Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model - PubMed (original) (raw)

Necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model

Daniel M Rosenbaum et al. J Neurosci Res. 2010.

Abstract

Necroptosis is programmed necrosis triggered by death receptor signaling. We investigated whether necroptosis contributes to neuronal damage and functional impairment in a model of retinal ischemia.

Methods: Sprague-Dawley rats were subjected to raised intra-ocular pressure for 45 min and received intravitreal injections of the specific necroptosis inhibitor, Nec-1, its inactive analogue (Nec-1i) or vehicle. Seven days after ischemia, ERGs were performed and then the eyes were enucleated for histological analysis. In other animals, retinas were subjected to propodium iodide, TUNEL staining or Western Blotting and probed with anti-LC-3 antibody.

Results: Retinal ischemia resulted in selective neuronal degeneration of the inner layers. Pretreatment with Nec-1 led to significant preservation in thickness and histoarchitecture of the inner retina and functional improvement compared with vehicle-treated controls. Pretreatment with Nec-1i did not provide histological or functional protection. Post-treatment with Nec-1 also significantly attenuated the ERG b-wave reduction compared with ischemic vehicle controls. Nec-1 had no effect on the number of caspase or TUNEL-labelled cells in the ischemic retina but did inhibit the induction of LC-3 II and reduced the number of PI-labelled cells after ischemia.

Conclusion: Necroptosis is an important mode of neuronal cell death and involves autophagy in a model of retinal ischemia.

(c) 2009 Wiley-Liss, Inc.

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Figures

Fig. 1

Fig. 1

Chemical structures of Nec-1 and its inactive analogue, Nec-1i.

Fig. 2

Fig. 2

Necrostatin-1 reduces retinal injury and protects neurons in the inner retinal layers A, B: Following 45 min of retinal ischemia of the right eye, animals treated with Nec-1 led to significant preservation in thickness and histoarchitecture of the inner retina compared with vehicle-treated controls or inactive-Nec-1 treated animals (n = 6 per group, P < 0.05). A: Representative photomicrographs of the different treatment groups. B: Percent retinal thickness of the inner retina normalized to the untouched contralateral eye. C, D: The bar graphs represent the percentage of neurons normalized to the non-ischemic control. The control group shows a 25% decrease in neurons. In the INL (C), there was significant neuronal protection when the eyes were treated with Nec-1 as compared to the inactive Nec-1 analogue or the DMSO control group (n = 6, *P < 0.05 compared with vehicle control). In the GCL (D), there was a trend towards a decrease in the number of cells that had undergone cell death in the Nec-1 treated group (n = 6, p = 0.0621 compared to vehicle control). [Color figure can be viewed in the online issue, which is available at

www.interscience.wiley.com

.]

Fig. 3

Fig. 3

Pre-treatment with Nec-1 reduces propidium iodide (PI)-positive cells at 6 hr after retinal ischemia in the injured inner nuclear and ganglion cell layer. A: Representative photomicrographs showing reduced numbers of PI-positive cells in the INL and GCL after retinal ischemia in Nec-1 and vehicle-treated rats. Magnification ×40. B, C: Quantitation of PI-positive cells in injured INL (B) and GCL (C). *P < 0.05 vs. vehicle treated animals (n = 6). Left eye is untouched, non-ischemic control.

Fig. 4

Fig. 4

There is no effect of Nec-1 on caspase-3 (A) or TUNEL (B) stained cells after retinal ischemia. Rats were pretreated with 4 mM Nec-1 or DMSO by an intravitreal injection at the time of and 2 hr after ischemia. TUNEL or caspase-3 staining was then performed after 24 hr of reperfusion. Cell counting in the GCL is shown. N = 4 per group.

Fig. 5

Fig. 5

Nec-1 inhibits autophagy after retinal ischemic injury. Rats were pretreated with an intravitreal injection at the time of and 2 hr after ischemia with DMSO, Nec-1, or its inactive analogue (Nec-1i). After 8 hr of reperfusion, the eyes were enucleated, sectioned and exposed to an antibody against LC-3. A: Representative blots of 4 samples. The results show in the DMSO group two bands: autophagy is characterized by the induction of LC3-II (16kD: lower band) from LC3-I (18kd: upper band). Nec-1 partially inhibited the induction of LC-3 (II). However, there was no inhibition when Nec-1 was substituted for Nec-1i. Equal loading was confirmed by ERK protein. B: Densitometry revealed a significant reduction in the 16kd LC-3 (II) in the Nec-1 treated samples compared with the Nec-1i treated samples (P < 0.05; N = 4). Ex = experimental group; Con = untouched control eye.

Fig. 6

Fig. 6

Nec-1 leads to functional protection in both pre and post-treatment paradigms. A, B: Representative ERGs of animals pre-treated with Nec-1 or vehicle or Nec-1i. C is the baseline ERG prior to ischemia. S and E were pretreated with an intravitreal injection at the time of and 2 hr post ischemia. S was treated with Nec-1 (4 mM) while E was treated with an inactive analogue of Nec-1 (Nec-1i). C: The mean ERG b-wave at 7 days after retinal ischemia was reduced to 13% of baseline in the vehicle treated group while the administration of Nec-1 led to a significant preservation of the ERG b-wave; 27% of baseline (*P < 0.05, n = 8). D: Animals were treated at 2 and 4 hr after retinal ischemia and ERG b-wave was significantly higher compared to the vehicle-controlled eyes (*P < 0.05, n = 8).

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