BritishJournal ofOphthalmology 1994; 78: 449-453

Optic nerve head blood flow using a laser Dopplervelocimeter and haemorheology in primary openangle glaucoma and normal pressure glaucoma

P Hamard, H Hamard, J Dufaux, S Quesnot

AbstractOptic disc blood flow velocity was measured inhealthy patients, those with primary openangle glaucoma (POAG), and patients withnormal pressure glaucoma (NPG). The velo-city of the red blood cells (RBCs) in thecapillaries of the optic nerve head (ONH) hasbeen measured with a laser Doppler veloci-meter (LDV), and blood viscosity has beenevaluated notably by determining the aggreg-ability of the RBCs with an erythroaggrega-meter. Our results in POAG patients and NPGpatients showed that their optic nerve bloodflow velocity was reduced and that the aggreg-ability of the RBCs was increased. The hyper-aggregability ofthe erythrocytes is responsiblefor the increase of the local viscosity in thepapillary capillary network. These haemo-dynamic modifications observed in patientswith glaucoma support the hypothesis of avasogenic mechanism that could impair theoptic nerve in glaucoma patients.(BrJ Ophthalmol 1994; 78: 449-453)

Centre HospitalierNationald'Ophtalmologie desQuinze-Vingts, 28 rue deCharenton 75012 Paris,FranceP HamardH HamardS Quesnot

LBHPURA 343 CNRSUniversity ofParis VII,2 place Jussieu, 75005Paris, FranceJ DufauxCorrespondence to:Dr P Hamard.Accepted for publication8 February 1994

The pathogenesis ofoptic nerve injury and visualdefects in glaucoma is not well known. Twodifferent theories have been proposed to explainthe optic nerve damage. The mechanical theoryemphasises that excavation of the optic nervehead is due to elevated intraocular pressure(IOP), with interruption of axoplasmic flow andlate optic nerve fibres degeneration. 2 However,optic nerve excavation and glaucoma visual fielddefects have been described without elevatedIOP, leading to the 'low tension' concept ofglaucoma. The vasogenic theory supports thehypothesis that hypoperfusion of the papilla isresponsible for optic nerve head impairment.3This approach is backed up by the fact thatglaucomatous field losses are also encountered incases of untreated systemic hypertension orhypotension, positive histories of major vascular

4crisis, coagulopathy, hyperviscosity, andvascular inflammatory diseases. Histological andfluorescein angiographic studies have also sup-ported this theory.35

In order to assess optic nerve head circulation,the blood flow velocity can be measured in agiven area of the papilla with a non-invasivetechnique using the laser Doppler velocimeter(LDV).6 It determines the maximum velocity ofthe moving cells in the papillary capillaries.However, to achieve the circulation in a capillarynetwork, blood viscosity must be taken intoaccount since it is known that the blood viscosityis proportional to the rate of flow of the blood,which is a non-Newtonian fluid; thus, the blood

flow rate is influenced by the blood viscosity.Blood viscosity itself depends on numerousfactors. However, in the microcirculation, it isessentially the capacity of the red cells to aggre-gate and to change form which controls theviscosity of the blood.78 Abnormality in theaggregability of the red blood cells has beendescribed in numerous diseases such as arterialhypertension,9 diabetes mellitus,'0 coronaryinsufficiency," and ocular venous thrombosis.'2

In an earlier study,'3 we showed that patientswith primary open angle glaucoma (POAG),whose IOP was controlled with topical hypotonictherapy, had a decrease in their papillary capil-lary blood flow velocity measured with LDV,and that they had an increase in their erythrocyteaggregability. These two findings suggested thattheir optic nerve head perfusion was impaired.

In this present study, we wished to investigatewhether patients with normal pressure glaucoma(NPG) had the same haemodynamic changes intheir optic disc as was shown in POAG patients,independently of IOP and of any glaucomatherapy. We measured the velocity of the redblood cells on the papillary area with an LDVand the aggregability of the red blood cells inNPG patients. The results were compared withcontrol and POAG patients.

