In this pilot study, there was no significant difference in StO2 between brain-injured and normal neonates, suggesting that StO2 alone may be insensitive to evolving brain injury. However, CBV and estimates of rCMRO2 were significantly increased in the brain-injured group compared with all other clinical groups despite the diversity of brain injury types. In this small study, combining CBV and rCMRO2 resulted in encouraging sensitivity and specificity values for FD-NIRS for detecting brain injury within the first two weeks of life.
Many neonatal NIRS studies have focused on StO2
(Austin et al, 2006
; Benaron et al, 1992
; Isobe et al, 2000
; Toet et al, 2006
; Tsuji et al, 2000
; Wong et al, 2008
; Wyatt et al, 1986
), following the assumption that tissue oxygenation is a good marker of brain health. In a study of severe hypoxic-ischemic injury, increased cerebral tissue oxygenation in the first 48 h of life was correlated with poor outcomes (Toet et al, 2006
). We also observed an increase in StO2
in the brain-injured group, but this was not significant. Although the lack of significance in our study may be due to the later time periods and more heterogeneous nature of the brain insults, it is also possible that StO2
is not a sensitive marker of evolving injury. We have observed earlier that StO2
is not a sensitive marker of brain development in the first year of life, showing little change as CBV increases with age (Franceschini et al, 2007
). In this case, we hypothesized that CBV increases to meet increasing neuronal demands in such a way that StO2
remains relatively stable. Here, we hypothesize that StO2
is useful for monitoring the severity of the primary insult. However, after the insult and during the injury evolution, cerebral perfusion increases in an effort to stabilize StO2
, as during development. Future studies with more patients and multiple time points starting closer to birth are needed to monitor the temporal evolution of StO2
and test this hypothesis in humans.
Increased CBV after neonatal brain injury in the first week of life has been reported in prior NIRS studies (Meek et al, 1999
; Wyatt et al, 1990
). Here, we extend the time window to 2 weeks and show that the increase in CBV is associated with relative increases in CMRO2
. These increases in CBV and CMRO2
suggest increased neuronal activity (Huppert et al, 2006
; Uludag et al, 2004
). In fact, the trend of an increase in StO2
in the brain group is in keeping with increased neuronal activity. Functional MRI, positron emission tomography, and NIRS studies have shown us that normal brain function is associated with a local increase in oxy-hemoglobin and a decrease in deoxy-hemoglobin concentrations, and an overall increase in local CBV and CBF (Fox et al, 1988
; Malonek and Grinvald, 1996
Increased neuronal activity in neonatal brain injury has two important implications: (1) increased cerebral perfusion after cerebral injury may be due to increased neuronal activity and not loss of vascular control; (2) increased neuronal activity may persist for days after injury despite standard anti-seizure medication and a lack of EEG and clinical signs of seizures. A seizure is defined as a transient symptom of excessive or synchronous neuronal activity in the brain (Fisher et al, 2005
). This abnormal synchronous neuronal activity can be detected with EEG and is associated with increased neuronal activity. Unlike many epilepsy syndromes, seizure activity in neonatal brain injury occurs secondary to injury and excitotoxic cascades. Therefore, we hypothesize that neuronal synchrony may not occur and EEG results may be misleading. Thus, we hypothesize that increases in CBV and rCMRO2
can be seen without frank clinical or electrographic seizure activity due to incoherent increased neuronal activity resulting from excitotoxic injury. Another possibility for elevated CBV and rCMRO2
in the absence of EEG seizure activity is that the neurons are unable to generate an action potential due to injury but are continuing to consume large amounts of oxygen due to excitotoxic stress. Further studies correlating FD-NIRS directly with EEG at the time of FD-NIRS measurement are needed to explore this relationship further.
Although there have been previous reports of CMRO2
in neonates, these studies required radioactive tracers, invasive procedures or ventilated subjects (Altman and Volpe, 1991
; Elwell et al, 2005
; Skov et al, 1993
; Yoxall and Weindling, 1998
). Therefore, the numbers in these studies are small and no normal neonates are included. Higher CMRO2
rates were reported in term and near-term neonates compared with premature neonates. However, the term and near-term neonates often suffered from hypoxic-ischemic insults, whereas the premature neonates had other types of injuries, including hyaline membrane disease, respiratory distress syndrome, or intraventricular hemorrhage. It is, therefore, impossible to determine whether the term neonates had higher CMRO2
due to increased maturity or to evolving cerebral injury.
We averaged results from all locations as only three subjects (19, 30, and 35) had marked asymmetry to the MR imaging findings and 12 out of the 43 did not have high quality FD-NIRS results bilaterally to allow accurate lateralization. We could not assess laterality of injury in subject 19, as only middle forehead measurements were acceptable for analysis. For the remaining 28 subjects for whom high quality bilateral data were available and markedly asymmetric injury was not present on MRI, the average difference between left and right temporal/parietal regions was 17±11% for CBV and 3±2% for StO2. These left and right asymmetry values are within our reproducibility errors (11±1% for CBV and 4±1% for StO2) and consistent with the mean COV calculated across all head locations (18±8% for CBV and 4±2% for StO2). For patient 30 with the large left frontal hematoma due to an arterio-venous malformation (AVM) rupture, differences in left and right temporal/parietal hemispheres were 1% for CBV and 0% for StO2. However, the diffusion weighted images (DWIs) in this patient showed evidence of a bilaterally symmetric profound hypoxic-ischemic injury that was confirmed on pathology. Therefore, this subject had both an AVM that ruptured and profound global hypoxic-ischemic injury. The large left frontal hematoma was over 2 cm below the cortex within the deep white matter of the left frontal lobe. As the depth penetration of FD-NIRS is only 1 cm, it is likely that the FD-NIRS measurements were dominated by the global symmetric HII affecting cortical neuronal health. In subject 35, the asymmetries were higher than those in any other subject, with left-sided CBV 46% larger than right and left-sided StO2 24% larger than right. This correlated with the large left MCA arterial ischemic stroke identified on MRI. The next subject with CBV asymmetry larger than the average was subject 41 with 38% left/right difference (StO2 asymmetry only 3% in this subject). This subject had asymmetric partial hypoxic-ischemic injury on DWI with the left hemisphere more severely affected than the right. The next subject with StO2 asymmetry larger than the average was subject 43 with 10% left/right difference (CBV asymmetry only 7%). This subject also had asymmetric HII on DWI with more cortical low apparent diffusion coefficient (ADC) on the left. Therefore, as these few cases suggest, FD-NIRS has the potential to lateralize injury, although better determination of normal laterality and confirmation with more cases of asymmetric injury is needed.
