Retinal and Optic Nerve Head Vascular Reactivity in Primary Open Angle Glaucoma
Trichy Venkataraman, Subha
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The global aim of this thesis was to assess retinal vascular reactivity in glaucoma patients using a standardised hypercapnic stimulus. There is a suggestion of disturbance in the regulation of retinal and optic nerve head (ONH) hemodynamics in patients with Primary Open Angle Glaucoma (POAG), although much of the work to-date has either been equivocal or speculative. Previous studies have used non-standardised hypercapnic stimuli to assess vascular reactivity. To explain, hypercapnia induces hyperventilation which disturbs arterial oxygen concentration, an effect that varies between individuals resulting in the non-standardised provocation of vascular reactivity. Therefore, a normoxic hypercapnic provocation was developed to avoid additional and potentially uncontrolled vasoconstriction in what is thought to be a vasospastic disease. The development of a safe, sustained and stable normoxic hypercapnic stimulus was essential for the assessment of retinal arteriolar vascular reactivity so that repeated hemodynamic measurements could be obtained. Furthermore, most techniques used to measure vascular reactivity do not comprehensively assess retinal hemodynamics, in terms of the simultaneous measurement of vessel diameter and blood velocity in order to calculate flow. In this respect, this study utilized a technique that quantitatively assesses retinal blood flow and vascular reactivity of the major arterioles in close proximity to the ONH. The stimulus and vascular reactivity quantification technique was validated in healthy controls and then was clinically applied in patients with POAG. Newly diagnosed patients with untreated POAG (uPOAG) were recruited in order to avoid any confounding pharmacological effects and patients with progressive POAG (pPOAG) were also selected since they are thought to likely manifest vascular dysregulation. Finally, the results of the functional vascular reactivity assessment were compared to those of systemic biochemical markers of endothelial function in patients with untreated and progressive POAG and in healthy controls. Overall summary A safe, sustained, stable and repeatable normoxic hypercapnic stimulus was developed, evaluated and validated. In terms of the physiology of retinal vascular regulation, the percent magnitude of vascular reactivity of the arterioles and capillaries was found to be comparable in terms of flow. The new stimulus was successfully applied in POAG and in healthy controls to assess vascular reactivity and was also compared to plasma levels of ET-1 and cGMP. In terms of the patho-physiology of POAG, the study revealed a clear impairment of vascular reactivity in the uPOAG and pPOAG groups. There were reduced levels of plasma ET-1 in the uPOAG and ntPOAG groups. In addition, treatment with Dorzolamide improved vascular reactivity in the ntPOAG group in the absence of any change in the expression of plasma ET-1 or cGMP. Future work will address this apparent contradiction between the outcome of the functional vascular reactivity assessment and the biochemical markers of endothelial function in newly diagnosed POAG patients treated with Dorzolamide. Aims of chapters Chapter 3: To determine the effect of hypercapnia on retinal capillary blood flow in the macula and ONH using scanning laser Doppler flowmetry (SLDF) in young healthy subjects. Chapter 4: To describe a new manual methodology that permits the comprehensive assessment of retinal arteriolar vascular reactivity in response to a sustained and stable hypercapnic stimulus. The secondary aim was to determine the magnitude of the vascular reactivity response of the retinal arterioles to hypercapnic provocation in young healthy subjects. Chapter 5: To compare the magnitude of vascular reactivity of the retinal arterioles in terms of percentage change of flow to that of the retinal capillaries using a novel automated standardized methodology to provoke normoxic, or isoxic, hypercapnia. Chapter 6: To determine the magnitude of retinal arteriolar vascular reactivity to normoxic hypercapnia in patients with untreated POAG (uPOAG), progressive POAG (pPOAG) and controls. The secondary aim was to determine retinal vascular reactivity in newly treated POAG (ntPOAG, i.e. after treatment with 2% Dorzolamide, twice daily for 2 weeks). Chapter 7: To compare plasma endothelin-1 (ET-1) and cyclic guanosine monophosphate (cGMP) between groups of patients with