Permanent bilateral occlusion of the common carotid arteries is a model of chronic cerebral hypoperfusion leading to a progressive loss of hippocampal neurons, cholinergic dysfunction and cognitive impairment. It also affects the retina and optic nerve, thus providing a useful model for investigating the mechanisms of and therapy for the retinal degeneration associated with vascular defects . In this study we demonstrated that a single intravitreal injection of NGF has a protective effect on retina and optic nerve degeneration involving the early regulation of pro- and anti-apoptotic gene expression. Exogenous NGF seems to strengthen the effect of endogenous NGF, the synthesis of which is increased by vascular defect and also by the mechanical lesion associated with NGF or even vehicle intraocular delivery.
Animals and model characterization
Permanent bilateral ligation of the carotid arteries causes rapid optic nerve lesion and loss of the pupillary reflex, followed by retrograde dysfunction and apoptosis of RGCs [9, 16]. Only a subset of 2VO rats (60%) loses the pupillary reflex and suffers visual system pathology. This is probably due to the high individual variability of the Willis circle structure, which allows reverse perfusion of the retina through internal carotid and thence pterygopalatine arteries that branches from the internal carotid and supplies blood to the optic nerve [17, 8]. Rats without this characteristic may suffer more severe ischemia and lose their pupillary reflex [8, 17, 9]. We included only affected eyes in our study. Moreover, we analyzed the retina and the intra-orbital portion of the optic nerve, that is, areas both supplied by the central artery of the retina, which derives from of the internal carotid through the ophthalmic artery [18, 19].
According to the hallmarks of the model [20, 10] retinal degeneration is characterized by a severe reduction of retinal layer thickness, particularly in the OPL and IPL layers, already 8 days after 2VO, whereas the number of RGC nuclear profiles decreases 75 days after ligation. The marked disaggregation of microtubules and myelin disintegration in the optic nerve is accompanied by microglial activation [16, 21, 22]. Degeneration of the optic nerve fibers accompanies the progressive death of RGCs.
In our experimental conditions, two pathogenic events might influence degeneration of RGCs: the first one is direct retina and optic nerve injury caused by a vascular defect (hypoperfusion); the second one is eye puncture and/or injection. In fact, according to Sobrado-Calvo et al. , eye puncture itself or the increase in intraocular pressure due to injection of substances can induce eye damage. An increase in vitreous humor volume of about 5%  induced by an injection volume of 3 μl could contribute to retinal degeneration. Optic nerve fibers could be compressed by high intraocular pressure, producing a disruption of retrograde transport of neurotrophins from the superior colliculus, along the optic nerve, to the RGC soma [23, 24, 3, 26]. We actually found an increase in NGF mRNA expression 8 days after 2VO ligation. At this time, a rapid and transient activation of OPCs (NG2-IR cells) and microglial cells is observed in the optic nerve, probably reflecting inflammation .
NGF protects retina and optic nerve from vascular lesion
NGF is a potent stimulant of the trophism and wound healing process in the eye  and basically all eye structures are able to produce NGF and even to up-regulate NGF synthesis in the case of lesion . In our study we found that NGF-injection, but also vehicle injection, strongly stimulates NGF synthesis in the retina, thus confirming that endogenous NGF plays a critical role in retina and optic nerve protection, also in vascular lesion. Moreover, this increase, observed after 8 days, is further stimulated by hypoperfusion and even more by exogenous NGF. NGF injected intravitrealy has a great capability to diffuse and to reach the optic nerve itself , as also indicated by the brain diffusion of NGF corneal drops . A single NGF injection induced the regulation of Bax/Bcl-2 gene expression balance and also the expression of the IEG c-jun in the retina at the earlier time studied (8 days after 2VO ligation), and the effect of this single dose of NGF was long lasting, as indicated by preservation of the optic nerve diameter and myelination and by RGC profile counting.
There are several possible stages at which NGF could interfere with retinal and optic nerve degeneration and different cellular targets, including: direct effect on RGC degeneration, indirect effect on RGC retrograde degeneration through axon protection, myelin repair promotion, neoangiogenesis and vasculogenesis promotion.
NGF counteracts RGC degeneration by affecting Bax/Bcl2 balance- and c-jun- expression, suggesting a direct effect of NGF on retinal cells. This effect is early and long-lasting. Axotomy and glaucoma cause apoptosis of RGCs, as has been demonstrated by expression studies of caspases, Bcl-2/Bax and c-jun [31–33]. RGC death begins 3 days after axonal lesion and is preceded by an early activation of Bax . Moreover, vascular injury also increases the expression of stress-related proteins, including death-shock proteins as c-fos and DNA nuclear fragmentation in the retina . In these conditions, neurotrophins support survival of injured RGCs [12, 29, 22]. A similar anti-apoptotic effect of exogenous NGF has also been described in experimental retinal detachment [35, 29].
NGF could also regulate demyelination/remyelination balance after ischemic injury in the optic nerve, as indicated by the MBP expression regulation. At low concentrations, NGF enhances survival of the oligodendrocyte, the myelinating and remyelinating cell of the optic nerve , favouring fiber regeneration and proliferation  while at high concentrations it elicits apoptosis of mature cortical oligodendrocytes in vitro . NGF stimulates oligodendrocyte differentiation, as defined by the elevation of myelin basic protein (MBP) in postmitotic cells . NGF also regulates axonal signals that control myelination, promoting myelination by Schwann cells but reducing myelination by oligodendrocytes [40, 41]. Finally, hypomyelination of the optic nerve has been observed in mice lacking BDNF [42, 25]. In sum, the picture of the possible involvement of neurotrophins and related receptors in myelination is still partial and in some instances, controversial .
NGF has also been indicated as a novel angiogenic molecule that may contribute to the maintenance, survival, and function of endothelial cells by autocrine and/or paracrine mechanisms [44, 45] and the reciprocal regulation between VEGF and NGF might also result in neuroprotection . As expected, VEGF and Flt-1 mRNA expression is up-regulated in 2VO animals, whereas NGF administration is ineffective in this lesion-induced regulation. However, time course experiments are to be recommended in order to explore possible cross talk between NGF and VEGF signalling.
We also explored NGF high (TrkA) and low (p75NTR) affinity receptor mRNA expression regulation, observing a decrease in TrkA expression in all groups compared to the sham-operated group (the only one without any eye damage), and a slight decrease in p75NTR mRNA expression in NGF-injected eyes. No up-regulation of p75NTR mRNA expression was observed in 2VO animals. We studied NGF receptor expression at only one time-point, and might thus have missed earlier and late expression regulation. In fact, p75 NTR mRNA expression increases in retina during experimental glaucoma starting at 20 days, corresponding to increased Bcl-2/Bax ratio and apoptotic cell death .
Indeed, the contribution of the NGF/TrkA/p75NTRp75 signalling pathways to cellular activities is complex and still largely unknown [48, 2, 49]. NGF also plays a crucial role also during nerve injury and in the regulation of immune and inflammatory responses . According to this, the expression regulation of NGF receptors is a distant reflexion of possible cellular action by NGF. P75NTR is normally scarcely expressed in the retina, and its expression is differentially regulated also according to the chronology of the lesion. For example, p75 expression increases 3–5 days after ischemia, and then decreases further . Conversely, ocular hypertension induces a late increase in p75 expression in the retina, i.e. after 28 days  and apoptosis is associated with a consistent p75 expression [3, 51].