2.1.

Phosphorescence Quenching by O ₂

For in vivo measurements of pO _2,blood , metalloporphyrin molecules capable of phosphorescing are bound to serum albumin before being injected intravenously into the blood stream. A brief flash illuminates the tissue of interest and excites the porphyrin molecules to a triplet state. The excited molecules return to their ground state either through a radiative process (phosphorescence) or by transfer of energy to other molecules (quenching). When measured from a solution containing a large number of quenching molecules, the phosphorescence intensity decays exponentially with a time constant (τ), which represents the time at which the initial intensity is reduced by 1/e. The only significant quenching agent present in the blood being²⁸ O ₂, τ is related to pO _2,blood through the Stern–Volmer equation: τ₀/τ=1+k_Qτ₀pO _2,blood , where τ₀ is the probe lifetime in the absence of a quenching agent, and k_Q the quenching constant.

2.2.

Phosphor

The oxygen probe used for PQ measurements is a Pd-meso-tetra (4-carboxyphenyl) porphine (Oxygen Enterprises, Pennsylvania). This compound must be bound to serum albumin before systemic injection. Therefore, blood is drawn from the animal (∼4 ml/kg) and the serum is extracted by centrifugation at 8000 rpm for 10 min. The probe (20 mg/kg of animal weight) is mixed with the serum, then the pH is adjusted to 7.4, and the serum/probe solution filtered with 0.2-μm sterile filter. The solution is infused intravenously (∼100 ml/h) and allowed to equilibrate with the blood before measurements proceed. The binding of the phosphorescent probe to albumin ensures that the parameters τ₀ and k_Q are constant over the range of physiological pH and temperature. The probe’s sensitivity to O ₂ is also enhanced and its self-quenching is reduced. Since albumin does not diffuse across the wall of the retinal and ONH blood vessels due to the blood retinal barrier, PQ measurements from these tissues provide only data on O ₂ dissolved in blood.

In saline solution, the probe absorbs maximally at 412 and 528 nm and its emission spectrum is centered at 706 nm. In the physiological range of pH and blood temperatures (i.e., 7.2 to 7.4 pH unit and 36 to 38 °C, respectively), τ₀ decreases by ∼0.8/°C and ∼1 per 0.1 pH unit, whereas k_Q increases by ∼3/°C and ∼1 per 0.1 pH unit.²⁹ Thus, keeping the pH and temperature within the ranges already specified induces a maximum error of ∼6 when evaluating pO _2,blood based on phosphorescence lifetimes greater than 25 μs (pO _2,blood <100 mm Hg). In this study, we used²⁹ the values of τ₀ and k_Q corresponding to a pH of 7.4 and a temperature of 38 °C (i.e., 637 μs and 381 mm Hg⁻¹ s⁻¹, respectively).

2.3.

Ocular Fundus Phosphorometer

Figure 1 schematically shows the phosphorometer built to measure pO _2,blood in the fundus of the eye. The optics of this device have been mounted on a fundus camera (Zeiss, Germany). To illuminate the fundus with the excitation light, the flash lamp normally supplied for fundus photography was replaced by a flash lamp (F) with shorter pulse width. This unit consisted of a 60-W, 2-μs flash width at half-maximal intensity (Oxygen Enterprises, Ltd., Pennsylvania). An interference filter (IF, XM-530, Corion™, United States; center wavelength=530 nm, FWMH=25 nm) placed at the lamp output transmitted the spectral portion of the flash light that matched the absorption spectrum of the phosphorescent probe. Each flash delivered an energy of ∼25 μJ/cm² over a 30-deg area at the posterior pole. Diffuse illumination of the fundus for alignment or light adaptation of the eye was achieved through the conventional illumination path of the camera starting at the 50-W tungsten lamp (L) and traversing the beamsplitter (BS). The irradiance of the white illumination light measured at the eye fundus was 2 mW/cm².