untreated primary open angle glaucoma (uPOAG), progressive POAG (pPOAG), newly treated POAG (ntPOAG) and controls. The effect of normoxic hypercapnia on plasma ET-1 and cGMP was also assessed. The functional measures of retinal blood flow and vascular reactivity were correlated with systemic biochemical markers of endothelial function. Methods Chapters 3, 4 and 5 were conducted on young healthy control subjects, where as Chapters 6 and 7 were conducted on patients with glaucoma and healthy controls. Chapter 3: Subjects breathed unrestricted air for 15 minutes (baseline) via a sequential gas delivery circuit and then the fractional (percent) end-tidal concentration of CO2 (FETCO2) was manually raised for 15 minutes by adding a low flow of CO2 to the inspired air. For the last 15 minutes, FETCO2 was returned to baseline values to establish a recovery period. Heidelberg Retina Flowmeter (HRF) images centered on both the ONH and the macula were acquired during each phase. Chapter 4: Subjects breathed air via a sequential gas delivery circuit for 15 minutes and the air flow was then manually decreased so that subjects inspired gases from the rebreathing reservoir until a stable 10-15% increase in FETCO2 concentration was achieved for 20 minutes. Air flow rate was then manually elevated so that subjects breathed primarily from the fresh gas reservoir to return FETCO2 back to baseline for the last 15 minutes. Retinal arteriolar hemodynamics was assessed using the Canon Laser Blood Flowmeter (CLBF) during all three breathing phases. Chapter 5: Normoxic, or isoxic, hypercapnia was induced using an automated gas flow controller (RespirActTM, Thornhill Research Inc. Toronto, Canada). Subjects breathed air with PETCO2 normalized at 38 mmHg. An increase in PETCO2 of 15% above baseline, whilst maintaining normoxia, was then implemented for 20 minutes and then PETCO2 was returned to baseline conditions for 10 minutes. Retinal and ONH hemodynamic measurements were performed using the CLBF and HRF in random order across sessions. Chapter 6: Retinal arteriolar vascular reactivity was assessed in patients with uPOAG, pPOAG (defined by the occurrence of optic disc hemorrhage within the past 24 months) and controls during normoxic hypercapnia. Using the automated gas flow controller, patients breathed air for 10 mins and PETCO2 was maintained at 38mmHg. Following this normoxic hypercapnia (a 15% increase in PETCO2 while PETO2 was maintained at resting levels) was induced for 15 mins and then for the last 10 mins PETCO2 was returned to baseline (post-hypercapnia) to establish recovery blood flow values. Retinal arteriolar diameter, blood velocity and blood flow was assessed using the CLBF in both patient groups and controls. A similar paradigm was repeated in the newly treated POAG group (ntPOAG, i.e. after treatment with 2% Dorzolamide, twice daily for 2 weeks). Chapter 7: Blood samples were collected from the cubital vein of all participants (uPOAG, pPOAG, ntPOAG and controls) during baseline conditions (PETCO2=38mmHg) and then during normoxic hypercapnia (i.e. a 15% increase in PETCO2 relative to the baseline) using the paradigm described for Chapter 6. ET-1 and cGMP was assessed using immunoassay. Results Chapter 3: The group mean nasal macula capillary blood flow increased from 127.17 a.u. (SD 32.59) at baseline to 151.22 a.u. (SD 36.67) during hypercapnia (p=0.028), while foveal blood flow increased from 92.71 a.u. (SD 28.07) to 107.39 a.u. (SD 34.43) (p=0.042). There was a concomitant and uncontrolled +13% increase in the group mean PETO2 during the hypercapnic provocation of +14% increase in PETCO2. Chapter 4: Retinal arteriolar diameter, blood velocity and blood flow increased by 3.2% (p=0.0045), 26.4% (p<0.0001) and 34.9% (p<0.0001), respectively during hypercapnia. There was a stable ¬+12% increase in PETCO2 during hypercapnia and a concomitant -6% decrease in PETO2. Chapter 5: Using an automated gas flow controller the co-efficient of repeatability (COR) was 5% of the average PETCO2 at baseline and during normoxic hypercapnia. The COR for PETO2 was 10% and 7% of the average PETO2 at baseline and during normoxic hypercapnia, respectively. Group mean PETCO2 increased by approximately +14.4% and there was only a +4.3% increase in PETO2 during hypercapnia across both study sessions. Retinal arteriolar hemodynamics increased during hypercapnia (p<0.001). Similarly, there was an increase in the capillary blood flow of the temporal rim of the ONH (p<0.001), nasal macula (p<0.001) and foveal areas (p<0.006) during hypercapnia. A