Material and methodsWe selected three groups of patients: (1) a controlgroup of 14 healthy subjects (eight men and sixwomen); (2) a second group of 17 POAG patients(11 men and six women); (3) a third group of 18NPG patients (three men and 15 women).The patients in the study were a subset of

a larger population followed in the NationalOphthalmology Hospital Centre in Paris, for achronic open angle glaucoma or for a normalpressure glaucoma. Healthy volunteers with theappropriate inclusion criteria and without thepredefined exclusion criteria were chosen fromthe hospital staff.

Patients were excluded if they had any diseaseknown to modify the aggregability of the redblood cells or the blood flow velocity such as:arterial hypertension, hyperlipidaemia, diabetesmellitus, cardiovascular disease (in particularcarotid artery stenosis), smoking; and anypatients with other ocular disease excludingglaucoma. Patients were excluded if they hadpigmentary dispersion or exfoliation. Alsopatients with secondary glaucoma were excluded.The only concurrent treatments allowed werelocal hypotonic agents in POAG patients (1Badrenergic blockers for all patients except onetaking anticholinergic agents). Also patients

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Hamard, Hamard, Dufaux, Quesnot

were excluded who had undergone lasertrabeculoplasty or filtering surgery. All womenwere post-menopausal and none was receivingsubstitution therapy.

Furthermore, as it is difficult to focus the laserbeam correctly on the papillary area in patientswith ametropia (more than 3 dioptres myopia orhyperopia) or in patients with lens opacities,these patients have also been excluded.

Patients were included if they fulfilled thefollowing criteria:

(1) Control group: bilateral IOP less than 19mm Hg without treatment, normal visual field(automated perimeter, Octopus Gl program),and normal papilla (cup/disc ratio vertical andhorizontal (C/D0 5).During the study, all POAG patients had IOP

less than 20 mm Hg under local hypotonicagents.

(3) NPG patients: a diagnosis of bilateralnormal pressure glaucoma - that is, bilateral IOPless than 20 mm Hg without therapy (the IOPwas measured every 2 hours for 24 hours),bilateral glaucoma visual field defects, andglaucoma papillary excavation.The number of patients in the glaucoma

groups was insufficient to allow determination ofsubgroups according to the increasing severity ofvisual field loss.A velocimeter assessment was carried out in

each patient at the right and the left optic nervehead (ONH), and baseline measurements offibrinogen, plasma protein, a packed cellvolume, blood viscosity, and aggregability of theerythrocytes were carried out. All 4ata wereanalysed in a blinded manner as to whetherthe source was from patients or volunteers.Informed consent was obtained from all patients.

THE LASER DOPPLER VELOCIMETERWe use a prototype laser Doppler14 based on themodel described by Riva et al6 and built in alaboratory of the University of Paris VII(LBHP).A weak laser beam (power of0 025 mW on the

cornea, from an HeNe laser, wavelength 632 nm)is focused for 2 to 3 seconds on a papillary area of50 ,um, at a depth of between 100 and 200 iim asfar as possible from visible vessels. The light ispartly scattered by the tissue and the moving redblood cells, having undergone a shift in itsfrequency (Af), owing to the Doppler effect.This frequency shift is proportional to thevelocity of the red cells in the volume exposed tothe laser. The collected signal is then analysed. AFourier transform reconstitutes the frequencieswhich compose the spectrum and allows deter-mination of the maximum frequency shift of thelight (Afmax). The maximum velocity ofthe redblood cells (Vmax) in the volume exposed to thelaser can be calculated using the formula:

V(max)= 1/2.k. Af (max)

with V(max)=maximum velocity of the redblood cells; X=wavelength of the laser beam (632nm); Af (max)=maximum frequency shift.

