with diabetes [24, 187]. Later, Skovborg and coworkers reported that the calibre of retinal arteries is also increased in diabetic subjects [170]. The Þrst direct empirical evidence of altered retinal blood ßow in persons with diabetes came from Kohner et al. in the mid-1970s coinciding with the development of methods for the measurement of mean retinal circulation time from ßuorescein angiograms [118]. Their results showed that retinal blood ßow was increased in diabetic subjects with absent or mild retinopathy but not in those with moderate or severe diabetic retinopathy. Since then, the haemodynamics of the retina in persons with diabetes, as well as diabetic animal models, has attracted considerable research interest. Several comprehensive review articles have appeared in recent years that provide an overview of retinal perfusion abnormalities in the different stages of diabetic retinopathy [46, 49, 163]. There is signiÞcant discrepancy between the results obtained in the various clinical and experimental studies of ocular blood ßow in diabetes. This may be attributable to the variety of techniques used to measure retinal haemodynamics, the use of differing sites to measure retinal blood ßow and the fact that in many studies, relatively little attention has been paid to the demographic and metabolic parameters (blood glucose, lipids, insulin, blood pressure, diabetes duration, etc.) of the study cohorts. Although there are some conßicting reports, the majority of studies suggest that in patients with a short duration of diabetes (<5 years), there is a constriction of the major arteries and arterioles [115, 192], and retinal blood ßow is decreased [29, 36, 73]. With longer durations of diabetes and the presence of clinical retinopathy, arterial vessels begin to dilate, and bulk retinal blood ßow increases in proportion to the severity of retinopathy [47, 72, 74, 91, 152].

15.3Retinal Hypoperfusion

A decrease in retinal blood ßow is one of the earliest abnormalities observed in the diabetic retina. Bertram et al. [29] measured the arteriovenous passage time in patients with type 1 diabetes using video ßuorescein angiography (VFA). The

arteriovenous passage time is deÞned as the interval between the Þrst inßux of ßuorescein into a retinal artery and its Þrst appearance in the corresponding retinal vein. They found that the arteriovenous passage time was increased in diabetic patients with no retinopathy, indicative of reduced retinal blood ßow. Similarly, Bursell et al. [36] reported that the mean circulation time, a parameter closely related to the arteriovenous passage time, is increased in patients with type 1 diabetes and no retinopathy. Laser Doppler techniques have also been used to quantify blood ßow in the major retinal vessels of diabetic patients with no retinopathy. Feke et al. [73] found that arterial blood speeds were reduced by ~30% in type 1 diabetic patients prior to the appearance of overt diabetic retinopathy. Several studies have investigated perimacular capillary perfusion in early diabetic retinopathy using confocal scanning laser ophthalmoscopy. A reduction in ßow velocities has been observed in both type 1 and type 2 diabetic patients with absent or mild retinopathy [16, 17, 191]. Impairment of retinal blood ßow has also been reported in diabetic rodents up to 12-week disease duration [37, 49, 101].

15.3.1 Mechanisms of Hypoperfusion

15.3.1.1 Glycaemic Control

Studies in both humans and animals have suggested that hypoperfusion in diabetes is closely associated with poor glycaemic control. Indeed, several reports have shown that retinal perfusion is inversely correlated with HbA1c in patients with both type 1 and type 2 diabetes [28, 29, 47]. Furthermore, normal retinal haemodynamics have been reported in well-controlled diabetic patients (HbA1c £7.5%) with no or minimal retinopathy, even after several years of diabetes [86, 133]. In experimental studies, normalisation of retinal blood ßow in streptozotocin (STZ)- induced diabetic rats has been accomplished by primary intervention with insulin therapy [48, 101]. Reversal of abnormal retinal haemodynamics in diabetic rats has also been reported using acarbose, an a-glucosidase inhibitor that reduces blood glucose concentration [178]. As outlined

below, two major mechanisms have been proposed to explain how hyperglycaemia decreases retinal blood ßow in early diabetes, namely, protein kinase C (PKC) activation and ion channel dysfunction in the contractile mural cells of retinal microvessels.

15.3.1.2 Protein Kinase C

Diabetes causes an increase in diacylglycerol (DAG) concentrations in vascular tissues associated with diabetic complications, including the retina [121, 167]. The molecular species of these pathophysiological DAGs are consistent with them being produced by the shunting of excess glucose through the de novo synthesis pathway [194]. These in turn activate several conventional and novel isoforms of PKC, including PKCa, PKCb, PKCd and PKCe [121]. PKC activation in diabetes may also arise through oxidative stress or increased concentrations of free fatty acids [56]. In the diabetic rat retina, the PKCbII isoform is preferentially activated [167]. There is now good evidence to suggest that PKC activation contributes to the impaired retinal blood ßow observed in experimental and human diabetes: Intravitreal injection of phorbol dibutyrate, a PKC activator, or R59949, a DAG kinase inhibitor that elevates total retinal DAG levels, have been shown to decrease retinal blood ßow in nondiabetic rats [38]. Also ruboxistaurin, a speciÞc PKCb inhibitor, can improve retinal blood ßow in diabetic animals [103], and diabetic PKCb knockout mice have been reported to exhibit no abnormalities in retinal blood ßow [49]. In clinical studies, increases in the mean retinal circulation time were ameliorated by ruboxistaurin in type 1 and type 2 diabetic patients with no or very mild diabetic retinopathy [9].

