2.1.

In Vitro Experimental Protocol

To provide a model for in vivo measurement of Hb in the piglet brain, we filled a 1.000-mL glass beaker with 0.1-M phosphate buffer (pH 7.4) containing 0.7% (w/vol) dry yeast in 1% intralipid and glucose (2% w/vol) to produce a solution with a light scattering condition equivalent to that of blood-perfused brain tissue. A magnetic stirrer was used to prevent particle settling. The measurement receiver fiber was set at a distance of 30 mm from the light source fiber, and the fibers were immersed directly in the suspension. The oxygen content was also monitored at the same time with a conventional gas analyzer (ABL5, Radiometer, Copenhagen, Denmark). In the first stage of the experiment, human blood was added gradually to the beaker to achieve blood volume ranging from 0 to 100 μM, and the resulting changes in the reemission profiles were recorded. Each model was bubbled with oxygen. In the second stage of the experiment, we used a model solution with a 80-μM Hb suspension, and the oxygen concentration in the solution was reduced to simulate anoxia in the brain. A mixture of O ₂ and N ₂ gases was prepared and bubbled through the suspension to manipulate the oxygen saturation of Hb from 0 to 100%.

Once μ_a had been determined at three wavelengths, the oxyHb and deoxyHb concentrations were calculated from the absorption coefficients of oxyHb and deoxyHb using the same law as that described in Sec. 2.4 with the assumption that background absorption is due to only 100% (by volume) water and that the refractive index of water is 1.3.

2.2.

Animal Study Protocol

Eleven newborn piglets, less than 48 h old and weighing 1.9 to 2.4 kg, were each anesthetized with an intramuscular injection of sodium pentobarbital (2 mg/kg). The umbilical vein and artery of each piglet were cannulated. After cannulation, each piglet was paralyzed with pancronium bromide at an initial dose of 0.1 mg/kg followed by infusion at 0.1 mg/kg h⁻¹ and then anesthetized with fentanyl citrate at an initial dose of 10 μg/kg followed by infusion at 5 μg/kg h⁻¹. The animals were then intubated and mechanically ventilated with an infant ventilator. A 24-gauge cannula was inserted into the sagittal sinus through a burr hole in the anterior fontanel for venous blood sampling.

The umbilical artery was used for blood pressure monitoring and arterial blood sampling. Infusion of maintenance solution was continued at a rate of 4 mL/kg h⁻¹ via the umbilical vein. Respiration and acid-base balance were checked by arterial blood gas analysis at each step of FI _{O 2} . A negative base excess lower than 5.0 mmol/L, which was caused by hypoxia, was corrected as much as possible by sodium bicarbonate infusion to maintain pH between 7.3 and 7.5, thus minimizing the shift in the Hb dissociation curve. The hematocrit of arterial blood was in the normal range, and mean arterial pressure was maintained above 60 mm Hg except near zero FI _{O 2} . Arterial Hb oxygen saturation (SaO ₂) and sagittal sinus venous Hb oxygen saturation (SvO ₂) were measured using a blood co-oximeter (OSM-3, Radiometer, Copenhagen, Denmark). To obtain aerobic and anaerobic conditions, FI _{O 2} was changed by mixing pure nitrogen and oxygen gases with continuous monitoring by an oxygen electrode. We first set FI _{O 2} at 27% and then at 100% to obtain hyperaerobic conditions. Then, to obtain anaerobic conditions, FI _{O 2} was decreased in steps from 100 to 4%. The hair on the scalp of the parietal region was removed with an epilating agent. Two optical fibers were brought into contact with the skin on the head of each piglet. Throughout the experiment, rectal temperature was monitored and maintained between 38.0 and 39.0 °C using a heated-water mattress. At the end of the experiment, animals were sacrificed with intravenous pentobarbital (100 mg/kg). The protocols for animal care were in compliance with institutional guidelines.

2.3.

Near-IR TRS System and Analysis

We used a portable three-wavelength TRS system (TRS-10, Hamamatsu Photonics K.K., Japan) and attached a probe to the head of each piglet with a light source fiber and light detector fiber separation of 30 mm. In the TRS system, a time-correlated single-photon-counting technique is used for detection. The system is controlled by a computer through a digital I/O interface consisting of a three-wavelength (761, 795, and 835 nm) picosecond light pulser (PLP) as the pulsed light source, a photon-counting head for single photon detection, and signal-processing circuits for time-resolved measurement. The PLP emits NIR light with a pulse duration of about 100 ps and an average power of at least 150 μW at each wavelength at a repetition of 5 MHz, and the input light power to the subject was around 20 μW because of the loss in an incident optical system of the TRS system.

