Caspase-related apoptosis in chronic ischaemic microangiopathy following experimental vein occlusion in mini-pigs (2008)

Purpose: Acute brain ischaemia (stroke) causes a central area of coagulation necrosis. Peripheral to it and after a few hours, apoptosis causes neurons throughout the entire area to die progressively. However, this sequence of events is related to the reperfusion of regenerated capillaries or collateral circulation, and is considered to be potentially salvageable. Similar findings have been reported in the retina after ischaemia−reperfusion injury in rats. In the present study, we intended to investigate whether delayed cell death is involved in neuronal injuries to the inner retina during chronic retinal ischaemia. Methods: Experimental branch retinal vein occlusion (BRVO) was induced in miniature pigs using indirect argon laser. The eyes were prelevated at 4, 24 and 48 hours and at 1 and 3 weeks following BRVO. The caspase inhibitor Z-VAD was injected intravitreally 24 hours after BRVO. Affected retinas were examined 24 hours later for any protective effect from apoptotic cell death. Histological examination with cresyl violet staining and TUNEL (TdT-mediated dUTP−biotin nick-end labelling) was performed on the samples. Results: A progressive oedema of the nerve fibre, ganglion cell and inner plexiform layers, related to a widely diffused cell necrosis, was observed in the affected territory within 4–24 hours after BRVO. This was followed by a wave of apoptosis localized at the periphery of the affected territory, which peaked approximately 48 hours after BRVO and was associated with a diffuse oedema of the inner nuclear layer. A progressive atrophy of the inner retina was observed 1–3 weeks after BRVO. Injection of the caspase inhibitor Z-VAD (24 hours after BRVO) decreased the amount of apoptotic cell bodies 48 hours after BRVO. Conclusions: This study shows that although necrosis is the predominant form of neuronal death in the early phase, massive delayed neuronal cell death caused by apoptosis occurs on a widespread basis as a result of chronic ischaemia after BRVO in the retina. Further studies are needed to evaluate the possibility of rescuing retinal neurons from death by neuroprotective treatments. Along with diabetic retinopathy and age-related macular degeneration, retinal ischaemia secondary to arterial or retinal vein occlusion constitutes the leading cause of blindness in developed countries. Acute brain ischaemia (stroke) causes a central area of coagulation necrosis. Peripheral to it and after a few hours, a zone of considerable heterogeneous tissue lesions appears, according to the degree of reperfusion or collateral circulation. This is considered to be potentially salvageable and is called the ‘penumbra’. Recently, it has been shown in animal models that apoptosis in this zone causes a gradual loss of neurons; thus injury accounts for neuronal death, which suggests that both neuronal necrosis and apoptosis may occur in parallel after ischaemic injury (Barinaga 1998). Similar findings have been reported in the retina after ischaemia−reperfusion injury in rats. Indeed, the features observed in the ischaemia−reperfusion model closely resemble ischaemic insults secondary to complete abolition of retinal perfusion as it occurs in central retinal artery occlusion in the human (Lam et al. 1999; Zhang et al. 2005). However, cases of central retinal vein occlusion (CRVO) or branch retinal vein occlusion (BRVO) lesions are of an experimental nature and fail to demonstrate the gradual loss of neurons caused by apoptosis in the penumbra zone. Branch retinal vein occlusion profoundly impairs a retinal metabolic pathway that produces nitric oxide (NO) and which is therefore responsible for the maintenance of the vascular tone (Donati et al. 1995, 1997). As the structure of retinal cells, and more particularly of Müller cells, appears to be well preserved in histological sections taken throughout the retina, despite swelling, it seems unlikely that the decrease in NO release 1–2 hours following BRVO is caused by a massive reduction of the retinal cellular volume (Pournaras et al. 1990). In another study, we showed that the arteriolar constriction could be reversed with NO donors (Donati et al. 1998). As a result of a decrease in blood flow caused by venous stasis, moderate chronic ischaemia of the retina and tissue hypoxia, rather than the abolition of retinal perfusion, was demonstrated (Pournaras et al. 1990). However, a recent study using inducible nitric oxide synthase (iNOS) knock-out mice showed that iNOS expression induces apoptosis in the inner nuclear layer of the avascular retina (Sennlaub et al. 2002). Therefore, NO seems to play a dual role in chronic retinal ischaemia, with a putative protective role in the initial phase (preventing the arteriolar vasoconstriction associated to BRVO and a proapoptotic role in a second phase resulting from the activation of iNOS. Experimental BRVO in monkeys and pigs has led to the development of a clinical picture more closely resembling that found in humans after CRVO or BRVO (Kohner et al. 1970; Pournaras et al. 1990). Experimental BRVO in the mini-pig showed a horizontal homogeneity