THE HAEMORHEOLOGICAL PARAMETERSVenous blood samples were drawn from theantecubital vein and collected in various plastictubes according to the different assays - that is,anticoagulated with EDTA salt for packed cellvolume, with trisodium citrate for plasma fibrin-ogen, and heparinised for the erythrocyte aggre-gability study. Fibrinogen was assayed using achronometrical method, following the additionof thrombin. 15 The capacity of the red blood cellsto aggregate and disaggregate was determinedusing the following apparatus:

A Couette viscometer'6This viscometer enables measurement of theviscosity coefficient of a given blood sample indifferent flow conditions, at constant tempera-ture (37°C). The Couette system consists of twoconcentric cylinders, with an annular gap of 0 5mm. The shear rate is generated across the gapby rotating the outer cylinder, the inner cylinderremains stationary. The shear rate can vary from0-017 s 1to 128 s-l in 30 discrete steps. For eachshear rate, the viscometer calculates the corres-ponding in the value of blood viscosity.

An erythroaggregameter (Sefam)'7This apparatus uses a modified Couette visco-meter. The blood sample (1-5 ml) is placedbetween the two cylinders. The external cylinderis transparent and rotates at variable speedscontrolled by a microcomputer which allows theblood suspension to be exposed to varying shearrates between 7 and 400 s-1. An infrared laserbeam illuminates the blood sample through thetransparent external cylinder. The backscatteredlight is collected on a photodiode. The reflect-ivity signal is directly related to the mean bridg-ing area of the red blood cells per unit volume ofthe suspension at a given shear rate. The blood isfirst subjected for 10 seconds to a high shearing(400 s- ) which leads to a break up of anyaggregate and disorientates the red blood cells.The shearing is suddenly stopped, allowingaggregate formation. This corresponds to adecrease in the backscattered light. The blood isthen subjected to 40 decreasing shear rates,starting at 400 s-1 and ending at 7 s-i. Eachshear rate is applied for 5 seconds and suddenlystopped for 5 seconds. When the shear rateis insufficient to dissociate the packed redblood cells, the backscattered light amplitudediminishes. This given point corresponds to thelowest shear rate beyond which the red cells willno longer disaggregate and is called the disaggre-gation shear rate. This parameter along with thecorresponding viscosity is used to calculate thedisaggregation shear stress (T dyne/cm2) whichreflects the forces binding the erythrocytestogether as packed red cells.The Mann Whitney test and Student's two

tailed t test were used. Significance was con-sidered ifp

Optic nerve head bloodflow usinga laserDopplervelocimeter and haemorheology inprimary open angle glaucoma and normalpressure glaucoma

Table I Patient characteristics

Controls (n=14) POAG (n=17) NPG (n= 18)

Mean age (years) (SD) 44-8 (12 7) 51-6 (11-6) 57-4 (5 4)tMean packed cell volume (SD) 42-7 (2 9) 42-2 (4 2) 39-2 (3 5)Mean plasma protein (g/l) (SD) 68-3 (30) 70-6 (4 4) 70-2 (3 2)Mean fibrinogen (g/l) (SD) 2-7 (0 3) 3-1(0 8) 3-1(0 6)

*Not significant. tp

Hamard, Hamard, Dufaux, Quesnot

viscosity with four parameters of blood viscosityobtained at four shear rates chosen between0 03 s-1 and 400 s-1. The use of such a modelrequires matching the curve to four points, andwe have to extrapolate the values of the viscosityat shear rates inside the reference values. Thus,this model only provides approximate values andnot the real viscosity at a given rotation point.Furthermore, the curve starts at a very low valueof rotation (003 s- 1), a point at which false lowvalues for viscosity can be interpreted: this is oneof the limitations of the use of the viscometer.

