1.

Introduction

The survival rate of preterm infants has improved dramatically in recent decades due to advances in perinatal and neonatal care. However, the neurodevelopmental outcome of NICU infants remains a major clinical concern, with a significant proportion displaying cognitive and behavioral deficits during childhood.¹ The ability to noninvasively assess neural function may provide early recognition and evaluation of cerebral dysfunction in high-risk infants, enabling targeted early intervention. Functional neuroimaging is commonly performed using hemodynamic contrasts [e.g., functional MRI (fMRI) or near-infrared spectroscopy (NIRS)], in which local neural activity is mapped through the neurovascular response. While fMRI is the most widely used technique in adults and has a well-established methodology, its disadvantages include potential safety concerns in transporting unstable infants to the fixed scanner, indirect surrogates of hemodynamics [e.g., blood oxygenation level dependent (BOLD) signal], low temporal resolution, and high cost. NIRS is an alternative neuroimaging modality with the advantages of portability, low cost, and more comprehensive hemodynamic contrasts.^{2, 3} These benefits may allow NIRS systems to be used for longitudinal, bedside clinical monitoring of neonatal brain function.

Despite its inherent advantages, the clinical application of NIRS has been limited by technological challenges. Stringent instrumentation requirements have restricted the ability to distinguish cerebrovascular signals from superficial hemodynamic signals arising from scalp and skull. Recently, a tomographic approach using high-density NIRS imaging arrays has been shown to provide improved measurements of brain function in adults.^{2, 4, 5, 6} The high-density fiber arrangement also provides multiple distance measurements within the array, therefore allowing a regression approach to minimize spurious or systemic signals contaminating true cerebral signals. In this study, we aim to extend the use of our high-density NIRS arrays² to evaluate healthy term-born infants.

The extension of functional neuroimaging (for fMRI or NIRS) to infants has been hindered by a lack of understanding about the neurovascular coupling linking neural activity to the hemodynamic response. In the healthy, mature brain, increased neural activity drives an increase in both cerebral blood volume (CBV) and tissue oxygen saturation $\left(\mathrm{St}{\mathrm{O}}_2\right)$ . In healthy adults, the balance of these two factors results in a net localized decrease in deoxyhemoglobin (HbR): a “positive BOLD response.”^{7, 8} When this balance is altered (e.g., due to injury), stimulation may result in “negative BOLD responses.”^{9, 10, 11, 12, 13} During early development, the brain’s metabolism is complex and varying, resulting in the possibility of an immature neurovascular coupling.¹⁴ fMRI studies of infants have found both negative and positive BOLD responses in response to sensory stimuli.^{15, 16, 17, 18, 19, 20} In contrast to BOLD-fMRI, NIRS can measure oxy- $\left(\mathrm{Hb}{\mathrm{O}}_2\right)$ , deoxy- (HbR),²¹ and total hemoglobin (HbT), providing a more comprehensive view of brain metabolism,^{22, 23, 24} even in situations of altered neurovascular coupling. HbT provides a relatively robust indicator of CBV, while the ratio of $\mathrm{Hb}{\mathrm{O}}_2$ and HbT reflects $\mathrm{St}{\mathrm{O}}_2$ . In addition to imaging technique issues, previous NIRS and fMRI studies of infants have also suffered from a broad range of study protocols (e.g., a diversity of stimulus conditions, sleep/sedation states, and ages). Thus, the nature of the cerebral hemodynamic response to increased neural activity in healthy infants remains unclear.

In this study, we constrain the stimulus protocol variables by evaluating a relatively well-studied sensory stimulus (visual) within the first three days of life in healthy term infants. This study provides the youngest cohort of subjects in which the hemodynamic response to visual stimulation has been assessed. This feasibility study of high-density NIRS and investigation of the healthy neonatal hemodynamic response intends to strengthen the foundation for the development of NIRS as a clinical tool.

2.

Methods

2.2.