in tissue lesions according to the formation of peripheral venous collaterals (Kohner et al. 1970; Pournaras et al. 1990; Pournaras 1995), which may be similar to the penumbra area described in the brain (Barinaga 1998). In the present study, we investigated whether delayed cell death by apoptosis could be involved in neuronal injuries of the inner retina that occur after BRVO in the mini-pig model. All experiments were conducted in compliance with the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmic and Vision Research. Miniature pigs (10–12 kg; Arare Animal Facility, Geneva, Switzerland) were prepared for experiments as previously described (Pournaras et al. 1989). Briefly, after injection of 5 ml Stresnil® (Azaperone; Janssen Pharmaceutica, Beerse, Belgium), 1 ml Atropine sulphate (Grosse Apotheke dr. G. Bichsel AG, Interlaken, Switzerland) and 0.5 ml Dormicum® (Midazolamum; Roche Pharma, Reinach, Switzerland), neuroleptanalgesia was maintained during the experiment by continuous perfusion of 100 mg/hour of Hypnodil™ (Metomidate; Janssen Pharmaceutica, Beerse, Belgium) through an ear vein catheter under spontaneous ventilation. Then, BRVO was performed as described here Enucleation was performed under anaesthesia at 4, 24 and 48 hours and at 3 weeks after BRVO. (In cases where enucleation was performed 4 hours after BRVO, anaesthesia was maintained throughout the period between BRVO and enucleation). Immediately after the enucleation of both the affected and control eyes, the animals were killed with an intravenous injection of a bolus of KCl (10 ml, 150 g/l). Branch retinal vein occlusion was performed with an indirect argon dye laser (argon green, wavelength 514 nm). One of the main retinal veins was photocoagulated. Briefly, vein occlusion was usually achieved using 100 shots with an energy intensity of 250 mW, with a pulse duration of 0.2 seconds and a spot diameter of about 500 μm. After occlusion was completed, the vein segment proximal to the occlusion site was constricted and the distal vein segment was dilated. Immediately after removal of the anterior segment, the posterior segment was prepared as an eyecup. The anterior and central vitreous were gently removed with a cotton tip. The posterior segment was divided into eight strips, two from each from the inferior and superior nasal and temporal quadrants. Samples were fixed for 1 night and processed for paraffin embedding. Sections of 7 μm thickness were used for cresyl violet staining for morphological examination and TUNEL (TdT-mediated biotin−dUTP nick-end labelling) detection. Semi-quantitative evaluation of the degree of inner retinal oedema was performed by two independent observers and graded from − to ++++. The 7-μm paraffin sections were used. TUNEL detection was performed as previously described (de Bilbao & Dubois-Dauphin 1996) and an in situ apoptosis detection kit (Apop Tag®; Oncor,) was used. Semi-quantitative evaluation of the labelling was performed by two independent observers and graded from − to ++++. The effects of Z-VAD-FMK (Z-Val-Ala-Asp[Ome]-FMK), a specific caspase III inhibitor, on apoptotic and tissue responses were evaluated. It was dissolved in dimethylsulphoxide (DMSO) 0.2% to obtain a stock solution of 20 mm of Z-VAD. Either 40 μl or 80 μl of the above solution was combined with aqueous humour to obtain a 200-μl solution of either 4 mm or 8 mm that was injected intravitreally 24 hours after BRVO. The animals were killed 48 hours after BRVO and histology was performed. Histopathological examination and TUNEL were performed at 4, 24 and 48 hours and at 1 and 3 weeks after experimental BRVO. Four hours after BRVO, the affected inner retina showed diffuse oedema of the ganglion cell layer (GCL). The inner nuclear layer (INL) showed no marked oedema. Near to the occlusion site, a few neurons in the GCL underwent marked necrosis. This was made apparent by irregular scattered condensation of nuclear chromatin associated with various degrees of cytoplasmic swelling. TUNEL did not significantly differ from that of a normal retina (Fig. 1B). Retinal veins appeared dilated and red blood cells were visible outside the vessels in the optic nerve fibre layer. (A) Histology of a normal miniature pig retina (cresyl blue) showing the normal retinal structure (GCL = ganglia cell's layer; INL = inner nuclear layer; ONL = outer nuclear layer; OS = outer segment layer). (B) Cresyl blue staining of an affected retina 4 hours after BVO showing edema of the GCL associated to necrotic cell death. Note the integrity of the INL and ONL as the absence of any TUNEL labelled apoptotic bodies. (C) Cresyl blue staining combined with tunel labelling of an affected retina 24 hours after BVO showing edema of the GCL extending to the INL. There is still widely diffused necrotic cell death, but note the presence of sparse tunel labelled picnotic nuclei both in the inner nuclear- and the ganglia cells layers suggesting the onset of some apoptotic cell death. (The separation of the cone and rod layer from the RPE is an artefact of fixation.) (D) Tunel labelling of an affected retina 48 hours after BVO showing diffuse edema of the GCL and INL. Note the absence of necrotic cell death and the