Figure 1

Zeiss fundus camera modified to incorporate the various optical components necessary for PQ and laser Doppler flowmeter (LDF) measurements: flash source for dye excitation (F), interference filter (IF) for spectral selection of the excitation light, fundus illumination lamp (L), beamsplitter (BS) in the illumination pathway, 670-nm laser diode (D) for LDF measurements, motorized mirror (M) to select PQ or LDF detection pathway (M), long-pass filter (LP) for spectral selection of the phosphorescent light, pinhole (PH) for spatial detection of the phosphorescent light, photomultiplier tube (PMT) for detection of the phosphorescent light, optical fiber (OF) for spatial selection of the laser light, avalanche photodiode (APD) for detection of the laser light.

The phosphorescent emission was collected from a localized area (Ø≅200 μm) of the optic disk through the optics used for fundus observation. This area was spatially defined by a pinhole (PH) placed in a retinal image plane of the fundus camera. The phosphorescence was detected by a photomultiplier (PMT) having its maximum sensitivity in the red portion of the spectrum (R928, Hamamatsu, Japan). A long-pass filter (LP, RG630, Schott, Germany) was mounted in front of PH to enhance the phosphorescence detection. Its transmission range was 650 to 800 nm.

The signal at the output of PMT was amplified, then digitized at 1 MHz and fed into an IBM PC compatible computer. Labview (National Instrument, Austin, Texas) software was developed to control the flash triggering, acquire and analyze the phosphorescence data and calculate the pO _2,blood . To eliminate potentially undesirable contributions to the pO _2,blood resulting from the tail of this emission, the recording of the digitized photocurrent started 40 μs after and stopped 2360 μs after triggering the excitation flash. After removing all data points below the dark noise level of the detector, the pO _2,blood was obtained based on the average of 10 to 30 successive phosphorescence decay curves obtained at rate of 30 Hz. A linear regression optimized for noise correction (weighted linear fit)³⁰ was applied to the natural logarithm of all data points to calculate τ and pO _2,blood , which were then displayed on the Labview stripchart. The total acquisition and computation time for each value of pO _2,blood was approximately 2.5 s.

2.4.

Blood Flow Measurements

For the quasisimultaneous measurement of blood flow and pO _2,blood at the same location of the optic disk, an LDF was incorporated into the phosphorometer. The LDF technique has been previously described in detail.³¹ Briefly, a laser beam (670 nm) with long optical coherence is focused on a small area (Ø≅150 μm) of the optic disk, away from the larger vessels. A photomultiplier detects the portion of the laser light scattered by the red blood cells and the surrounding tissue that exits the eye pupil. The photocurrent is amplified, digitized and analyzed by a computer that calculates the relative flow (F _ONH ) of the red blood cells in the ONH volume sampled by the laser beam.

The LDF system was incorporated into the phosphorometer by having the beam from a visible laser (LDM115, Imatronic, United Kingdom, wavelength=670 nm) follow the optical path of the fundus illumination system of the camera and focusing it precisely at the center of the area from which the phosphorescence was detected. The light scattered by the red blood cells and surrounding tissue in the volume sampled by the laser beam was collected by an optical fiber (OF) placed at the image plane of the illuminated site and guided to an avalanche photodiode (APD). The photocurrent was analyzed using dedicated software running on a NeXT™ computer system to calculate the LDF parameters.³¹

A mirror (M) could be flipped from position 1, which reflected the phosphorescence toward the PH, to position 2 that enabled the scattered laser light to reach the optic fiber. Both PH and OFs were positioned in conjugated retinal image planes and “looked” at the same retinal location. Thus, the tip of the optical fiber indicated the location where pO _2,blood was measured. The proximity of laser and peak phosphorescence wavelengths did not allow simultaneous LDF and PQ recordings.

2.5.

