and propidium iodide (PI) was used to evaluate the effect of hydrocortisone on cell viability, whereas dichlorodihydrofluorescein diacetate (DCH2FDA) was used to visualize reactive oxygen species (ROS) generation in neurons.

Histological evaluation of the neural tissue of chick embryos exposed to 0.137M and 0.685M hydrocortisone showed reduced neuron density and morphological changes associated with cell death. Glutaraldehyde with added magnesium chloride (MgCl2) as stabilizing chemical and potassium permangenate were two fixatives that caused minimal disruption to neural tissue. These two fixating methods were applied to control neural tissue as well as tissues exposed to 0.137M and 0.685M hydrocortisone. When evaluated by TEM, the control tissue appeared to be intact with no displacement. Exposure of neurons to 0.137M hydrocortisone appeared to have severe effects on the morphology of the mitochondria, endoplasmic reticulum (ER), nuclear and plasma membranes. More extensive damage was noted with 0.685M hydrocortisone, leaving almost no cellular structure. Both concentrations of hydrocortisone indicated cell death associated with apoptosis and necrosis. In vitro studies using primary cultures of chick neurons indicated that hydrocortisone is non-toxic at low concentrations (26.3nM V 3.8M) with the percentage viability ranging between 73% and 88%. A more toxic effect was seen at high concentrations (22.8M). Cell death at the higher concentrations (22.8M and 3.8M) of hydrocortisone occurred due to ROS generation, as indicated by DCH2FDA fluorescence

In conclusion, hydrocortisone indicated neurotoxicity at high concentrations of exposure. Although cell death could be detected, the exact mechanism (apoptosis or necrosis) still needs to be investigated. Since the developing brain is so susceptible to chemical insults care should be taken when administering this drug to pregnant mothers or young children.

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