Klaver," Trope,'9 and Foulds2" showed anincrease in blood viscosity in patients sufferingfrom glaucoma, without giving any definiteexplanation for the exact cause. There is nopublished evidence that the increase in bloodviscosity could be explained by an anomaly in theaggregability of the erythrocytes. However,Fouds's findings20 suggested that the aggreg-ability of the red blood cells could be increasedsince he showed that blood viscosity measuredwith a Contraves viscometer tended towardshigh values at low shear rates. It is well estab-lished that the aggregability of the red cells isresponsible for the values of the blood viscosityat low shear rates,7 but the aggregability of thered cells was not measured directly as this can bedone with an erythroaggregameter.'7

It is known that blood viscosity depends onseveral parameters: packed cell volume, plasmaviscosity,22 especially the level of fibrinogen andproteins,23 and capacities of the red cells toaggregate and to deform. In our study, we foundno difference beween the level of packed cellvolume in the control group or in patients withglaucoma. Elsewhere, we found no increase inplasma viscosity in patients with glaucoma, sinceplasma protein levels, notably the level of fibrin-ogen, were similar to those in control subjects.This agrees with previous studies'9 but it is incontradiction with the results of Garcia24 whoshowed an increase in the level of proteins inPOAG patients. The increase in blood viscosityat low and high shear rates could be explained,therefore, by modifications directly linked withthe erythrocytes - that is, modification in thecapacity of the red cells to disaggregate and todeform. In a recent study, Mary25 showed thatthe deformability of the erythrocytes wasimpaired in POAG patients. However, this studyhas failed to detect a modification in the aggreg-ability of the erythrocytes in POAG patients.This discrepancy with our results has beendiscussed elsewhere.'3

Erythrocyte hyperaggregability and decreasein the blood flow velocity in the optic disc seem tobe common factors in both types of glaucomastudied. But we could not prove the connectionbetween the hyperaggregability of the red cellsand the decrease in the papillary blood flowvelocity. This means that factors other thanblood viscosity are reponsible for the blood flowvelocity in the ONH capillaries. One possibilityis that the decrease of the ONH blood flowvelocity depends on the level of the IOP. Wefound that the mean IOP in thePOAG group wasslightly higher compared with the mean IOPin the healthy group. However, we found nostatistical difference between the mean IOP in

the NPG group and the healthy group althoughthe decrease in the maximum frequency shiftswas the same in the POAG and the NPG group.The effect of IOP on the decrease in the bloodvelocity in the papillary capillaries cannot beexcluded but the maximum threshold IOPbeyond which the ONH circulation could beaffected should be determined. A second poss-ible explanation for the decrease of the ONHblood flow velocity could be the pathologicalstructure of the optic nerve head caused bycupping in optic atrophy. If the nutrient capil-laries that are located within the laminar sheetsare compressed and their direction modifiedbecause of the misalignment of the pores of thelamina cribrosa, the blood velocity can decrease.The consequence is that the local shear rates willdecrease and will become insufficient to dissoci-ate the red cell from each other especially if thereis an impairment, even minor, in the aggreg-ability of the red blood cells. The packed redcells will in turn reduce the blood velocity. Thisis the haemorheological vicious circle.There are other factors which could modify

the blood flow velocity in response to an haemo-rheological anomaly, in particular the levelof papillary autoregulation, which might bedifferent depending on the severity of theglaucoma.2"2" Thus, the blood flow velocityresponse to the same haemorheological anomalycould be different in two patients. Finally, thelack of correlation between the decrease in theONH blood flow velocity and the increase in theaggregability of the red cells in these glaucomapatients could be explained by the presence ofanother haemorheological anomaly - namely,impaired deformability of the red cells. Indeed,in our study, the hyperaggregability of the redcells was not linked with modification of theplasma environment, but might be caused bymodifications of the coating of the red blood cells- that is, modification in the capacity of the redcells to deform as shown by Mary et al.25Even if the hyperaggregability of the erythro-

cytes that we observed in our glaucoma patientscannot alone explain the decrease in the papillaryblood flow velocity, this does show that theremust be a vascular pathology which adds to orhelps the degeneration of the nervous fibres inglaucoma.

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