We have demonstrated the potential of NIRS to be used in longitudinal studies to monitor brain hemoglobin oxygenation changes with brain maturation. Our results have shown reproducible measures of brain optical properties in 14 neonatal rabbits. Currently there are no methods that can be used to measure tissue optical properties in vivo
, and there are no published NIRS data of brain optical properties in rabbits. Nevertheless, our measured optical properties are consistent with optical properties measured in piglet and in human brain.53–55
In this study, we used the diffusion equation applied to the model of a semi-infinite homogeneous medium to calculate the optical properties of brain tissue. The head of the rabbit is small, curved, and layered and is not a semi-infinite homogeneous medium, for which this model is valid, and as such there are errors that are inherent in our approximation, e.g., we expect that the surface layers of the head (skin, bone) could introduce some error in the measured optical properties of the brain. However, phantom studies reported in the literature48
have shown that when the FD multidistance method is used, superficial layers of thickness, 4 mm and smaller, do not affect the measurements of the deeper layers. In addition, we performed additional experiments using layered phantoms, with similar optical properties to brain tissue (data not shown), with the range of distances used in our experiment, to further verify that our optical signals were being measured from brain tissue.
From the anatomical MRI measurements we verified that the overall thickness of the superficial layers of skin, scalp, and skull (on the rabbits’ heads) was never larger than 3 mm, and therefore sufficiently small to be neglected. Moreover, by using only two source-detector distances of the four available to calculate the optical properties in the brain, we obtained the same results. Specifically, using any of the following three source-detector distance combinations 0.5 to 0.8, 0.8 to 1.1, and 1.1 to 1.5 cm, we obtained optical measurements that were within experimental error. The 0.8- to 1.1-cm set yielded results closest to those obtained by using the combination of all the four distances. The results reported in this paper are those obtained using all source detector distances.
Loss of light laterally, due to the small size of the head of the animal, can effectively result in increased measured absorption coefficients. This problem was minimized by using a small range of source-detector distances (0.5 to 1.5 cm) and our data do not seem to be have been affected by this problem. In fact, the increasing absorption coefficients in the first few days of life, when the head size also increases, is opposite to what we would expect. The combined contribution of lateral loss of light and increased measured μa should be maxi- mum when the head is smaller, i.e., at P3 and P6, not at P13, where the absorption peaks (see ).
The inhomogeneity of the rabbit’s head and the use of small source-detector distances can cause crosstalk between the absorption and reduced scattering coefficients. In our measurements, the crosstalk between μa
does not seem to be significant. In fact, μa
at 690 nm are not correlated (r
=0.28) and μa
at 830 nm are weakly correlated (r
=0.57), while, as expected,
at the two different wavelengths are correlated (r
=0.82). In contrast, during brain development, absorption at different wavelengths is not necessarily correlated and we found μa
at 690 nm and μa
at 830 nm to be weakly correlated (r
=0.47). This results because the independent changes in blood volume and oxygenation influence the absorption at these two wavelengths.
The value of cerebral hemoglobin oxygenation during the first 15 days of life is lower than what is usually measured in piglets56
or term human neonates.57
At birth, the rabbit brain is immature and is comparable to the brain of a premature human infant (24 to 32 weeks gestation). In fact, the piglet brain at birth is more mature than that of a term human neonate. As such, the data measured in the term human and the piglet brains are not directly comparable to that measured in the neonatal rabbit brain. In the following paragraphs, we outline some reasons for the low optically measured StO2
within the first 30 postnatal days () in neonatal rabbit brain.
One possible explanation for this low StO2 value could have been the use of the extinction coefficients of hemoglobin derived from human studies, which were used to compute the hemoglobin concentrations in rabbit brain. However, this is unlikely because our measurements of blood samples taken at various ages from P3 to adult showed no differences between the hemoglobin absorption spectra of neonatal rabbits, adult rabbits, and humans. Moreover, the values of the peripheral arterial oxygenation measured with the pulse oximeter on the neonatal rabbits were, as expected, 97 to 100%. If the neonatal hemoglobin spectra differed substantially from that of human hemoglobin, the SaO2 measured by the pulse oximeter would also be affected, possibly resulting in lower than normal readings. Another possible reason is the presence of an additional, unknown absorber in the neonatal brain that affects the quantification of the hemoglobin concentration resulting in low StO2. This issue cannot be addressed in this study and may be addressed in future in another NIRS study that uses more than two wavelengths of light.