presence of foci of TUNEL labelled apoptotic cell bodies around the vessels and in the INL (arrows). (E) Cresyl blue staining combined eith TUNEL labelling of an affected retina (periphery of the affected territory) 48 hours after BVO showing less edema of the inner retina and less TUNEL labelled apoptotic cell death located around the vessels and in the INL (arrows) then in fig. D. Z-VAD-DCB was injected intravitreously 24 h after BVO. (F) Cresyl blue staining of an affected retina 3 weeks after BVO showing marked thinning of the inner retina with no more visible GCL and INL. OS is absent because of fixation artefact. Twenty-four hours after BRVO, the inner retina showed marked oedema of the GCL and, to a lesser degree, of the INL. An increasing number of neurons in the GCL and the INL underwent marked necrosis, which was made apparent by irregular scattered condensation of nuclear chromatin related to various degrees of cytoplasmic swelling. A few neurons in both the GCL and INL also showed cell body shrinkage associated with condensation of nuclear chromatin suggestive of apoptosis. TUNEL showed scarce positive nuclei scattered in the retinal GCL and INL (Fig. 1C). At 48 hours after BRVO, even more substantial oedema of the GCL and INL was seen. Some neurons that had undergone necrosis were still present in both the GCL and INL of the affected retina. However, the prominent finding involved the appearance of massively increasing numbers of neurons that showed cell body shrinkage associated with condensation of nuclear chromatin suggestive of apoptosis in both the GCL and INL. It is noteworthy that no features typical of necrosis were present at the periphery of the affected area, but great amounts of TUNEL-marked neurons showing apoptotic features were present in the GCL, around the vessels and in the INL (Fig. 1D). Retinal veins appeared dilated and red blood cells were visible outside the vessels in the optic nerve fibre layer. One week after BRVO, there was marked atrophy of the GCL and inner plexiform layer (IPL), with few nuclei of normal appearance remaining in the GCL. The INL was markedly thinned. A few TUNEL-marked nuclei were still present (Fig. 1E). Three weeks after BRVO, even more marked atrophy of the GCL and INL was observed, with only a few nuclei of normal appearance remaining in the GCL and INL. The outer nuclear layer (ONL) was also damaged as it showed some picnotic nuclei; however, no significant TUNEL markers remained in either the inner or outer retina (Fig. 1F). As the above results provide evidence for a delayed apoptotic cell death that peaks 48 hours after the onset of the BRVO, we examined the possibility of modulating the apoptotic process by intravitreal injection of Z-VAD in DMSO 2% at 24 hours after BRVO. Injection of 40 μl of the stock solution combined with aqueous humour to obtain a 200-μl solution (4 mm) showed no significant differences in the degree of oedema and TUNEL markers in the centre of the affected area, but a significant decrease in the oedema and in the number of TUNEL-marked apoptotic cells was observed in the INL at the periphery of the affected area compared with the control eye (Table 1). Injection of 40 μl of the vehicle (DMSO 0.2%) combined with aqueous humour to obtain a 200-μl solution of aqueous humour showed no effect (picture similar to that observed without injection) (Table 2). Thereafter, to obtain a neuroprotective effect in the centre of the affected area, we increased the doses of Z-VAD. Doubling the doses of Z-VAD caused a transitory whitening of the retina that occurred minutes after the injection and resolved about 1 hour later. Histopathological examination performed 24 hours after the injection (48 hours after BRVO) showed a picture similar to that observed without injection (Table 2). Injection of 80 μl of the vehicle (DMSO 0.2%) combined with aqueous humour to obtain a 200-μl solution showed a picture similar to that observed without injection (Table 2). This suggests a probable toxic effect of the higher doses of DMSO used, which counteracts the neuroprotective effect of Z-VAD. There is increasing evidence indicate that both apoptosis and necrosis account for neuronal death after ischaemia in the retina (Joo et al. 1999). Indeed, it has been recently shown that in rats that retinal neurons may undergo two distinct morphological patterns of degeneration after ischaemia−reperfusion (Joo et al. 1999) a model that closely resembles that of arterial retinal occlusion in humans. However, the present study suggests that neuronal cell death as a result of apoptosis also occurs during chronic retinal ischaemia after experimental BRVO. It has been recently suggested that the gradual loss of neurons observed in the region outside the stroke’s core in the brain, where the oxygen supply is reduced but not abolished, occurs because some of these cells may recover from the ischaemia but die because injury triggers their suicide programmes (Lam et al. 1999). The idea that apoptosis is one of the leading mechanisms of death for neurons during stroke was recently supported by the finding in rats and mice that caspase inhibition may decrease the damage to the stroke area in the brain by up to 