This study encompassed a large range of brain injuries, including hypoglycemic, metabolic, hypoxic ischemic, and focal arterial ischemic injuries (). We allowed this diversity to determine whether FD-NIRS measures of StO2
, CBV, and/or rCMRO2
could detect a unique response to primary brain injuries distinguishing them from normal neonates and neonates with non-brain issues. Using the sensitivity and specificity values in Supplementary Figure 3
, 12 neonates, had increased CBV and rCMRO2
with 11 out of the 12 from the brain injury group. All nine patients who had an MRI within 10 days of the NIRS study and at least one site with low ADC (subjects 9, 12, 17, 19, 30, 31, 35, 41, and 43) were in this group with elevated CBV and rCMRO2
. Arterial patterns of hypoxic-ischemic injury (HIE, HII, and arterial stroke) were present on MRI in 6 out of the 9 of these patients with low ADC associated with high CBV and rCMRO2
(subjects 9, 12, 30, 35, 41, and 43). However, elevated CBV and rCMRO2
were observed in subject 10 and 23 even though no ADC abnormalities were present on recent DWI. Subject 10 had a mitochondrial disorder and subject 23 had bilateral choroids plexus bleeds with IVH on MRI after respiratory distress and seizures at birth. We hypothesize that all these disorders share the common mechanism of hyperperfusion due to increased neuronal activity, with the increased neuronal activity possibly due to excitotoxic cascades. Although brain injuries with high CBV and rCMRO2
are most commonly associated with low ADC, the absence of low ADC in two subjects suggests that neuronal dysfunction can occur in the absence of low ADC. In addition, in patients 9, 12, 17, 19, and 30, the regions of low ADC were deep and, therefore, unlikely to be directly sampled by FD-NIRS. These findings suggest that if the brain injury is severe enough to cause ADC to decrease in any region of the brain, it may be severe enough to result in global changes in cortical CBV and rCMRO2
. We hypothesize that CBV and rCMRO2
increases may be due to neurons that are functioning abnormally but are not necrotic. Neuronal dysfunction could be secondary to primary insults, disrupted connections to deep gray nuclei or deep gray nuclei injury.
Not all brain injury subjects exhibited increases in CBV and rCMRO2. For example, one neonate 10 days after hypoxic-ischemic injury (20), one with vasogenic edema that resolved (38), and one with a mitochondrial disorder (11) had CBV and rCMRO2 results within the normal rage. As other subjects with more acute HII showed increased CBV and rCMRO2, we expect that hypoxic-ischemic injuries evolve over time, eventually normalizing and probably going on to have lower rCMRO2 than normal. The lack of increased CBV and rCMRO2 in neonates with vasogenic edema and mitochondrial disorder suggests that FD-NIRS may help distinguish different types and severity of brain injury. However, larger studies are needed to determine the role of FD-NIRS in distinguishing different types of neonatal brain injury and the role of FD-NIRS in monitoring the evolution of neonatal brain injury.
Variations in HGB may have contributed to errors in CBV calculations as we did not have HGB values for all infants. Values were available for all unstable neonates and most with brain injury. We did not have IRB approval to do blood work if no values were available in the clinical chart. In these cases we used standard normal values for age. To estimate the magnitude of the error and bias on CBV and rCMRO2 values due to use of table values, we repeated the statistical analysis using table values for all neonates and with a value of 15 g/dL for all neonates. In both cases increased CBV and CMRO2 in the brain injury group compared with all others maintained significant ANOVA but with higher P-values. In summary, the errors introduced by using table values for HGB are not likely large enough to alter the findings in this study.
Also, the error in rCMRO2
is unknown because the value of β
is unknown. We show, however, that over a large physiological range of β
remains significantly elevated in brain injured compared with all other groups (Supplementary Figure 3, online
). Direct measurements of CBF in the future will improve the accuracy of rCMRO2
estimates and better understanding of neurovascular coupling in injury and development.
In summary, this pilot study suggests that NIRS measures of StO2 alone may be insensitive to evolving neonatal brain injury. However, increased CBV and increased relative CMRO2 on bedside FD-NIRS may be useful for detecting or monitoring evolving neonatal brain injury as these increases seem to distinguish brain injured from all other neonates. The observed increase in rCMRO2 suggests that the increased perfusion seen in neonatal brain injury may be related to increased neuronal activity, despite the lack of EEG or clinical signs of seizures. Larger prospective longitudinal studies with age-matched controls and neonates with each subtype of brain injury are required to confirm these findings and better determine the sensitivity and specificity of FD-NIRS measures of CBV and rCMRO2. In particular, the sensitivity and specificity of these FD-NIRS measures in the critical first few hours of life is still unclear.