2.3.1 Choroidal blood flow: Choroidal blood flow is about 10 times higher than the flow in the grey matter of the brain and four times that of the kidney[54, 55], with flow estimates ranging from 500 to 2000 ml/min/100g [56-63], even though a corresponding difference in metabolic requirements does not exist.
This high rate of blood flow[64] is probably attributable to the low resistance of the choroidal vascular system, a consequence of the unusually large caliber of the choriocapillary lumen. Eighty-five of the total blood flow to the eye is distributed to the choroid and only 4% to the retina. The remaining flow perfuses the ciliary body (10%) and the iris (1%).[54, 57]
The more obvious function of high choroidal blood flow is the delivery of oxygen and nutrients and the removal of metabolic waste; the high blood flow would optimize the partial pressure and concentration gradients for efficient metabolic exchanges between the choroid and the retina. Consequently, the oxygen extraction from the choroidal blood is very low: the arteriovenous difference is only about 3%.
[57, 58] As a result of the low oxygen consumption from the choroïd, high tissue oxygen partial pressure (PO2) values (superior to 80 mmHg) are recorded at the choroïd.[65-67] High choroidal PO2 values are essential to assure about 65mmHg PO2 decrease between the choriocapillaries and the inner segments of photoreceptor.[68]
In pigs, about 60% of oxygen and 75% of glucose are delivered to the retina by the choroidal circulation, despite the low oxygen extraction from the choroidal blood.[69]
The oxygen content of the retinal venous blood in humans[70] and pigs [69] is about 38% lower than of arterial blood.
In man and other primates, the choroidal metabolic support of the retina is supplemented by the retinal vessels, present only in the superficial layers, and retinal blood flow is relatively small, i.e., 25 to 50 ml/min/100g.[57, 59, 60, 71, 72]
2.3.2 Systemic vascular factors and AMD: A great number of risk factors for the development of AMD are identical to them of cardiovascular pathologies. Systemic hypertension [73-75] history of coronary, carotid and peripheral vascular disease, [76]
serum cholesterol[77], dietary fat intake[78, 79], body mass index [80] and smoking[81, 82] are considered to be relevant risk factors. Among these potential risk factors, epidemiological studies have shown that only smoking has consistently been associated with AMD. [82]
As a consequence of these associations, patients of 49 to 73 years old presenting early clinical manifestations of AMD could be associated to higher risk of cerebrovascular strokes after 10 years follow-up.[83] Others added that AMD could be linked to higher risk of cardiovascular mortality.[84]
There have been reports linking statin use to a lower risk of AMD.[24, 85-87]
Several authors suggested that hypertension could increase the potential risk for the development of AMD based on the effects on choroidal circulation [88, 89]
Observational [74, 90] and prospective studies [73, 75] pointed out the association between hypertension and AMD risk. In the ‘Beaver Dam Eye Study’, the incidence of pigment abnormalities and the risk of AMD manifestation at 10 years was increased in the presence of high systolic blood pressure and pulse pressure at the initial examination[77]. A twofold to threefold risk of neovascular AMD in 10 years was associated with controlled or uncontrolled hypertension under medication at initial examination. A threefold risk of incident late stage AMD over the next 5 years was observed in people whose systolic blood pressure had increased more than 5 mmHg from baseline to the 5 year follow up examination, compared with people presenting stable systolic blood pressure values during the same period. The australian
‘Blue Mountains Study’ demonstrated that focal arteriolar narrowing was associated to the incidence of several AMD signs, [91] underlying the association with systemic hypertension.
As studies demonstrated an overlap of risk factors for AMD and cardiovascular disease, suggestion arises that a common disease mechanism may be operative in both AMD and cardiovascular disease and atherosclerosis, resulting in the deposition of lipid in the sclera and in Bruch’s membrane. The increasingly rigid sclera would act to encapsulate the ocular vasculature in a more incompressible compartment, leading to a greater degree of systolic-diastolic variation in the blood
velocity during the cardiac cycle.[1, 6, 92] This working hypothesis gives us the correlation between the alterations observed and systemic factors. Further investigations are needed to know whether AMD-related haemodynamic abnormalities are due to increased sclera rigidity, increased systemic vascular rigidity, or both.