Instrumentation

We previously developed a multichannel, continuous-wave, high-density diffuse optical imager for use in adults.² The simple and modular instrumentation made it easily scalable to a portable system for bedside use in the nursery. For this study, we assembled a system with 13 source positions and 15 detector positions. Each source position consisted of two near-infrared wavelengths (750 and $850\mathrm{nm}$ ) of light emitting diodes (LEDs). Each detector position had a dedicated avalanche photodiode and analog-to-digital converter. Light was carried to and from the scalp via flexible optical fibers with a diameter of $2.5\mathrm{mm}$ (optodes). The optodes were held onto the scalp by a silicone pad, an elastic neoprene band, and hook-and-loop strapping. For recording over the visual cortex, we arranged fibers in a high-density $4\times 10$ configuration of four sources and ten detectors [Fig. 1 , overall dimensions $2\times 6\mathrm{cm}$ ]. For recording simultaneously over the motor cortex, we assembled a second similar pad with a $7\times 5$ configuration.

Fig. 1

High-density scanning grid on an infant subject. (a) Schematic of our high-density scanning grid. Blue dots are light source positions; red dots are detector positions. Each detector is capable of distinguishing light signals originating from multiple source positions (the interconnecting lines). (b) Visual response data being taken on a sleeping baby. The LCD screen is typically placed $20\mathrm{cm}$ away from the infant’s face (not shown). Observe the soft neoprene cap and flexible optical fibers placed over the occipital cortex. (c) A schematic diagram showing a detector and two source positions arranged 1 and $2.3\mathrm{cm}$ apart. Note that signals coming from the $1\text{-}\mathrm{cm}$ source position likely sample tissue from the superficial layer of the head, while signals from the second-nearest source position likely sample tissues from a combination of superficial and brain tissues. (Color online only.)

3.

Results

We recruited a total of 11 healthy (five male and six female) full-term neonates during the first three days of life. The mean gestational age of the infants was $395∕7\text{weeks}$ (range $371∕7\text{to}411∕7\text{weeks}$ ), and the mean postnatal age at the time of the scan was $2\text{days}$ (range $1\text{to}3\text{days}$ ). The prenatal and postnatal courses were uncomplicated for all of these infants. Each infant was examined by a physician and none showed any abnormal findings. The clinical details of the subjects are outlined in Table 1 . The presence of the cap did not disturb the infants, and all infants laid quietly with eyes closed (visual inspection by data collectors) during the scanning session [as in Fig. 1]. Infant 3 was excluded from analysis due to significant noise, likely due to excessive motion (strong pacifier sucking was observed). Of the remaining ten infants, eight were imaged with the standard visual cap and stimulus. The final two were imaged with the combined motor and visual cap, and their data are presented separately.

Table 1

Clinical characteristics for each study subject. ( M=male ; F=female ; C∕S=Cesarean-section ; Vag=vaginal delivery; C=Caucasian ; B=Black ).

In the eight infants who were presented with the visual stimulus, we found the same hemodynamic response pattern: increases in oxyhemoglobin $\left(\mathrm{Hb}{\mathrm{O}}_2\right)$ and total hemoglobin (HbT) and a decrease in deoxyhemoglobin (HbR). The hemodynamic response averaged across all selected blocks from all infants is shown in Fig. 2 . Note the number of statistically significant deviations from baseline. For comparison, Fig. 2 shows the typical hemodynamic response measured in a single healthy adult. The results for each infant individually are shown in Table 2 . The peak changes were significant in $\mathrm{Hb}{\mathrm{O}}_2$ for six of eight infants, in HbR for four of eight infants, and in HbT for five of eight infants. The mean $\mathrm{Hb}{\mathrm{O}}_2$ peak was $0.28\pm 0.16\mu \mathrm{mol}∕\mathrm{L}$ (± standard deviation), the mean HbT peak was $0.27\pm 0.18\mu \mathrm{mol}∕\mathrm{L}$ , and the mean HbR trough was $-0.11\pm 0.11\mu \mathrm{mol}∕\mathrm{L}$ . Note that since the peak changes occur at different times, the sum of the peak values for $\mathrm{Hb}{\mathrm{O}}_2$ and HbR do not equal the peak values for HbT. The last three columns (5, 6, and 7) of Table 2 detail the ratio between the contrast-to-noise ratio (CNR) in the data with and without superficial signal regression of the data. The CNR improvements are evident in all three contrasts.

