Measurement of Local Partial Pressure of Oxygen in the Brain Tissue under Normoxia and Epilepsy with Phosphorescence Lifetime Microscopy

Phosphorescent probe Oxyphor G4

Phosphorescence lifetime microscopy has been used previously to measure pO₂ changes through phosphorescence of exogenous probes. Early phosphorescent probes, based on Pd porphyrins [13,28], required pre-binding to a macromolecular carrier (e.g. albumin) in order to enhance their aqueous solubility and bring their quenching parameters into the range compatible with physiological oxygen concentration [13,29]. Moreover, the albumin was a potential source of toxicity. Recently, Esipova et al. [30] developed a new probe, Oxyphor G4, which is free of these limitations. Oxyphor G4 is derived from Pd-meso-tetra-(3, 5-dicarboxyphenyl)-tetrabenzoporphyrin (PdTBP) and belongs to the group of dendritic oxygen probe [31]. It is highly soluble in aqueous environments and does not permeate biological membranes. It can operate in either albumin-rich (blood plasma) or albumin-free (interstitial space) environments at all physiological oxygen concentrations, from normoxic to deep hypoxic conditions. Oxyphor G4 used in these studies was obtained from Oxygen Enterprises Ltd (University of Pennsylvania, Philadelphia, PA 19104–6059, USA). Received Oxyphor G4 was calibrated before the experiments, first equilibrated with room air (21% O₂) and then with a completely deoxygenated solution at various temperatures (results of calibration are shown in Fig 1). In vivo the measured parameters (K_q and τ₀) were selected at temperature ~37°C. The phosphorescence lifetimes of Oxyphor G4 range from ~23 to ~215 μs in the physiological pO₂ range (160 mmHg-0 mmHg).

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Fig 1

Temperature dependencies of oxygen quenching constants (Kq) and lifetimes (τ0) for G4 (a and b).

The measurements were performed using 50 μM solutions of the probes, pH 7.23.

Animal preparation

Animals were used according to the recommendations of the Canadian Council on Animal Care, and all procedures were approved by Animal Research Ethics Committee of the Montreal Heart Institute (Permit Number: 2013-32-01). Eight male C57BL/6 mice (8 weeks old, 20-25g weight) were anesthetized by injection of urethane (2 mg/g body weight) in a 10% (wt/vol) saline solution. Body temperature was maintained at 37°C with controlled heating blanket. Mice were ventilated via a tracheotomy using ambient air. After positioning mice on a stereotactic frame, the scalp was retracted, and 5 × 5mm sections of the skin were removed over the coronal suture around bregma (AP: -1.5 mm DV: +1.5 mm). The somatosensory cortex was exposed and the bone was removed over a region along the coronal suture closer to the bregma. Following brain exposure, 500nL of 50μM solution of Oxyphor G4 was injected in the tissue via a 34G bevelled syringe with a microsyringe pump controller (uMC4, World Precision Instruments, Sarasota, FL) over a period of 10 min. The syringe was lowered to a depth ~300μm for injections. A glass coverslip window (5mm in diameter) was then installed using agarose gel and fixed with dental acrylic cement.

For epilepsy experiments, epileptiform activity was induced in 6 mice by injecting of 500nL of the K⁺-channel blocking agent 4-AP (A78403, Sigma-Aldrich, St. Louis, MO) solution (100mM), mixed with Oxyphor G4, through a syringe pump controller, similar to the injection of Oxyphor G4 alone described above. A higher concentration of 4-AP was required in this study to generate regular seizures, mixing with G4 may reduce the potency of 4-AP thus requiring these higher dosages. Following injection, a tungsten microelectrode (0.5–2 MΩ) was placed around the glass coverslip and inserted ~500μm into the cortex with an angle ~30° (leading to a depth of 250μm) in order to record extracellular local field potential (LFP). At the end of experiments, animals were sacrificed by cervical dislocation while under anesthesia.