2.3.3 Retinal vascular changes and retinal blood flow in AMD: Retinal vascular changes such as focal arteriolar narrowing, arterio-venous (AV) nicking, and generalized retinal arteriolar and venular narrowing were recently associated with cerebrovascular and cardiovascular outcomes such as stroke[93] and coronary heart disease,[94] and may be markers of microvascular damage from disease processes including prolonged hypertension and chronic inflammation[95]. Less is known regarding the associations of focal and generalized retinal arteriolar narrowing, AV nicking and retinopathy with AMD.
A limited number of population-based cohort studies have investigated the association of retinal vessel diameter and retinal vascular structural changes with the incidence of early (indistinct soft or reticular drusen or combined distinct soft drusen and retinal pigmentary abnormalities) and late (geographic atrophy or neovascularization) AMD.
In the Beaver Dam Eye Study, retinal vascular characteristics appeared to be weakly related and inconsistently associated with AMD.[73] Arteriole-to-venule ratio was only associated with the incidence of soft indistinct drusen and the incidence of RPE depigmentation. Focal retinal arteriolar narrowing was not associated with the incidence of AMD while AV nicking was associated with the incidence of early AMD. The Beaver Dam Eye Study also showed that retinal arteriolar diameter was not related to the 10-year incident early or late AMD and narrower arteriolar diameter was associated only with incident RPE depigmentation. [73]
Similarly, the Rotterdam Study found no association between incident AMD and retinal vessel (artery or vein) diameter.[96] This latter finding is consistent with data from the Atherosclerosis Risk in Communities Study [97] and the Blue Mountains Eye study.[91, 98]
In the Blue Mountains Eye study, focal arteriolar narrowing and AV nicking at baseline were weakly associated and of borderline significance with the 5-year
incidence of both early and late AMD.[91] In the Atherosclerosis Risk in Communities study, focal retinal arteriolar narrowing was associated with RPE depigmentation, although it was not associated with other AMD changes.[97]
In the 10-year incidence of early and late AMD and following adjustment for age, gender, smoking, and mean arterial blood pressure, the Blue Mountains Eye study found that the presence of focal arteriolar narrowing was associated with an increased risk of incident neovascular AMD and geographic atrophy, although only the latter reached statistical significance.[98] Focal arteriolar narrowing was not significantly associated with incident early AMD, whereas the presence of moderate/severe AV nicking at baseline remained significantly associated with incident early and late AMD. No significant association was found between baseline arteriolar or venular caliber and the 10-year incidence of late or early AMD, although a weak association, was observed between vessel calibers and pigment abnormalities.[98]
Taken together, these population-based data suggest that retinal arteriolar changes are inconsistently related to the incidence of AMD. Arterio-venous nicking seems to be more often associated with AMD than any other retinal vascular sign.
In stratified analyses, this association was found to be present in persons without hypertension, indicating that the AV nicking–AMD link may be independent of hypertensive processes.[98] On the contrary, there seems to be no association between the retinal vessel diameter and incident AMD. The significance and reproducibility of these findings is yet to be determined and deserve further research.
In recent years, a few studies have also investigated retinal blood flow in AMD.
Its relation to the disease has not been fully understood. Applying the laser Doppler technique to retinal arteries of eyes with various degrees of AMD, Sato et al found increasing pulsatility but constant blood flow, with increasing severity of AMD.[99]
This can be interpreted as an effect of reduced compliance proximal to the eye and not as increased distal vascular resistance, suggesting a more generalized systemic vascular pathology rather than an intraretinal vascular pathology. In another study, changes in retinal capillary blood flow in AMD were assessed with the Heidelberg Retinal Flowmeter.[100]These results indicated no change in blood flow in eyes with non-exudative AMD compared with healthy controls. However, reduced capillary blood flow in the disciform stage of late AMD and increased perimacular capillary blood flow was found in the exudative form of late AMD.[100] Such hemodynamic
data in late AMD need to be interpreted with caution as it is yet not clear whether abnormalities in retinal capillary blood flow are secondary to an autoregulative reaction or whether they are a primary mechanism in the pathogenesis of AMD.
Subsequent studies did not confirm the previous results and found reduced retinal capillary blood flow in eyes with non-exudative AMD [101]and no changes in retinal capillary blood flow in eyes with exudative AMD comparing to normal controls.[102]
More data about the changes in retinal blood flow in AMD are warranted.