Activation of PKC contracts retinal arterioles by sensitisation of the contractile apparatus to Ca2+ (Fig 15.1) [55], and this represents the most likely mechanism through which PKC activation in diabetes impairs retinal blood ßow. Another mechanism by which PKC has been proposed to enhance microvascular constriction in the diabetic retina is through up-regulation of the vasoconstrictor peptide, endothelin-1(Et-1) [177, 197]. Increased Et-1 immunoreactivity

has been reported in the retina of STZ-induced diabetic rats and the spontaneously diabetic BB/W rat [39, 40, 43]. The increased expression of Et-1 is thought to be partly due to the activation of the b and d isoforms of PKC [151]. However, it should be stressed that the effects of elevated Et-1 in the retina during diabetes are offset by an increased resistance of the retinal microvessels to this vasoconstrictor [56], and therefore, it may not play a primary role in mediating arteriolar dysfunction in this disease.

15.3.1.3 Ion Channel Dysfunction

Ion channels in plasma membranes of retinal vascular smooth muscle cells and capillary pericytes play a central role in the regulation of vascular tone and blood ßow in the retina [157, 165]. There is growing evidence that disruption of ion channel function may contribute to retinal vasoconstriction and decreased retinal blood ßow during early diabetes.

The retinal arterioles constitute the main site of local blood ßow regulation within the retinal microvascular tree [102]. Retinal arteriolar smooth muscle cells express several kinds of plasma membrane ion channels, including volt- age-gated K+ channels [137], large-conductance Ca2+-activated K+ channels (BK channels) [136], Ca2+-activated Cl− channels [135] and L-type Ca2+ channels [135, 164], all of which may be involved in the regulation of retinal vascular tone [165]. Among these channels, BK channels are known to play a critical role because their blockade with the speciÞc inhibitor Penitrem A causes vasoconstriction in pressurised, isolated retinal vessels [136]. Normally, the opening of these channels in response to localised intracellular Ca2+ transients (Ca2+ sparks) results in spontaneous outward currents (STOCs), which cause membrane hyperpolarisation. This closes voltage-dependent Ca2+ channels, which decreases Ca2+ inßux, thereby leading to vasorelaxation. BK channels are composed of pore-forming a-subunits and accessory b-subunits [117]. The b1 subunit is preferentially expressed in vascular smooth muscle [33], and this subunit increases the sensitivity of the BK channels to Ca2+ [52]. Recent studies from our own laboratory suggest that impairment of BK

Fig. 15.1 PKC activation induces retinal arteriolar vasoconstriction by sensitising the contractile apparatus to Ca2+. (a) An original recording of [Ca2+]i and vessel diameter in a rat retinal arteriole exposed to the PKC activator phorbol myristate acetate (PMA). PMA reduced basal [Ca2+]i but caused the arteriole to constrict (photomicrographs below).

Scale bars = 5 mm on photos (From Curtis et al. [55] with permission). (b) Bar charts showing mean [Ca2+]i and outer vessel diameter for arterioles before and 5 min after exposure to 100 nM PMA (n = 9). Error bars are SEMs; * and *** denote

p < 0.05 and p < 0.001, respectively

a

b

10

0

100 nM PMA

*

Basal PMA

60 sec

channel activity may contribute to retinal hypoperfusion in early diabetes [136]. We found that STOCs were smaller (Fig 15.2a), but the amplitude of Ca2+ sparks was larger in retinal arteriolar myocytes from STZ-induced diabetic rats. This was explained by a reduced Ca2+ sensitivity of the BK channels to Ca2+ associated with a decreased abundance of the b1-subunit at the mRNA and protein level (Fig. 15.2b). This effect is early in onset, occurring within 1 month of diabetes induction [138]. It appears, therefore, that this downregulation decreases BK channel activity in retinal arteriolar smooth muscle cells, reducing negative feedback by intracellular Ca2+ and promoting contraction. This effect also appears to be highly selective for BK channels since no changes were observed when other conductances were compared between diabetic and age-matched control tissues [138].

While arterioles play a major role in regulating blood ßow, recent studies have suggested that

retinal perfusion may also be actively regulated at the capillary level. Retinal capillaries are richly endowed with contractile pericytes on their abluminal surface, and in vitro work has shown that these cells can modulate capillary luminal diameters in response to retinal neurotransmitters [155, 193]. Electrophysiological recordings have shown that retinal pericytes express a range of functional ion channels, including inward rectiÞer potassium channels (KIR channels) [134, 157]. KIR channels are believed to play an important role in establishing the resting membrane potential, and thereby the contractile tone of retinal pericytes. Diabetes has been reported to reduce outward KIR currents in pericytes located at proximal sites within the retinal microvascular network [134]. These diabetes-induced changes seem to be mediated by the polyamine, spermine, which is elevated in the diabetic eye [147]. Consistent with the reduced K+ efßux through KIR channels, proximal pericytes on diabetic