Fabry disease is an X-linked disorder resulting in reduced activity of the lysosomal enzyme α-galactosidase A [1, 2]. This results in the systemic accumulation of D-galatosyl conjugates, particularly globotriosylceramide (Gb3) [3, 4]. Although the disorder is heterogeneous, patients suffer renal, cardiovascular and cerebrovascular complications [4]. Enzyme replacement therapy has recently shown to have an effect on resting cerebral blood flow abnormalities and on other organ systems functions [5, 6]. In the cerebrovascular system there is an increased incidence of stroke together with vessel ectasia [7]. This may be related to the progressive accumulation of storage material in the cerebral vessel or possibly, may be due to an abnormality of vessel function [8, 9]. The cerebrovascular complications are distributed predominantly in the posterior circulation with a progressive increase over time in the posterior distribution of small vessel disease as found on T₂-weighted images or fluid attenuated inversion recovery (FLAIR) MR [7, 10, 11]. It likely that such high signal lesions on MR images represent ischemic small vessel disease, however, there are no neuropathological-radiological correlative studies confirming this assumption. Since ischemic brain lesions are irreversible, and accumulate over years, demonstrating a reduction in incidence is likely to require prolonged therapy in a relatively large cohort of patients following this disease end-point. Previously, in Fabry disease, we have reported resting cerebral hyper-perfusion in a predominantly vertebro-basilar distribution together with abnormalities of vascular nitrotyrosine staining. This was reversed by enzyme replacement therapy [5, 10]. The current study examines the cerebrovascular system in Fabry disease in response to functional and pharmacological activation.

We hypothesized that the cerebral vasculopathy of Fabry disease is associated with abnormal cerebrovascular responses, and furthermore, that these would normalize with repeated intra-vascular infusion of α-galactosidase A [5, 6]. We compared the functional cerebrovascular responses in Fabry disease to those in age and sex matched controls using both visual stimulation and acetazolamide challenge by [¹⁵O]H₂O and positron emission tomography (PET) to measure rCBF. The patients were then re-examined following a 6-month double-blind placebo-controlled trial of enzyme replacement therapy (ERT). Functional activation by visual stimulation was chosen to examine the vertebro-basilar diathesis of Fabry disease. Acetazolamide, on the other hand, was hypothesized to identify areas of decreased cerebral perfusion following maximal cerebral vasodilation in Fabry disease compared to controls. Such an abnormality, if present, might then be used to assess the therapeutic efficacy of enzyme replacement therapy.

We define our usage of the term cerebral vasomotor reserve as (ΔCBF_res) the CBF response at 20 or 30 minutes post-acetazolamide challenge minus the resting CBF for each subject. We define cerebral vascular reactivity as the functional CBF response to a stimulus minus the resting CBF (ΔCBF_react) while the maximal response to acetazolamide challenge provides an index of the cerebrovascular capacity. The CBF response curve to acetazolamide can be considered like a high pass filter with an onset time constant to a steady state τ₁ and an offset time constant of τ₂. Alteration and prolongation of τ₂ will correspond to prolonged cerebral vascular response time. An important concept of a high pass filter is that the system response is dependent on both the resistive (R) and capacitative (C) elements. Hence, the steady state value as well as time constants τ₁ and τ₂ depend on both the R and C components. Therefore the value of ΔCBF_res also depends on the RC component of the cerebral vascular system so that if there is an increased R component then ΔCBF_res will be reduced, a situation seen in cerebral vaso-occlusive disease. If on the other hand the RC components are approximately equal in the comparison groups, similar steady state values of CBF and ΔCBF_res values will be seen. The magnitude of the steady state response by itself gives an index of the cerebrovascular capacitative component (C).

Our data indicate the presence of an abnormal functional response to visual stimulation both in global and regional CBF analysis in Fabry disease. This however was not due to an alteration in vascular reactivity. Previously, we demonstrated that the resting rCBF in Fabry disease is elevated compared to controls particularly in the posterior circulation [5]. In view of the normal visual area reactivity in Fabry disease, these findings indicate that the Fabry group response to visual activation is largely explained by the elevated resting rCBF. However, the more generalized cerebrovascular response noted in both the carotid and vertebro-basilar circulation suggests stimulus spread in Fabry disease, possibly due to a decreased response threshold in non-visual cortex vasculature. Following ERT, this resting rCBF abnormality was reversed [5].