2.3.4 Anatomical changes in choriocapillaris/Bruch's membrane/RPE in dry AMD: In geographic atrophy (GA), the photoreceptors and RPE degenerate in a horse shoe-shaped pattern surrounding the fovea; the loss of choroidal vasculature appears to be a secondary event.[103]
Quantification of CC (choriocapillaries) number and lumen diameters in cross sections, indicate a decrease with age and a further decline in both in AMD,[42] an increase in capillary density and a decrease in large blood vessel diameter [104] and a narrowing of CC lumen and loss of CC cellularity in AMD.[105]
In case of GA with areolar RPE atrophy, a severe 53% reduction in vascular density in the area from disk to the submacular region was observed, associated to an almost complete RPE atrophy. The border of the RPE defect was clearly delineated and coincided closely with the area of decreased choroidal vascular density. Surviving CC in the area of RPE atrophy had significantly smaller luminal diameters than CC in control subjects and in normal areas of the GA eyes. The study of GA subjects demonstrated that the RPE cells atrophied first followed by degeneration of the CC.[106] Therefore, in this form of AMD at least, the loss of choroidal vasculature appears to be a secondary event suggesting that GA is not of vascular etiology.
Interestingly, even in areas with complete RPE atrophy, some CC segments remained viable but severely constricted, contradicting the data found in animal models, suggesting that RPE are essential for survival of CC[107]
The association of surviving RPE cells with CNV in GA specimens suggest that RPE cells may furnish a stimulus for new vessel formation or stabilization.[108]
2.3.5 Anatomical changes in choriocapillaris/Bruch's membrane/RPE in exudative AMD: In subjects with wet AMD characterized by fluid and hemorrhage beneath the RPE or neurosensory retina due to choroidal neovascularization (CNV),
significant attenuation of CC around active CNV and disciform scars were observed.
The surviving CC and CNV lumens were always associated with viable RPE cells, and areas without RPE, had greatly reduced areas of viable CC as well.
According to the study of Lutty GA et all[109] it was apparent that RPE were present over areas with extremely attenuated CC, but the RPE were hypertrophic. In areas with active CNV, the CC was not viable and had extremely hypertrophic RPE covering the CNV, every example of active CNV was associated with surviving RPE[109]
2.3.6 Choroidal watershed zones and neovascularisation: In vivo studies have shown the choroidal vascular bed to be a segmental and an end-arterial system [110-113] with each choriocapillaris lobule being a functional independent unit with no anastomoses with the adjacent lobules in the living eye.[111]
Observed areas of lobular ICG-dye filling were viewed as evidence that choriocapillaris blood flow can be at least functionally segmented.[114]
The choroid is therefore an end-arterial tissue and as such, has watershed zones located at the border between the areas of distribution of any two or more end-arteries. The apical parts of the various segments supplied by the short PCAs (Posterior Ciliary Arteries), meet each other in the center of the macula. Multiple watershed zones meet in the macula, and consequently are most vulnerable to ischemic disorders. Thus, the submacular choroid, is predisposed to chronic ischemia and neovascularisation more that any other part of the posterior choroid.
In patients with exudative AMD and choroidal watershed zone(s), three patterns were identified. The stellate pattern of the watershed zone was the most common one (60%), followed by the vertical (36%) and the angled one (4%).
Choroidal neovascularisation arose within the watershed zone in 88% of cases.[115]
Since the center of the watershed zone is the one that receives blood at lower pressure, it follows that a decrease in the perfusion pressure of the choroid causes a decrease in blood flow that is most severe in the center of the watershed zone. In the event of such a reduction in the choroidal perfusion pressure in association with the altered vascular regulation seen in patients with AMD [116], and/or with the AMD-related rarefaction and dysfunction of the choriocapillaris,[117] the resulting hypoxia-ischemia could disturb the physiologic balance between angiogenic and
anti-agiogenic factors predisposing to choroidal neovascularisation[118, 119] arising within or from the margin of the macular watershed zone(s).
2.3.7 Laser Doppler Flowmetry evaluation: Grunwald et al., using laser Doppler flowmetry, showed that there is a systematic decrease in choroidal circulatory parameters with an increase in the severity of AMD features associated with risk for the development of CNV, suggesting a role for ischemia in the development of CNV.[120]
2.3.8 Retrobulbar hemodynamic modifications: Friedman et al found reduced flow velocities and increased pulsatilities indices in the central retinal artery and short posterior ciliary arteries in a mixed group of patients with exudative and non-exudative AMD, as compared with healthy, age-matched control subjects, suggesting increased resistance of the choroidal vasculature. [7] Related to these results, they proposed a hemodynamic model of the pathogenesis of AMD and suggested that this disorder was caused by a progressive decrease in the compliance of the sclera and the choroidal vessels, leading to an increase in the resistance of the choroid to flow of blood.[1, 6, 92]
2.3.9 Modifications of vascular blood flow and alterations cascade: In the healthy or in AMD human eye, the choroidal blood flow evaluation and regulation experimentations allow a better understanding of the correlation between the pathological conditions and the choroidal circulation changes.