Phosphorescence lifetime microscopy setup and pO₂ estimation

The confocal lifetime system is depicted in Fig 2. The excitation light is provided by a laser diode at 637 nm (Thorlabs: HL63133DG), controlled by a data acquisition (DAQ) board (National Instruments USB-6343). A galvanometer mirror positioning system (Thorlabs: GVS002) guides the excitation beam to selected points in the focal plane with a telescope consisting of two convex lens (f₁ = 50 mm and f₂ = 125 mm). The response time of galvanometric scanners was 3ms. A 10×magnification objective lens (Olympus PLN10x NA = 0.25) is used to focus the light onto the sample. The phosphorescence light travels back through the telescope to be separated by a beam splitter (BS2, Semrock: FF685-DI02-25×36) and a band-pass filter (Semrock: FF02-809/81-25, 768.5 nm ~ 849.5 nm), so that only the emitted phosphorescence signal is collected by a photon-counting avalanche photodiode (APD) (Micro Photon Devices: PDM series), whose active area (50 μm in diameter) functions as a pinhole. The APD amplifier outputs a TTL pulse for each detected photon and the pulsed are counted by the DAQ board. A second beam splitter (BS1, Semrock: FF520-DI02-25×36) is placed between the objective lens and the telescope to be used in combination with a camera (Thorlabs: DCC1545M) to gather anatomic references during experiments using LED illumination. The system is controlled by a computer running custom-designed software written in Matlab (The MathWorks, Natick, MA). The software allows adjustment of the length of the excitation pulse (temporal gate), and selection of points at which pO₂ values are measured. The in-plane resolution of the system in non-diffusive media is ~1.9 μm and axial resolution ~20.1μm. Assuming a Gaussian beam, the radius of each measured region is ~1.5 μm. In all experiments below, the excitation power of laser diode after the objective was kept below 6.5mW.

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Fig 2

(a) Schematic of the confocal lifetime imaging system. Excitation light is provided by a laser diode (λ = 637nm, 170 mW maximum power, which is collimated by a convex lens (L1) and travels through the objective for illumination. It is focused onto the cranial window by a 10×magnification objective (Obj), which is directed to the specific points using galvanometric scanners (xy). Emitted phosphorescence light is separated from excitation light using a beam splitter (BS2) and filter (F) and detected with an avalanche photodiode (APD). The system is controlled by a computer through a data acquisition card (DAQ). (b) In vivo measurements of pO₂vs mean counts per millisecond as controlled by the diode laser power. Higher laser powers correlate with higher consumption of O₂ leading to a significant decrease of pO₂ estimates over time (seen in the first point when average counts exceed 10000). When limiting to 3000 average counts, no significant decrease in pO₂ could be measured over time. (c) Example of phosphorescence decay profiles under conditions where photo-consumption is negligible. Higher O₂ concentration causes more quenching of phosphorescence signal, and consequently a faster decay (red profile).

Experiments are performed in two steps. Focus is first achieved on the cortex and the animal is moved towards the objective to measure tissue at a depth of ~100 μm. Initially, a few points of interest are selected for measuring pO₂. Survey scan phosphorescence recordings are obtained by scanning these points and summing photon counts at each point using an excitation pulse duration and phosphorescence detection window of 100 μs and 500 μs respectively. From the survey scan counts of these points, the power of the laser diode for each point is adjusted for ensuing scans to minimize the photo-oxidative damage to tissue and limit consumption according to the number of measured counts in the survey. A value of counts around 3000 was sought based on Fig 2(B). Thus following survey scans, the same points are measured while averaging 100 times to increase the signal-to-noise ratio at varying laser power. Representative excitation and decay profiles are provided in Fig 2(C). Consequently, each pO₂ measurement at a given location required around 250 ms.

All data was processed using software custom-written in Matlab. Using a weighted least squares fitting routine, the resulting time decay curves at each point are fit with a single-exponential function:

where I(t) represents the light intensity at time t and I₀ is the initial value of light intensity at time t = 0. Here τ is the phosphorescence lifetime at the point being measured and c is the magnitude of the baseline I₀. Following the estimated parameter τ, the Stern-Volmer formula was applied to calculate the pO₂ values.

Normal and variable F_iO₂ experiments

The system was first tested in vivo by monitoring pO₂ in cortical tissue near an artery and by varying the fraction of inspired oxygen (F_iO₂). In the first mouse, pO₂ values were measured in cortical tissue near the artery during the normal atmospheric gas fraction, which the F_iO₂ is 21%. In a second mouse, the inspired gas fractions was changed from normal to higher F_iO₂ from 21% to 40% for 10 minutes at t = 260s. During each experiment, pO₂ values were measured at multiple selected positions.