The cerebral visual stimulation paradigm used in the current study is known to induce maximal cerebral blood flow [12] and is therefore a useful stimulus for the study of neuro-vascular coupling. Visual stimulation was chosen in particular because of the known predilection of the cerebrovascular abnormalities in Fabry disease for the posterior circulation [7, 10]. The lack of an increase or decrease in visual rCBF reactivity in Fabry disease suggests normal neuro-vascular coupling mechanism [18, 19].

Acetazolamide-induced maximal cerebral vasodilatory response is often used to assess cerebrovascular reserve [20]. The greatest difference between our patients and controls occurred at the 30-minute time point rather than at the 20-minute time point of the acetazolamide challenge. This finding is consistent with a prolongation of the cerebral vascular response time in the Fabry disease. ERT significantly decreased the prolonged cerebral vascular response time. The vasodilator effect of acetazolamide on the cerebral vasculature has been characterized by both PET and transcranial Doppler (TCD) studies [21, 22]. At a dose of 15 mg/kg the effect of acetazolamide is thought to be supramaximal with maximal cerebral vasodilation induced within about 20 minutes. By TCD a maximal dose-response was found to occur with an acetazolamide infusion of between 15 and 18 mg/kg with maximal vasoreactivity occurring between 10 and 30 minutes post acetazolamide [21]. Using [¹⁵O]H₂O and PET, a further study of rCBF following 1 g of acetazolamide demonstrated the peak response to occur at 10 minutes while the plateau phase occurred during the twenty to thirty minute time period post acetazolamide [22]. The measurements in the current study obtained at twenty and thirty minutes post acetazolamide injection therefore, probably corresponds to the plateau phase of reported acetazolamide responses.

The CBF response to supra-maximal acetazolamide challenge consists of a phase of increasing CBF followed by a plateau phase with a terminal recovery phase. This can be considered in terms of cerebrovascular resistance and capacity, analogous to a high pass filter where high frequency components of the system response are removed. The importance of this analogy is that the plateau response is dependent on both the system's resistive and capacitative components. In the present study it was seen that the vasomotor reserve in Fabry disease was within normal limits despite the elevated resting rCBF. The cerebrovascular capacity in Fabry disease was also within normal limits, suggesting absence of clinically significant vaso-occlusive disease in the cohort of Fabry patients examined. This finding is supported by the absence of abnormalities on magnetic resonance angiography in the same group. Reduced ΔCBF_res would be expected when the resistive component is increased reducing the amplitude of the acetazolamide induced plateau response.

The hyperemic cerebrovascular response to hypercapnia or acetazolamide is not inhibited by nitric oxide synthase inhibitors such as N ^G-monomethyl-L-arginine (L-NMMA) suggesting that NO does not mediate vasoreactivity [23, 24]. The mechanism of acetazolamide vasodilation, beyond smooth muscle intracellular and extracellular acidosis due carbonic anhydrase inhibition, is unclear. Abnormalities of the peripheral vasculature responses to acetylcholine have been previously described in Fabry disease together with nitric oxide pathway dysregulation [5, 9] suggesting participation of non-NO regulation of vascular tone. A similar vascular hyper-responsiveness has been noted in caveolin-1 knock-out mice [25, 26] raising the possibility that the accumulation of Gb₃ results in over-activity of uncoupled endothelial nitric oxide synthase within endothelial cell caveolae generating superoxide and other reactive oxygen species [27]. The latter may have a vasodilatatory effect [28]. Predominance of vascular abnormalities in the vertebro-basilar system in Fabry disease suggests fundamental biologic differences between the anterior and the posterior circulation. Such difference might, for example, result from the neural crest origin of pericytes and vascular smooth muscle in the anterior circulation as opposed to paraxial mesoderm in the vertebro-basilar or posterior circulation [29].

The current study indicates that ERT normalized acetazolamide-induced prolonged cerebral vascular response time in Fabry disease. This finding suggests that the underlying abnormality is present at the luminal side of the blood-brain barrier (BBB) complex and is due to an abnormality of the cerebral vessels since it is unlikely that α-galactosidase A crosses the BBB to any appreciable extent. Further, the treatment effect appeared to be uniform across all cerebral vascular distributions.