non-significant trend for capillary blood flow to increase in the macula temporal area (+8.2%) was noted. In terms of percentage change of blood flow, retinal capillary vascular reactivity (i.e. all 4 analyzed areas = 22.4%) was similar to the magnitude of arteriolar (= 24.9%) vascular reactivity. Chapter 6: Retinal arteriolar diameter, blood velocity and flow did not increase during normoxic hypercapnia in uPOAG compared to controls. Diameter and blood velocity did not change in pPOAG during normoxic hypercapnia but there was a significant increase in blood flow (+9.1%, p=0.030). After treatment with 2% Dorzolamide for 2 weeks there was a 3% (p=0.040), 19% (p<0.001) and 26% (p<0.001) increase in diameter, velocity and flow, respectively, in the ntPOAG group. Group mean PETCO2 increased by approximately +15% in all the groups and there was only a +3% increase in PETO2 during hypercapnia. Chapter 7: Plasma ET-1 levels were significantly different across groups at baseline (one way ANOVA; p=0.0012) and this was repeated during normoxic hypercapnia (one way ANOVA; p=0.0014). ET-1 levels were lower in uPOAG compared to pPOAG and controls at baseline and during normoxic hypercapnia (Tukey’s honestly significant difference test). Similarly, ntPOAG group also showed lower ET-1 levels compared to the pPOAG and controls at baseline and during normoxic hypercapnia (Tukey’s honestly significant difference test). The cGMP at baseline and during normoxic hypercapnia across all groups was not different. In the control group, the change in ET-1 during normoxic hypercapnia was negatively correlated with change in retinal arteriolar blood flow (r = -0.52, p=0.04), that is, as the change in ET-1 reduced, the change in blood flow increased. A weak correlation was noted between change in cGMP during normoxic hypercapnia and the change in arteriolar blood flow (r = +0.45, p=0.08). Conclusions Chapter 3: Hypercapnia resulted in a quantifiable capillary vascular reactivity response in 2 of the 3 assessed retinal locations (i.e., nasal macula and fovea). There was no vascular reactivity response of the ONH. It is critical to minimise the concomitant change in PETO2 during hypercapnia in order to obtain robust vascular reactivity responses. Chapter 4: A technique to comprehensively assess vascular reactivity during stable and sustained hypercapnia was described. Retinal arteriolar diameter, blood velocity and blood flow increased in response to hypercapnia. The vascular reactivity results of this study served as a reference for future studies using the hypercapnic provocation and CLBF. Also, the concomitant change in PETO2 using the partial rebreathing technique was reduced compared to the manual addition of CO2 technique described in Chapter 3 but was still greater than optimal. Chapter 5: A new automated gas flow controller was used to induce standardised normoxic, or isoxic, hypercapnia. The magnitude of vascular reactivity in both retinal arterioles and capillaries in response to the new hypercapnic stimulus was robust compared to the previous stimuli. There was a clear ONH vascular reactivity response in this study, unlike the result attained in Chapter 3. Although theoretically it is predictable that the percent magnitude of vascular reactivity of the arterioles and capillaries should be similar, this is the first study to show that they are indeed comparable. The magnitude of hypercapnia was repeatable and the concomitant change in PETO2 was minimal and physiologically insignificant. Chapter 6: The normal response of the retinal arterioles and capillaries to normoxic hypercapnia is impaired in both uPOAG and pPOAG compared to controls. Short term treatment with 2% topical Dorzolamide for two weeks improved retinal vascular reactivity in ntPOAG. However, it is still unclear whether this improvement is a direct effect of Dorzolamide or as a secondary effect of the decrease in intraocular pressure (IOP). Chapter 7: We found a reduction in the plasma ET-1 at baseline and during normoxic hypercapnia in the uPOAG and in the ntPOAG groups. This is the first study to show a lower plasma ET-1 level in uPOAG. The fact that this finding was repeated after 2 weeks treatment with Dorzolamide in the ntPOAG group further validates these results. It also suggests that Dorzolamide treatment does not impact ET-1 and cGMP measures, although it clearly results in an improvement of vascular reactivity. Correlation results suggest that as the change in ET-1 reduced during normoxic hypercapnia, the change in blood flow increased in the controls.