Tissue oxygenation at the focus and in the surrounding area during seizure-like activity

Seizure-like activity was elicited with injection of 4-AP and recorded by local field potentials with a tungsten electrode, and were characterized by fast rhythmic spiking activity of increasing amplitude and decreasing frequency, evolving into rhythmic spikes and slow wave activity prior to gradual offset (see e.g. Fig 5). One hundred and twenty-one (121) seizure-like events were recorded in 6 mice with mean (±SE) duration of 68.9±35.1s.

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Fig 5

Electrophysiology of 4-AP induced epileptic activity.

Top: example of ictal discharges after the 4-AP injection. Middle: zoom on an ictal discharge. Bottom: expanded view of showing the onset of the discharge, the intermediate phase and the offset.

To study simultaneously changes of tissue oxygenation at the focus and surrounding areas, points were selected both adjacent and distant from injection site in 3 mice (Fig 6A). An example of change in pO₂ in the focus and surrounding areas from a single animal is shown in Fig 6B. At the focus, the typical pO₂ profile was biphasic with an early dip after ictal onset (deoxygenation), followed by a longer duration increase in pO₂ (hyperoxygenation). The early dip in the focus was described in previous papers during seizures [7] and was present in most seizures measured here. At a distance from the ictal focus, the pO₂ was monophasic and significantly increased, returning to the baseline at the offset of the seizure. These results were in agreement with tissue oxygen measured by oxygen microelectrodes [33]. To assess the spatial distribution of pO₂ around the focus, points were scanned near the focus in the form of a spiral during epileptic activity, where initial dips were measured. Fig 6C shows an example of measured percent of initial dip for different locations overlaid on a grayscale anatomical image. The higher values were obtained near the focus, and pO₂ decreased when points were farther away from the focus. These data indicated that the influx of blood into the focus was inadequate to perfuse the hypermetabolic neurons, after which there was a period of hyperperfusion and hyperoxygenation.

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Fig 6

Obtained pO₂ values in tissue near the focus and surround.

(a) Grayscale angiogram of cortical surface and locations for pO₂ measurement (red: focus; blue: surround). The artery was shown by the red arrows. Scale bar size: 0.2mm (b) Epileptic activity induced a transient dip in tissue pO₂ followed by an increase in pO₂ in the focus. A sustained increase in pO₂ was seen in the surround. The dashed vertical lines show the ictal onset (left) and offset (right). (c) Distribution of percent of initial dip at multiple locations (color dotted) during epileptic activity. The 4-AP injection site is shown by green circle. The artery was shown by the red arrows. Scale bar size: 0.2mm.

Correlation between the percent of initial dip and distance from the artery

To study oxidative metabolism near arteries during epileptic activity, 3 mice were recorded with measures at multiple locations near an artery that was close to the injection site. The percent pO₂ value changes adjacent to the artery were significantly lower than values located farther away (an example shown in Fig 7A) despite some points being closer to the focus. Fig 7B shows a linear relationship between arterial perpendicular distance and percent of initial dip which indicates a contribution of the vascular anatomy to define the focus-surround regions. Sites that were farther from an artery, located in the capillary bed, elicited a larger decrease in tissue pO₂ after onset. Extending data to the three mice, the slopes of these linear fit were combined over all seizures in Fig 7C, in all cases preserving the positive relationship. This data indicates that the vascular micro-environment contributes to oxygen consumption in the tissue during epileptic seizures.

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Fig 7

Correlation between percent change of the initial dip at multiple locations and distances from an artery (a) Measured pO₂ values of different points near an artery during the epileptic seizure (color dots), overlaid with a grayscale anatomy (with an artery shown by the red arrows). Scale bar size: 0.2mm (b) Relationship between distance from an artery for multiple points and initial dip during one epileptic seizure. The line of linear fit is y = 0.32x + 13.7, R² = 0.7182. (C) Boxplots of slopes of linear fits in 3 mice over 25 seizures.

