Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines - PubMed (original) (raw)

Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines

M Rabuffetti et al. J Neurosci. 2000.

Abstract

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

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Figures

Fig. 1.

Fig. 1.

Morphological evidence of infarct reduction after Ac-YVAD.cmk treatment. Ischemic brain coronal sections of representative animals injected with DMSO 0.6% (left) or with Ac-YVAD.cmk (right), analyzed 24 hr or 6 d after pMCA occlusion. Scale bar, 5 mm.

Fig. 2.

Fig. 2.

Ac-YVAD.cmk treatment reduces cerebral infarct volume. Total, cortical, and subcortical infarct volumes were measured on cresyl violet-stained coronal paraffin sections 24 hr and 6 d after pMCA occlusion in animals injected intracerebroventricularly 10 min after the ischemic insult with Ac-YVAD.cmk (white bar) or vehicle only (black bar). Data are expressed as mean ± SEM; *p < 0.05. Absolute values in cubic millimeters at 24 hr; total infarct volume, 111.1 ± 6.6 versus 80 ± 6.4; cortical infarct volume, 83.5 ± 5.7 versus 64.1 ± 5.5; striatal infarct volume, 27.6 ± 1.6 versus 15.9 ± 1.6. Values at 6 d (in cubic millimeters); total infarct volume, 77.6 ± 6.4 versus 55.3 ± 6; cortical infarct volume, 58.7 ± 4.8 versus 43.3 ± 4.5; striatal infarct volume, 18.9 ± 2.2 versus 13.7 ± 2.

Fig. 3.

Fig. 3.

Effects of Ac-YVAD.cmk on caspase-1 and -3 activity. Caspase-1 activity was determined by measuring cleavage of the fluorogenic substrate Ac-YVAD.amc in cortical homogenates 24 hr and 6 d after ischemic lesion in rats injected intracerebroventricularly with Ac-YVAD.cmk (white) or vehicle (black) 10 min after occlusion (A). Caspase-3 activity measured as described for caspase-1 activity using the fluorogenic substrate Ac-DEVD.afc (B). Data are expressed as mean ± SEM; **p < 0.01; *p < 0.05. Statistical analysis was performed by ANCOVA using right hemisphere as a covariate (n = 7–9 for evaluation of caspase-1 activity at 24 hr; n = 17 for evaluation at 6 d; n = 7–9 for evaluation of caspase-3 activity at 24 hr; n = 8–9 for evaluation at 6 d).

Fig. 4.

Fig. 4.

Evaluation of apoptosis after Ac-YVAD.cmk. Biochemical quantification of apoptotic cells assessed by monitoring histone-associated DNA fragments in cortical homogenates obtained 24 hr and 6 d after pMCA occlusion from animals treated with Ac-YVAD.cmk (white) or with vehicle (black). Data shown are expressed as mean ± SEM; **p < 0.01. Statistical analysis was performed by ANCOVA using right hemisphere as a covariate (n = 17–19 for evaluation at 24 hr; n = 7–9 for evaluation at 6 d).

Fig. 5.

Fig. 5.

Morphofunctional evaluation of apoptosis and microglia activation. In situ detection of necrotic and apoptotic cell by the TUNEL method (A–D, top panels) and identification of macrophage/activated microglia by lectin histochemistry using Griffonia simplicifolia B4 isolectin staining (E–H, bottom panels) 24 hr and 6 d after left MCA occlusion. Scale bars: A–D, 100 μm; E–H, 200 μm.

Fig. 6.

Fig. 6.

Effect of Ac-YVAD.cmk treatment on IL-1β and TNF-α level. A, Cerebral cortex IL-1β levels in animals treated with Ac-YVAD.cmk (white) or with vehicle (black) and killed 24 hr and 6 d after pMCA occlusion measured by ELISA assay and expressed as difference between left and right hemisphere. Data shown are expressed as mean ± SEM. Statistical analysis was performed by Mann–Whitney_U_ test (n = 6–10); *p < 0.05. B, Cerebral cortex TNF-α levels in animals treated as described above. Statistical analysis was performed by ANCOVA using right hemisphere as a covariate (n = 17–19 for evaluation at 24 hr;n = 7–9 for evaluation at 6 d); *p < 0.05.

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