40–50% (Loddick et al. 1996). The enrolment of apoptotic regulatory genes such as Bcl-2 and p53 in cerebral ischaemic injury has been demonstrated using transgenic mice (Martinou et al. 1994; Joo et al. 1999). Both apoptosis and necrosis also occur simultaneously after ischaemia−reperfusion in the rat retina (Joo et al. 1999; Lam et al. 1999) and upregulation of the p53 proapoptotic gene may be involved in the slowly evolving apoptosis and necrosis of neurons in the retina (Joo et al. 1999). In the present study, histopathological features typical of necrosis were observed within hours after BRVO in the GCL and with relative preservation of the INL. This was followed by a second phase of neuronal death showing typical apoptotic features 24 hours after BRVO, peaking at 48 hours and resolving 1 week after BRVO. This progressive cell death was characterized by shrinkage of the cell body and condensation of the nuclear chromatin of TUNEL-marked cells in both the GCL and INL. It is noteworthy that, at the periphery of the ischaemic retina, delayed neuronal cell death related to apoptosis was the prominent finding observed, in the absence of any significant necrosis. Although no electron microscopy (the gold standard test to confirm apoptosis) was performed in the present study, the presence of chromatic condensation and pyknotic nuclei definitely point toward an apoptotic nature of the DNA strands detected by the TUNEL technique. The above findings suggest that although necrosis is the predominant form of neuronal death in the early phase, apoptosis may also significantly account for neuronal death after retinal vein occlusion. Moreover, there seems to be a potentially salvageable penumbra area, similar to that observed after stroke in the brain (Lam et al. 1999), where apoptosis is the predominant mechanism of neuronal death that occurs within a 24–48-hour period. This may be of great clinical interest, because, if a significant proportion of the ischaemic lesions that occur after retinal vein occlusion are caused by apoptosis, there is potentially some time for treatment aimed at preventing damaged retinal neurons from committing cellular suicide. Such a treatment may reduce the extent of the area of chronic retinal ischaemia after CRVO or BRVO, thereby reducing the risk of preretinal neovascularization and neovascular glaucoma. However, the question of how to proceed in order to prevent apoptosis remains. Indeed, the mechanism underlying apoptosis in chronic ischaemic injury is yet to be revealed. Although excitotoxicity is a key cause of neuronal death by apoptosis in the retina (Olney 1982), it has been shown in vitro that, following oxygen–glucose deprivation, neurons undergo slowly evolving apoptosis even when glutamate receptors are blocked, indicating that ischaemic apoptosis may occur through non- excitotoxic mechanisms (Gwag et al. 1995). This was confirmed by in vivo studies in rats showing that intravitreal injection of the glutamate inhibitor MK-801 attenuates the early neuronal necrosis without interfering with the slowly evolving apoptotic cell death observed (Joo et al. 1999). Mitochondrial damage and the activation of proteases such as caspase have been proposed as key pathways leading to apoptosis in ischaemia (Loddick et al. 1996). Although the exact regulatory role of caspases in the apoptotic pathways is not clear, an increase in caspase II and III immunoreactivity in the inner rat retina after ischaemia−reperfusion and a protective effect of the caspase tetrapeptidyl caspase inhibitor YVAD.CMK have been reported (Lam et al. 1999). In the present study, we addressed the following research question: is the pan-caspases inhibitor Z-VAD-DCB as able to attenuate ischaemic damages following experimental BRVO in the miniature pig as it is following brain stroke in the rat (Loddick et al. 1996)? As apoptosis peaked at 48 hours after BRVO, Z-VAD-DCB was injected intravitreally 24 hours after BRVO. A protective effect was observed in the penumbra area, suggesting that caspase activation is probably one of the main causes of neuronal death in chronic retinal ischaemia after retinal vein occlusion. In addition to playing a direct role in apoptosis, pan-inhibition of the caspases by Z-VAD may also serve to modulate the inflammatory response. Indeed, caspase-I cleaves and activates interleukin-1β (IL-1β), a signalling molecule of the immune system that triggers inflammation, and it has been advocated that blocking of IL-1β reduces ischaemic brain damage by at least 50% in the rat brain after stroke (Hara et al. 1997). In summary, delayed neuronal cell death caused by apoptosis related to caspases activation seems to occur on a wide basis during chronic retinal ischaemia, as observed after experimental BRVO. Further research is needed to address the possibility of preventing neurons from dying using neuroprotective treatments. Intravitreal injection may constitute an efficient means for neuroprotective substances, but the need for to increase the permeability of the vitreous−retinal barrier (usually performed by using DMSO as a vehicle) should not remain unnoticed. This study was supported by the Swiss National Science Foundation (grant no. 32-49543.96).