Animals

Experiments were performed on 11 miniature pigs (∼10 kg). All procedures conformed to the Association for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in ophthalmic and vision research. After intramuscular injection of a tranquilizer for pigs (Stresnil™, Cilag Chemie AG, Switzerland), anesthesia was induced with 25 to 30 mg of Hypnodil™ (chlorhydrate of metomidate) injected into the ear vein. After arterial and venous catheterization, the pigs were curarized (Tubocurarine™, 1 mg/10 kg), intubated and artificially ventilated. During the experiment, anesthesia was maintained by continuous perfusion of Nabutal™, 4 ml/h, and Tubocurarine™, 0.1 mg/h. The animals were ventilated at approximately 18 strokes/min, with a continuous flow of 20 O ₂+80 N ₂ O using a variable volume respirator (Siemens™ volumeter). The head was secured to avoid movements. Systolic and diastolic blood pressures were monitored via the femoral artery using a transducer (Mingograph™, Siemens-Elema, Switzerland). The systemic arterial pO ₂, pCO ₂, and pH (pO _2,a, pCO _2,a, and pH _a, respectively) were measured intermittently from the same artery with a blood gas analyzer (AVL, automatic gas system 940). Adjustments of ventilatory rate, stroke volume, and the composition of the inspired gas maintained values of pCO _2,a of 30 to 35 mm Hg and pO _2,a of 100 to 110 mm Hg. A rectal thermometer was used to measure the animal’s temperature, which was maintained between 36 and 38°C by means of a thermoblanket with temperature control unit.

2.6.

Protocols

All PQ and LDF measurements were performed at a rim location of the optic disk and care was taken to avoid as well as possible the large retinal vessels visible at the disk surface.

In the 11 animals, pO _2,blood measurements were collected intermittently during 7:25±1:45 (standard deviation) hours after dye injection at an average of rate of ∼5 min⁻¹. Between PQ measurements that were performed in total darkness, the diffuse white fundus illumination (2 mW/cm²) was occasionally turned on for alignment.

The PQ and LDF techniques were employed alternatively to record the pO _2,blood and F _ONH response to three different ventilation mixtures, each of which was tested on one animal. After a recording of the baseline pO _2,blood and F _ONH with the animal ventilated with air (20 O ₂+80 N ₂ O) for approximately 1 min, the breathing conditions were changed. One animal was given 100 N ₂ O to breathe until death. The second animal received 100 O ₂ for 10 min and the third one breathed a CO ₂ enriched air mixture (20 O ₂+5 CO ₂+75 N ₂ O) for 12 min. The pO _2,blood and F _ONH were recorded alternatively.

3.1.

pO _2,blood Time Course

All pO _2,blood values collected in the 11 animals were grouped according to the time of their measurement after the phosphor injection (30-min groups). For each group, the mean pO _2,blood (± standard deviation) was calculated and plotted versus time (Figure 3). A significant decrease of pO _2,blood (ANOVA, p<0.0001) was observed, although during the first 120 min of the PQ measurements (period I), the pO _2,blood did not change (ANOVA, LSD post hoc test, p>0.05) and had an average value of 24±1.4 mm Hg. Thereafter (period II), it decreased steadily to a lower level of 7±0.9 mm Hg after 400 min (ANOVA, LSD post hoc test, p<0.05) . In most animals, observations of the eye fundus at this time of the experiment revealed constrictions and even closure of some arteries and veins in the area that had been repeatedly excited for PQ measurements. The retina and ONH tissues were edematous. A breakdown of the blood-retinal barrier was revealed by the leakage of fluorescein observed on angiograms (not shown). In one animal, the contralateral eye, which remained in complete darkness during the 375 min of the PQ investigations on the fellow eye, was prepared for PQ measurements. It appeared entirely normal and pO _2,blood averaged over different sites of the disk was 19±2.2 mm Hg. Fluorescein angiography performed on this eye did not show any early or late leakage of the dye.

Figure 3

All the pO _2,blood values measured in 11 animals were grouped according to their delay after the phosphor injection (30-min groups). The average value ± standard deviation is plotted here as a function of time. The pO _2,blood did not vary significantly [analysis of variance (ANOVA), p>0.05] during the first 100 min (period I) but then (period II) dropped significantly (ANOVA; p<0.001) .