The results obtained by laser Doppler flowmeter indicate significant reduction of the choroidal blood flow correlated to the age and to the presence of AMD disease, compared to control subjects in the same range of age.
Although RPE metabolic modifications due to senescence processes are considered as the principal cause of the early clinical observations of AMD, choroidal vascular modifications seems to have secondary effects affecting the RPE photoreceptors interactions.
Resistance increase at the level of choroidal circulation consecutive to generalized vascular sclerosis [7, 92] and/or scleral rigidity increase,[121] associated
to blood flow decrease, modify the osmotic gradient through RPE leading to metabolic waste accumulation and drusen formation.
Choroidal circulation alterations may affect the metabolic exchanges through RPE and Bruch membrane, important to maintain visual function. Electrical activity of RPE cells being very sensitive to systemic hypoxia,[122] choroidal circulation alterations can affect oxygen and metabolic substrats diffusion through RPE and Bruch membrane, leading to functional alterations essential for vision due to dysfunction of the metabolic interactions between RPE cells and photoreceptors.
Blood flow alteration at the level of retrofoveal choroidal circulation contributes to the process of neovascularization characteristic of AMD. The fact that patients with high risk of neovascularization present the most severe decrease of choroidal blood flow underline the role of choroidal ischemia in the development of neovessels in the evolution of AMD.[120] Pathophysiological mechanisms for the development of subretinal neovascular tissue implicate release of neovascular mediators, potentially related to metabolic changes at the level of the external layers of the retina secondary to the decrease of choroidal blood flow.
Anatomical alterations linked to AMD (thickening of Bruch’s membrane, drusen formation) increase diffusion distance between choriocapillaries and photoreceptors internal segments. These anatomical alterations associated to the reduction of choroidal blood flow should enhance tissue hypoxia at the area of the photoreceptors inner segments, essential stimulus of vasoproliferative factors which are upregulated by hypoxia.[123, 124]
Neovascularization in the eye is also considered to result from an imbalance between stimulatory and inhibitory angiogenic factors.[123-125] Vascular endothelial growth factor (VEGF) is a likely candidate for the angiogenic stimulus for CNV as VEGF is produced also by RPE [126] and so it has provided a therapeutic target for CNV [127, 128]
The balance between VEGF and pigment epithelial growth factor (PEDF) in aging and AMD had been suggested that could shift the angiogenic potential in retina.[125, 129] PEDF was purified from the conditioned media of human retinal pigment epithelial cells and was found to be a potent inhibitor of angiogenesis [130]
and a neurotrophic factor. [131] PEDF administered intravitreally inhibits aberrant blood vessel growth in VEGF-induced neovascularisation.[132]
In eyes with AMD, PEDF levels were found significantly reduced in RPE cells, Bruch's membrane and choroidal stroma in contrast to VEGF immunoreactivity which was not significantly reduced in the RPE/Bruch's membrane/CC complex.[133]
Several of the anti-angiogenic agents as endostatin, which is the proteolytically cleaved carboxyl terminus globular domain of collagen XVIII (coll XVIII) [134] or thrombospondin-1 (TSP-1), produced by RPE in culture [135, 136]
detected in vitreous and aqueous humor,[137] are potentially involved in AMD.
Endostatin inhibits endothelial cell proliferation and migration in vitro and potently inhibits angiogenesis in vivo,[138] inhibiting the mitogen-activated protein kinase (MAPK) activation in endothelial cells.[134] Endostatin is significantly reduced in CC, Bruch’s membrane, and RPE basal lamina in AMD compared with aged control choroids. [139] TSP-1 in Bruch's membrane declines with age and it is almost absent in AMD, being significantly reduced in Bruch’s membrane, CC, and walls of large choroidal blood vessels.[140] The decline in the endogenous anti-angiogenic substances potentially favours the development of CNV.
The contribution of the vascular disturbances in the pathogenesis of AMD is actually established. However their precise role is not determined yet; associated to multiple factors affecting the metabolism of the RPE and/or genetic factors, lead to the appearance of the various phenotypes of AMD.