The light from the PLP is sent to a subject by a source fiber with a length of 3 m, and the photon reemitted from the subject is collected simultaneously by a detector fiber bundle with a length²⁹ of 3 m. The light source fiber was a graded index (GI) type single fiber (GC200/250L Fujikura, Japan) with a numerical aperture (NA) of 0.25 and a core diameter 200 μm, and the light detector fiber was a bundle fiber with a diameter of 3 mm and NA of 0.21. Finally, a set of histograms of the photon flight time, which is called a reemission profile, is recorded. One temporal reemission profile includes 1024 time channels spanning about 10 ns with a time step of about 10 ps. In this study, the emerging light was collected over a period of 3 s to exceed at least 1000 photon counts at the peak channel of the reemission profiles, and the procedure was repeated 30 times for each measurement.

The instrumental response was measured with the input fiber placed opposite the receiving fiber through a neutral density filter. The instrumental response of the TRS system was around 150 ps FWHM at each wavelength.

The mean optical path length was calculated from the difference between the center of gravity of the measured reemission profile and that of the instrumental response. It was assumed³⁰ that the value for the refractive index of brain tissue is 1.4.

The reemission profiles observed at each measurement point were fitted by the photon diffusion equation proposed by Patterson et al.¹³ in order to calculate the values of absorption coefficient (μ_a) and reduced scattering coefficient (μ_s ^′) of the head at wavelengths¹⁸ of 761, 795, and 835 nm. In the fitting procedure, a nonlinear least-squares fitting method based on the Levenberg-Marquardt method was used. This is a method of iterative improvement. In each iterative calculation, the function from the photon diffusion equation in reflectance mode, which was convoluted with the instrumental response, was fitted to the observed reemission profile. The calculation regions were determined to include the observed profile data and the data of 600 channels were included into the fit.

2.4.

Estimation of Absolute Values of oxyHb and deoxyHb with Water-Only Background Absorption

After determination of the values of μ_a at three wavelengths, the oxyHb and deoxyHb concentrations were calculated from the absorption coefficients of oxyHb and deoxyHb using the following equations with the assumption that background absorption is due only to 85% (by volume) water.^{9 11}

First, the water absorption was subtracted from μ_a at each of the three wavelengths, and then the values of oxyHb and deoxyHb were estimated using the least-squares fitting method. The absorption coefficients for oxyHb, deoxyHb, and water shown in Table 1 were used.

Table 1

The cerebral total hemoglobin (total Hb) concentration and cerebral Hb oxygen saturation (SO ₂) were calculated as follows:

2.5.

Estimation of Absolute Values of oxyHb and deoxyHb with Absorption of the Piglet Head with Blood Exchange Transfusion by FC

For measurement of background absorption of the head without Hb, we used a piglet for blood exchange transfusion by FC. First, partial blood exchange was done by saline and then blood was replaced with perfluorotributylamine emulsion (FC43 Emulsion, Green Cross Corp., Japan) as an oxygen carrier. We used 500 mL of FC and the hematocrit was lowered to 0.5%. After replacing the blood with FC, oxygenated head TRS measurements were done while the piglet breathed 100% O ₂. The EEG showed normal activity during the period of ventilation with 100% O ₂. We used the μ_a of piglet head with blood exchange transfusion by FC at three wavelengths as the background absorption value. The values at 761, 795, and 835 nm were estimated to be 0.1190, 0.1008, and 0.1260, respectively (Table 1). The following equations were used for estimation of oxyHb and deoxyHb concentrations:

2.6.

Statistical Analysis

A Statview J 4.5 package for the Macintosh computer was used for statistical analysis. Values obtained at different levels of FI _{O 2} were compared from values at FI _{O 2} of 21% by Wilcoxon’s signed-rank test. The level of statistical significance was set at a probability of p<0.05 for all tests. Results at FI _{O 2} levels in the range 10 to 100% were obtained from measurements in 11 piglets, results at FI _{O 2} of 8 and 6% were obtained from 10 piglets and results at FI _{O 2} of 4% were obtained from 8 piglets. Values are expressed as means ± standard deviation (SD).

3.1.

In Vitro Experiment Results

Figure 1(a) shows the relationship between the actual blood volume and total Hb concentration of the model as measured by TRS. The relationship between the actual blood volume (X) and Hb concentration measured by TRS (Y) was Y=0.843X+2.21 (r=0.998, P<0.001). We can see that the relationship between these two parameters is linear over the experimental range of Hb concentrations. Figure 1(b) shows the relationship between the values of Hb oxygen saturation in the 80-μM Hb suspension measured by a conventional gas analyzer or TRS. The relationship between the values of Hb oxygen saturation measured by a conventional gas analyzer (X) and TRS (Y) was Y=0.960X+0.889 (r=0.991, P<0.001). Linear regression analysis demonstrated that a significant correlation existed between the respective values determined by the two methods. These results indicated that our TRS method enables accurate and noninvasive measurement of Hb content and oxygen saturation in a highly scattered medium such as the piglet brain.