Tissue pO₂ gradient near arteries in normoxia

Our work confirmed previous evidence that arteries are largely responsible for the heterogeneous oxygen distribution in the cortex. The observed variations in tissue pO₂ values near brain arteries showed a drop in the pO₂ values of points distant from an artery and located in the capillary bed during normoxia (Fig 3). Similar pO₂ gradients were also found by several investigators employing different methods [27,32]. For instance, near the pial artery in the rat cortex, a similar tendency was reported by Sakadzic et al. [27] with two-photon phosphorescence lifetime measurements. In addition, several theoretical models have predicted that steep pO₂ gradients arise in the vicinity of blood vessels [34,35]. Localized large tissue pO₂ values suggests that arteries provide a major source of O₂ to tissue [36] while our pO₂ values of locations far away from arteries (above ~60μm) most likely rely on capillaries which are invisible from the cortical surface [37].

Acute seizure activity leads to transient dip of pO₂ near epileptic focus

Some controversy remains in the literature as to whether local cerebral blood flow increases are adequate to meet supranormal oxygen demands throughout the ictus. Although the inadequacy of cerebral blood flow in addressing oxygen demands has been demonstrated by some investigators, there is also growing evidence of inadequate oxygenation at the onset or throughout shorter duration epileptic events [38,39]. In this study, a clear transient decrease in tissue oxygenation at multiple locations near the injection site after onset was shown, indicating that the increased metabolism of oxygen overwhelms the ability of the brain to provide oxygenated blood by increasing cerebral blood flow during the seizure-like activity in and near the focus. This observation may be useful at predicting the location of seizures. A significant increase in tissue oxygen consumption at multiple locations close to the focus was observed in the somatosensory cortex when mice had seizure-like activity (Fig 6C). Moreover, farther away from the focus there was increase in tissue oxygen with respect to baseline.

Previous literature using oxygen microelectrodes documented a transient dip in two locations: near the focus and an increase in the surround in epileptic rats during seizures [33]. In our present work, which focused on tissue oxygen changes at multiple locations (>2) during acute epileptiform events, we show a significant initial dip at multiple locations in our mice. With the ability of our system to gather spatial measures, our results indicate that the distance between the surround and the injection site (around 1.5mm) was a little smaller than what was measured in previous work (around 2mm) [33]. Whether the observed difference is due to changes in animal species, high metabolic demand at seizure locations near vessels remains to be investigated.

The relationship between the initial dip and distance from arterioles

Exploiting the spatial measures, we investigated how tissue oxygen pressure changed at different points near a surface arteriole located in the focus region during epileptiform activity. Despite increased consumption in the focus, arteriolar O₂ diffusion remained partially unaffected: a significant increase in the initial dip was observed with increased distance from the arteriole (see Fig 7) in three mice. These data are the first showing measurements of pO₂ gradients in brain arterioles during epileptic activity confirming that the arteriolar wall played a significant role in oxygen exchange between blood and tissue [32]. Our microscopic assessments thus paint a more complex spatial picture of oxygen consumption during epileptic seizures since arteries take part in diffusional exchange of respiratory gases (mainly oxygen) to tissue. This is in contrast to previous studies showing uniform cerebral blood volume (CBV) and cerebral blood flow (CBF) increase in the focus during epileptic events [33,40] to supply oxygen to meet demands of neuronal activity. The vascular architecture thus generates a microscopic structure to the epileptic focus, as our data showing increased initial dips far away from arteries, in capillary beds, suggest. Our results further suggest that the increased CBF and CBV will supply more oxygen to the tissue near an artery, but may not meet the demands of oxygen metabolism far away from an artery. Potential associated tissue and neuronal damage is thus more likely to occur, microscopically, in areas far from these feeding arteries.

The relationship between initial dip and seizure duration

Our data indicate that initial dips of greater amplitude are predictive of seizures of greater duration. Few study has investigated such a link between oxygen metabolism and seizure duration. However, some researchers observed that a biphasic deoxyhemoglobin (HbR) response to ictal events with an initial decrease in HbR followed by a longer increase in HbR measured by NIRS may be related to longer seizure duration [39]. These previous findings proposed that increased seizure duration could lead to increased oxidative metabolism. The etiology of tissue oxygen changes as the duration of seizures is increased remains unknown. The possibility is that longer seizures were induced by increased initial neuronal activities, which will lead to more decrease in tissue oxygenation [41,42]. Our study indicates that it might be possible to predict seizure duration from the initial dip amplitude.

We thank S. Vinogradov for discussions and generously sharing the G4 molecular probe and Marc-Antoine Gillis for his assistance in animal preparation.