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. 2003 Dec 10;23(36):11420-6.
doi: 10.1523/JNEUROSCI.23-36-11420.2003.

Estradiol Attenuates Programmed Cell Death After Stroke-Like Injury

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Estradiol Attenuates Programmed Cell Death After Stroke-Like Injury

Shane W Rau et al. J Neurosci. .
Free PMC article

Abstract

Estradiol is a known neurotrophic and neuroprotective factor. Our previous work demonstrated that replacement with physiological concentrations of estradiol protects the cortex against middle cerebral artery occlusion (MCAO)-induced cell death. The cerebral cortex exhibits caspase-dependent programmed cell death (PCD) in many models of focal cerebral ischemia. We hypothesized that estradiol attenuates PCD during stroke injury. The current study explored the temporospatial pattern of markers of PCD, their relationship to the evolution of injury, and their modulation by estradiol. Rats were ovariectomized and treated with either estradiol or vehicle. One week later, rats underwent MCAO, and brains were collected at 1, 4, 8, 16, and 24 hr. We assessed the temporospatial evolution of infarction volume, DNA fragmentation, and levels of spectrin cleavage products in ischemic cortex. Estradiol led to a delay and attenuation of injury-mediated DNA fragmentation as early as 8 hr after MCAO. Estradiol also dramatically reduced the level of the 120 kDa caspase-mediated spectrin breakdown product (SBDP120) at 4 hr but not at 8 or 16 hr. The SBDP150, produced by caspase and calpain, showed peak levels at 16 hr but was not altered by estradiol. These results strongly suggest that estradiol protects the ischemic cortex by attenuating PCD, thereby reducing caspase activity, DNA fragmentation, and subsequently, overall cell death. These studies deepen our understanding of the mechanisms underlying estrogen-mediated neuroprotection.

Figures

Figure 4.
Figure 4.
Representative images of sections from an oil- and estradiol-treated rat that underwent 24 hr of permanent middle cerebral artery occlusion. Infarcted tissue is light, whereas live tissue is dark. An adjacent 1 mm frozen coronal section was microdissected in anatomically equivalent areas (X) from oil- and estradiol-treated animals.
Figure 1.
Figure 1.
Estradiol (E2) protects the brain against ischemic injury. MCAO initiates an ischemic insult that develops into an extensive infarct over 24 hr. Data were analyzed using two-way ANOVA with dependent variables of time (1, 4, 8, 16, and 24 hr) and treatment (oil vs estradiol). There is an overall significant effect of time as well as an overall significant interaction between time and treatment. Using Newman-Keuls post hoc evaluation, there is a statistically significant difference between infarct volumes in oil- and estradiol-treated animals at 16 and 24 hr after MCAO (*p < 0.05; n = 8-10 per experimental group). Values represent mean ± SEM.
Figure 2.
Figure 2.
Composite of representative pictures from the ischemic cortex stained by the TUNEL technique and counterstained with Hoechst 33258. A, Microscope fields at 40× magnification showing cells that stained positive for TUNEL and the same field showing Hoechst counterstain. B, Representative 20× fields taken from within the parietal cortex of coronal brain sections stained by the TUNEL technique. These fields demonstrate TUNEL-positive cells from oil- and estradiol-treated animals at 4, 8, and 24 hr after the initiation of MCAO.
Figure 3.
Figure 3.
Estradiol (E2) delays and attenuates the number of TUNEL-positive cells in the ischemic cortex. The mean number of TUNEL-positive cells in the ischemic cortex rises dramatically after 4 hr in oil-treated animals and continues to rise through the remaining time points after ischemic injury. In estradiol-treated animals, the number of TUNEL-positive cells rises dramatically after 8 hr. ANOVAs were run for the early (1-8 hr; n = 8-10 per experimental group) and late (16-24 hr; n = 8-10 per experimental group) phases of injury. There is a main effect of treatment (oil vs estradiol) on the number of TUNEL-positive cells during early (#p < 0.05) and late (*p < 0.05) phases of ischemic injury. Values represent mean ± SEM.
Figure 5.
Figure 5.
Composite of Western blot results showing representative lanes from oil- and estradiol-treated animals killed 4 hr after the initiation of MCAO. Proteins extracted from area X (Fig. 4) were analyzed by Western blotting to detect intact spectrin (240 kDa) and 120 kDa and 150 kDa spectrin breakdown products.
Figure 6.
Figure 6.
Estradiol (E2) reduces the caspase-mediated (120 kDa) spectrin breakdown product at 4 hr after MCAO onset in the peri-infarct ischemic cortex (*p < 0.05). Two-way ANOVA demonstrated an overall effect of treatment (oil vs estradiol). Post hoc Newman-Keuls reveals an effect of estradiol specific to the 4 hr time point (*p < 0.05; n = 3 per experimental group). Estradiol has no effect on levels of the 120 kDa spectrin breakdown product at 8 hr or on the secondary rise in this product at 16 hr after MCAO onset (n = 3-4 per experimental group). Relative levels for each sample are expressed as a ratio of 120 kDa fragment to full-length spectrin (240 kDa) normalized to neurofilament 200 from the same sample. Values represent mean ± SEM.
Figure 7.
Figure 7.
Estradiol (E2) does not affect the 150 kDa spectrin breakdown product in the peri-infarct ischemic cortex. The 150 kDa spectrin breakdown product shows a dramatic rise at 16 hr after MCAO onset. Two-way ANOVA demonstrates a significant overall effect of time but no effect of treatment and no interaction (n = 3-4 per experimental group). Relative levels for each sample are expressed as a ratio of 150 kDa fragment to full-length spectrin (240 kDa) normalized to neurofilament 200 from the same sample. Values represent mean ± SEM.

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