Figure 1

(a) Relationship between actual blood volume and total Hb concentration of the model as measured by TRS. The relationship between actual blood volume (X) and Hb concentration measured by TRS (Y) was Y=0.843X+2.21 (r=0.998,P<0.001). (b) Relationship between values of Hb oxygen saturation in the 80-μM Hb suspension measured by a conventional gas analyzer and by TRS. The relationship between values of Hb oxygen saturation measured by a conventional gas analyzer (X) and by TRS (Y) was Y=0.960X+0.889 (r=0.991,P<0.001).

3.2.

Values of μ_s ^′ and μ_a

In Fig. 2, the values of μ_s ^′ measured concurrently at corresponding FI _{O 2} levels are plotted against FI _{O 2} . At FI _{O 2} of 21%, the values of μ_s ^′ of the newborn piglet brain at 761, 795, and 835 nm were estimated to be 8.70±1.23, 8.33±0.91, and 8.24±1.11/cm, respectively. There were no significant differences between the values of μ_s ^′ for each FI _{O 2} level in the range of 4 to 100% at all three wavelengths.

Figure 2

Values of μ_s ^′ at 761, 795, and 835 nm, which were measured concurrently at corresponding FI _{O 2} levels, are presented as a function of FI _{O 2} . The error bars show 1 SD of the mean. At FI _{O 2} of 21%, values of μ_s ^′ in the newborn piglet head at 761, 795, and 835 nm were estimated to be 8.70±1.23 (mean±SD), 8.33±0.91, and 8.24±1.11/cm, respectively.

In Fig. 3, the values of μ_a ^′ measured concurrently at corresponding FI _{O 2} levels are plotted against FI _{O 2} . The values of μ_a at 761 and 795 nm at FI _{O 2} levels below 15% were higher than the values at FI _{O 2} of 21% at each wavelength. When FI _{O 2} was decreased from 21 to 15%, the values of μ_a at 761 and 795 nm increased from 0.227±0.021 to 0.259±0.024/cm and from 0.189±0.016 to 0.203±0.013/cm, respectively. The values of μ_a at 835 nm at FI _{O 2} levels below 10% were higher than those at an FI _{O 2} level of 21%. When FI _{O 2} was decreased from 21 to 10%, the value of μ_a at 835 nm increased from 0.223±0.020 to 0.251±0.025/cm.

Figure 3

Values of μ_a at 761, 795, and 835 nm, which were measured concurrently at corresponding FI _{O 2} levels, are presented as a function of FI _{O 2} . The error bars show 1 SD of the mean. At FI _{O 2} of 21%, values of μ_a in the newborn piglet head at 761, 795, and 835 nm were estimated to be 0.227±0.021 (mean±SD), 0.189±0.016, and 0.223±0.020/cm, respectively. Here, #, P<0.001; ^*, P<0.01; and $, P<0.05, indicate significant differences from values at FI _{O 2} of 21% using Wilcoxon’s signed-rank test.

3.3.

Concentration of oxyHb and deoxyHb with Water-Only Background Absorption

As shown in Fig. 4(a), the relationship between oxyHb and deoxyHb changed reciprocally in a mirror-image manner when FI _{O 2} was changed. Total Hb showed an increase of 31.3% when FI _{O 2} was decreased from 21 to 6%, indicating that cerebral blood volume increased during hypoxia. The values of oxyHb, deoxyHb, and total Hb at FI _{O 2} of 21% were 53.5±8.1, 33.4±5.8, and 86.9±8.1 μM, respectively.

Figure 4

Concentrations of oxyHb, deoxyHb, and total Hb, which were measured concurrently at corresponding FI _{O 2} levels, are presented as a function of FI _{O 2} . The error bars show 1 SD of the mean. (a) Using the water-only background absorption, at FI _{O 2} of 21%, oxyHb, deoxyHb, and total Hb concentrations in the newborn piglet head were estimated to be 53.5±8.1 (mean±SD), 33.4±5.8, and 86.9±8.1 μM, respectively. (b) Using absorption of the piglet head with blood exchange transfusion by FC, at FI _{O 2} of 21%, oxyHb, deoxyHb, and total Hb concentrations in the newborn piglet head were estimated to be 25.5±7.3, 18.6±5.8, and 44.1±7.4 μM, respectively. Here, #, P<0.001; ^*, P<0.01; and $, P<0.05, indicate significant differences from values at FI _{O 2} of 21% using Wilcoxon’s signed-rank test.