3.2.

Measurements of pO _2,blood and F _ONH

In the animal that was ventilated with 100 N ₂ O. The pO _2,a dropped continuously, which produced a F _ONH increase of 75 and a pO _2,blood decrease of 60 (Figure 4).

Figure 4

In one animal, pO _2,blood (dots) and F _ONH (line) were simultaneously measured at the peripheral ONH during an acute hypoxemia. At time 0, a ventilation with 0 O ₂ was used. After 2 min, pO _2,blood was decreased by 60 while F _ONH increased by 75.

In the animal ventilated for 10 min with 100 O ₂, blood gases taken during normoxia and after 5 min of hyperoxia showed an increase of pO _2,a from 109 to 356 mm Hg. Consecutively, a 25 pO _2,blood increase and a 23 reduction of F _ONH were measured (Figure 5). Both pO _2,blood and F _ONH returned to baseline levels after the breathing was back to normal.

Figure 5

Recording of pO _2,blood (dots) and F _ONH (line) at the peripheral ONH during hyperoxia (one animal). After 90 s of baseline, the ventilation was switched to 100 O ₂. The F _ONH decreased by 22.8 while pO _2,blood increased by 25. The ventilation was switched to normal after 700 s and both parameters went back to their baseline levels.

The animal ventilated with 20 O ₂+5 CO ₂+75 N ₂ O became hypercapnic since the pCO _2,a increased from 34.3 to 45.6 mm Hg. Simultaneously, pO _2,blood and F _ONH increased by 24 and 12, respectively (Figure 6).

Figure 6

Recording of pO _2,blood (dots) and F _ONH (line) at the peripheral ONH during hypercapnia (one animal). After 2 min of baseline, the ventilation was switched to 20 O ₂+5 CO ₂+75 N ₂. The pO _2,blood increased by 12 and F _ONH by 25.

4.1.

Range of pO _2,blood

Several phosphorometers have been developed for studying pO ₂ in various biological tissues (muscles, brain, intestine, kidney, tumors).^{21 32 33 34 35 36 37} However, the device presented here is the first that enables focal measurements of pO _2,blood in the ocular fundus. As in the brain, the endothelial cells covering the inner surface of the retinal and ONH vessels in the healthy eye present tight junctions, which prevent the diffusion of soluble molecules such as albumin from the blood into the interstitial tissues (blood-retinal barrier). Therefore, our technique of phosphorescence quenching by O ₂ assesses only the phosphorescence emitted by the dye inside the vessels. Values of pO _2,blood in the ocular fundus were already measured in pigs and cats with an imaging technique using the phosphorescence quenching principle.^{22 24} These studies reported ONH pO _2,blood values of 35±9 and 33 mm Hg, respectively, which were higher than those obtained at period I with our phosphorometer in 11 miniature pigs (24±1.4 mm Hg). Even if these two imaging techniques and our phosphorometer applied the same phosphorescence quenching principle, specificity of each instrumental setup or data processing could account for the different pO _2,blood range. As pointed out by Golub et al.;,^{38 39} the temporal characteristic of the flash impulse function influences the shape of the phosphorescence emission (convolution) and consequently its lifetime. Because the excitation light delivered by our instrumental setup reaches its peak intensity after 2 μs, whereas the flash used in these two studies peaked at about 10 μs, the phosphorescence decay emitted by a given tissue sample certainly differs when obtained with different instruments. Furthermore, with the ideal impulse function for time-resolved phosphorescence measurements being a Dirac function, we believe that our pO _2,blood measurements obtained with a shorter flash impulse are more accurate than those obtained with flashes of longer duration. Difference in the processing of the phosphorescence emission may also explain the differences between the pO _2,blood values obtained with various devices. Whereas Blumenro¨der et al.; used a Levenberg–Marquardt fitting routine for calculating the lifetime of the phosphorescence decay²² and Shonat et al.; a linear fitting routine,²⁴ we applied a fast, weighted linear fit optimized for noise correction to extract³⁰ τ. Another important element that has to be considered when comparing the various pO _2,blood values is that these were obtained in different species (minipigs, pigs and cats).