It is also possible that at this particular stage of the brain’s development a low StO2
is perfectly normal given the immature state of the brain and the rigors of the birthing process. During the birthing process the fetus experiences “normal birth asphyxia” due to the repeated reduction in uterine blood flow as a result of uterine contractions. Hence, the fetus is born in a state of “physiological asphyxia,“ which is normally relieved after the newborn takes the first few breaths. Newborn rabbits, like other newborn animals and humans, must undergo certain essential adjustments directly after birth. The transition to extrauterine life involves the following two major events: (1) establishment of the neonatal circulation and (2) establishment of the pulmonary circulation. It has been reported in the literature that at birth there is globally decreased cerebral blood flow (CBF) and in newborn lambs, humans, and rabbits the cortex is preferentially supplied with blood. CBF is tailored to meet the energy and oxygen demands of the brain tissue.58
There is very little mylenation in the neonatal rabbit’s brain and the cortical vasculature is immature. CBF and glucose utilization (i.e., oxidative capacity) are low. Tuor31
and Tuor et al.35
using autoradiographic methods, showed that capillary density, oxidative metabolism, and local CBF change with development in the neonatal rabbit brain. Directly after birth (days 1 and 2), CBF is low and varies regionally. By day 17, there was markedly increased CBF (200 to 350%) compared to days 1 and 2, and CBF continued to increase in most brain regions up to 40 days postnatally. In these animals, capillary density increased from birth and matured to couple with cerebral metabolism at about postnatal day 17 then showed no significant change thereafter.
measured optically reflects the balance between oxygen delivery and consumption in the tissue microvasculature, i.e., chiefly in the capillaries. In neonatal rabbits, the immaturity of the brain at birth, the low cerebral blood flow and glucose utilization coupled with the adjustment to extrauterine life and the immaturity of the brain is the likely explanation for the low measured StO2
(on average 30% at P
3). Thereafter, StO2
increased with postnatal age (up to P
76) as the brain matures structurally and functionally and the animals become adjusted to postnatal life. This result is in keeping with data published on premature human neonates which reported low StO2
values at birth59
values within the first 1 to 3 days of life were 50, 66, and 76% respectively).
HbT measured optically reflects blood volume in the illuminated tissue and is indirectly correlated to capillary density. In our measurements, HbT peaked at P
17, consistent with an initial capillary density increase in the first 20 postnatal days reported by Tuor et al.35
After this period when the microvasculature matures, tissue volume and vasculature both increase, resulting in constant HbT. However, local cerebral blood flow autoregulation depends on the maturity of the tissue and is not optimum below P
17. These maturational changes support our optical data which showed a peak HbT between 15 and 17 days with no significant change thereafter, supporting a model of evolving, i.e., increasing, resting blood flow.
From our optical measurements, there were no differences, compared to normal controls, in tissue hemoglobin oxygenation or total hemoglobin concentration in brain tissue that suffered transient HI. Similarly, the longitudinal MRI data did not show appreciable differences in these two groups. However, the serial T2 and diffusion MRI data clearly demonstrated the major structural changes going on in the maturing tissue. The deep WM structures, subcortical WM, corpus callosum, and internal capsule, showed increased FA up to P31, indicative of continuing myelination (). This same process is also likely the cause of a sustained reduction in free water content in both gray and white matter, resulting in the progressively decreased T2 and ADC in these tissues.
Previous work in this HI model33
showed that there are selective, bilateral ischemic changes in the CA1/CA2 and CA3 cortical brain regions and in the periventricular WM, which were significantly greater on the side of ligation. These changes were not associated with inflammatory necrotic but rather apoptotic changes. The area of tissue measured in the HI and in the normal control animals may be somewhat dif- ferent due to some selective cell loss, i.e., we were measuring a population of cells with normal oxygenation. Our method may not be able to detect such small changes in the cerebral tissue oxygenation after HI. The fact that we measured these animals directly after (P
10) then 1 week later may also mean that we may have missed the window where the apoptotic cells were cleared swiftly and surgically from the surrounding brain tissue. Also, it has to be considered that this may in fact suggest that a moderate (as shown from previous histological data33
) and transient ischemic insult such as this may not have resulted in permanent changes in tissue oxygenation and blood volume.