In Fig. 5(a), levels of SO ₂, SaO ₂, and SvO ₂, which were measured concurrently at corresponding FI _{O 2} levels, are plotted against FI _{O 2} . At FI _{O 2} of 21%, the mean SO ₂ of newborn piglets was calculated to be 61.5±6.3%. The values of SaO ₂ and SvO ₂ were simultaneously determined to be 92.1±3.76 and 40.1±6.4%, respectively. The contributions of SaO ₂ and SvO ₂ to SO ₂ were 41.1 and 58.9%, respectively. However, values of SO ₂ at FI _{O 2} in the range of 10 to 4% were higher than those of SaO ₂.

Figure 5

Comparison of changes in SO ₂ in the brain, SaO ₂ in the artery, and SvO ₂ in the internal jugular vein during loading with stepwise decrease in FI _{O 2} using (a) water-only background absorption and (b) absorption of the piglet head with blood exchange transfusion by FC.

The SaO ₂ values also showed a positive linear relationship with SO ₂ [Fig. 6(a)]. The relationship between SaO ₂ and SO ₂ was SO ₂=37.9×10⁻²×SaO ₂+27.5 (r=0.862, P<0.001). From this regression equation, the values of SaO ₂ and SO ₂ were estimated to be 44.3% when SO ₂ was larger than SaO ₂ during a hypoxic condition.

Figure 6

Relationships between SO ₂ and SaO ₂ at various oxygenation levels: (a) using the water-only background absorption, the relationship between SO ₂ and SaO ₂ was SO ₂=37.9×10⁻²×SaO ₂+27.5 (r=0.862,P<0.001), and (b) using absorption of the piglet head with blood exchange transfusion by FC, the relationship between SaO ₂ and SO ₂ was SO ₂=59.0×10⁻²×SaO ₂+4.9 (r=0.843,P<0.001).

As shown in Fig. 7(a), there was a positive linear relationship between SvO ₂ and SO ₂. The relationship between SvO ₂ and SO ₂ was SO ₂=69.3×10⁻²×SvO ₂+31.8 (r=0.841, P<0.001).

Figure 7

Relationships between SO ₂ and SvO ₂ at various oxygenation levels: (a) using the water-only background absorption, the relationship between SO ₂ and SvO ₂ was SO ₂=69.3×10⁻²×SvO ₂+31.8 (r=0.841,P<0.001), and (b) using absorption of the piglet head with blood exchange transfusion by FC, the relationship between SvO ₂ and SO ₂ was SO ₂=108.6×10⁻²×SvO ₂+11.5 (r=0.829,P<0.001).

3.4.

Concentration of oxyHb and deoxyHb with Absorption of the Piglet Head with Blood Exchange Transfusion by Fluorocarbon

As shown in Fig. 4(b), the relationship between oxyHb and deoxyHb changed reciprocally in a mirror-image manner when FI _{O 2} was changed. The values of oxyHb, deoxyHb, and total Hb at FI _{O 2} of 21% were 25.5±7.3, 18.6±5.8, and 44.1±7.4 μM, respectively. The values of oxyHb, deoxyHb, and total Hb are significantly lower at each FI _{O 2} level than those in the case of water-only background absorption.

In Fig. 5(b), levels of SO ₂, SaO ₂, and SvO ₂, which were measured concurrently at corresponding FI _{O 2} levels, are plotted against FI _{O 2} . At FI _{O 2} of 21%, the mean SO ₂ of newborn piglets was calculated to be 57.6±12.3%. The contributions of SaO ₂ and SvO ₂ to SO ₂ were 33.7 and 66.3%, respectively. SO ₂ values were between those of SaO ₂ and SvO ₂ at all levels of FI _{O 2} . SO ₂ values are not significantly different from SO ₂ values using the water-only background at FI _{O 2} in the range of 15 to 100%, but the values are lower than the values using the water-only background at FI _{O 2} in the range of 4 to 12%.

The SaO ₂ values also showed a positive linear relationship with SO ₂ [Fig. 6(b)]. The relationship between SaO ₂ and SO ₂ was SO ₂=59.0×10⁻²×SaO ₂+4.9 (r=0.843, P<0.001). As shown in Fig. 7(b), there was a positive linear relationship between SvO ₂ and SO ₂. The relationship between SvO ₂ and SO ₂ was SO ₂=108.6×10⁻²×SvO ₂+11.5 (r=0.829, P<0.001).