As the diffusion of O ₂ from the blood vessels into the interstitial ONH tissue depends on (1) the intravascular blood O ₂ content, (2) the hemodynamics, (3) the O ₂ consumption of the vessel wall, (4) the architecture of the ONH microvascular network, and (5) the metabolic tissue consumption in the ONH, to establish the relation between pO _2,tissue and intravascular pO _2,blood a diffusion model is required. For the optic nerve, with its complex multilayered vascular systems, such a model is lacking. The pO _2,blood values obtained at the ONH in our study are not at odds with pO _2,tissue values obtained by others that used O ₂ -sensitive microelectrodes. Thus, Bouzas et al.;¹² found a prepapillary pO ₂ of 33.1±3.9 mm Hg at juxta-arteriolar locations and 16.6±1.4 mm Hg at intervascular locations, both with the probe tip positioned at 50 μm in front of the ONH surface. Stefansson et al.;,¹⁷ using large microelectrodes with a 100-μm tip, reported an intervascular prepapillary pO ₂ of 24.1±11.6 mm Hg. At 50 and 200 μm under the surface of the ONH cup, pO _2,tissue was 9.6±1.2 and 9.4±0.8 mm Hg, respectively.¹² At similar depths in the ONH rim, pO _2,tissue was¹² 10.8±1.9 and 8.2±1.8 mm Hg. In comparison, pO _2,tissue measured between 0- and 200-μm depth into the optic disk of the cat range¹¹ from 15 to 10 mm Hg. These values measured in the depths of the nerve, compared to the higher values found at the surface of the disc, suggest that our phosphorometer predominantly measures pO _2,blood in the retinal layer of the nerve.

4.2.

Origin of the Phosphorescence

Due to the topography of the ONH, the depth of tissue sampled with the PQ technique is difficult to evaluate. The green excitation light used for PQ measurements, because of its absorption and scattering by the ONH tissue and blood, should predominantly interact with the dye molecules located in the vessels close to the ONH surface. However, measurements by Koelle et al.; using sections of the cat ONH suggest that green light backscattered at a depth of 400 μm from the ONH surface still contributes to the signal detection.⁴⁰ In their paper on PQ in the pig, Blumenro¨der et al.;²² concluded from the fact that the lamina cribrosa cannot be seen by green light ophthalmoscopy that the light does not reach this layer. This argument, however, does not take into consideration that most of the light penetrating the ONH tissue is scattered, a process that may prevent the observation of a clear image of the lamina cribrosa. Regarding the vascular origin of the phosphorescence, placing the pin hole away from the larger vessels at the surface favored detection of phosphorescence from dye circulating in the small arterioles, capillaries, and venules, vessels that are usually not visible with a fundus camera. Therefore, the detected phosphorescence is a superposition of contributions from arterioles, capillaries, and venules. It is expected that, since the venous blood volume exceeds the arterial and capillary blood volumes, the major contribution of the measured pO _2,blood comes from venules. This contribution is further emphasized by the fact that quenching is weaker for the vessels with low pO _2,blood than for those with high pO _2,blood .

4.3.

Time Course of pO _2,blood during Prolonged PQ Measurements

The decrease of pO _2,blood during the period II of the PQ measurements is probably due to a decreased blood flow, as suggested by the marked constriction of the vasculature and white appearance of the ONH tissue in the region illuminated by the excitation light that was observed toward the end of this period. As the maximum irradiance used for each pO _2,blood measurements (30 flashes×25 μJ/cm ²=750 μJ/cm ² at 530 nm) is below the maximum permissible ocular exposure⁴¹ for a continuous exposure of 1 s (30 flashes delivered at 30 Hz), the damage observed at the ocular fundus is not the consequence of a thermic process, but likely results from the photoactivation of our phosphor. Light activation of intravascularly administered fluorochromes is known to generate toxic oxygen species, including singlet oxygen and superoxide anions. Various deleterious microvascular effects such as platelet thrombus formation, arteriolar constriction, or postcapillary venular leakage have been reported and attributed to the luminal generation of oxidant species.⁴² These effects provide the basis of the photodynamic treatment of tumors.⁴³ As the quenching of an activated phosphor by an O ₂ molecule also produces a singlet oxygen molecule, extended pO _2,blood measurements with the phosphorescence quenching technique may lead to vascular damage.

In one animal, the contralateral eye was also investigated at period II (direct observation, pO _2,blood measurements, fluorescein angiography). In this eye, which remained in darkness for hours before, the ocular fundus looked normal and pO _2,blood was approximately 19 mm Hg. It shows that the porphyrin dye, if not optically excited, does not affect the pO _2,blood level, which supports the preceeding hypothesis on alterations due to photoactivation of the phosphorescent probe.

In our system, two sources of phosphorescence excitation susceptible to induce photoactivation were present. One was the flash for excitation of the dye, the other the white light used for fundus observation. According to the data of Shonat et al.;,^{33 35} the energy density delivered to obtain a pO _2,blood measurement (∼750 mJ/cm²) should not affect the tissue. We presume that it is the visible light used to occasionally observe the fundus that caused the most severe photoactivation and produced tissue alterations resulting in the drop of the pO _2,blood in period II.

Future studies will benefit from any attempt to reduce the likelihood of alterations due to photoactivation, such as improving the phosphorescence detection sensitivity and reducing the number of flashes needed for the average phosphorescence decay curve, decreasing the dye concentration and removing the spectral component of the observation light likely to induce photoactivation.

4.4.

Modification of the Ventilation Conditions

Previous PQ or LDF measurements at the ONH during hypoxemia reported that pO _2,blood drops,²² and F _ONH increases⁴⁴ by 67 to 89. Our measurement, albeit in only one animal, is in agreement with these results. It also illustrates the feasibility of quasisimultaneous measurements at the ONH rim of pO _2,blood and F _ONH , since 100 N ₂ O breathing induced a 60 decrease and a 75 increase of these parameters, respectively. This measurement illustrates the regulation capabilities of the ONH vasculature, which compensates the drop of the arterial blood O ₂ content by increasing blood flow.

LDF measurements performed during hyperoxia (100 O ₂ breathing) showed blood flow decreases of 39 to 49 in cats⁴⁴ and 25 to 38 in humans.^{45 46} Using the PQ imaging technique, pO _2,blood was also measured at the optic disc during hyperoxia in 6 piglets.²² In that study, pO _2,blood rose from 35±9 to 44±14 mm Hg, while pO _2,a was increased from 100 to 120 mm Hg with an O ₂ -rich ventilation. Our measurement during hyperoxia (100 O ₂ breathing) shows that pO _2,blood increased by 25, whereas pO _2,a raised by 226. The difference between the increase of pO _2,a and pO _2,blood indicates a regulation mechanism that prevents excessive amounts of O ₂ to reach the ocular microcirculation.

CO ₂ has a vasodilating effect on the blood vessels, which can enhance the blood perfusion. Our F _ONH measurement is in accordance with a study by Harris,⁴⁶ who reported a similar 28 increase of the ONH blood flow when using the same gas mixture. A higher perfusion apparently contributes to increase the supply of O ₂ to the capillaries since pO _2,blood increased by approximately 12.

The good agreement of the results obtained during hypoxemia, hyperoxia, and hypercapnia with the literature shows that pO _2,blood and blood flow measurements can be reliably measured quasisimultaneously in the microcirculation of the prelaminar optic nerve. This instrument is specifically adapted for investigating with a good time resolution the time course of transient pO _2,blood changes that could occur during various physiological perturbations.