Fig. 2

Hemodynamic changes in response to visual stimulation ( $\mathrm{Hb}{\mathrm{O}}_2$ : red, HbR: blue, and HbT: green). The shaded gray areas denote our $10\text{-}\mathrm{s}$ visual stimulus. Error bars show mean and standard error for each five second interval. Asterisks show points that are significantly $(p<0.05)$ different from baseline using a two-tailed $t$ -test. (a) Hemodynamic response (averaged across eight infants) showing significant increases in $\mathrm{Hb}{\mathrm{O}}_2$ and HbT and a significant decrease in HbR. (b) Hemodynamic changes in response to visual stimulation in a single healthy young adult, showing the same pattern. (Color online only.)

Table 2

Average change and standard error in oxy-, deoxy-, and total hemoglobin concentrations in response to visual stimulation for each study subject. Deviations that are statistically different (p<0.05) from baseline are marked with an asterisk. The group mean and standard deviation of the mean are also shown. The effect of SSR on CNR is given for each individual. The group mean and standard deviation of the mean are also shown.

In the infant scanned with the additional sensorimotor cap and presented with the visual stimulus, the visual cortex showed the same changes as noted in the other infants [Fig. 3 ], while the sensorimotor region showed no significant changes [Fig. 3]. In the infant scanned without the presentation of a stimulus, neither the visual [Fig. 3] nor the sensorimotor cap [Fig. 3] showed any significant changes.

Fig. 3

The hemodynamic response is specific to both the visual stimulus and the occipital region ( $\mathrm{Hb}{\mathrm{O}}_2$ : red, HbR: blue, and HbT: green). The shaded areas denote our visual stimulus. Error bars show mean and standard error for each five second interval. Asterisks show points that are significantly $(p<0.05)$ different from baseline using a two-tailed $t$ -test. (a) and (b) Hemodynamic measurements in a single baby with simultaneous recording from both visual and sensorimotor cortices, showing a hemodynamic response in the visual but not the sensorimotor cortex. (c) and (d) Hemodynamic measurements taken from both visual and somatosensory cortices during $10\min$ of rest without a visual stimulus. Note the lack of any functional response. (Color online only.)

To determine whether the regression step improved the time courses, we also processed the data without the regression step (Fig. 4 ). With regression, the average contrast of the eight infants is 0.35, $-0.15$ , and $0.29\mu \mathrm{mol}∕\mathrm{L}$ for $\mathrm{Hb}{\mathrm{O}}_2$ , HbR, and HbT, respectively; noise (standard error) is 0.080, 0.055, and $0.11\mu \mathrm{mol}∕\mathrm{L}$ ; and contrast to noise is 3.8, 3.2, and 2.5. Without regression, the contrast is 0.50, $-0.22$ , and $0.45\mu \mathrm{mol}∕\mathrm{L}$ for $\mathrm{Hb}{\mathrm{O}}_2$ , HbR, and HbT, respectively; noise (standard error) is 0.45, 0.17, and $0.53\mu \mathrm{mol}∕\mathrm{L}$ ; and contrast to noise is 1.9, 2.7, and 1.5. Although there is a modest attenuation of the contrast, the regression provided a significant increase in the contrast-to-noise ratio due to the reduction of noise. The attenuation of the contrast is further considered in the discussion.

Fig. 4

Evaluation of the superficial signal regression (SSR) in infants. Raw data (a), (c), and (e) without the application of SSR show wide fluctuations (i.e., standard error bars) in all three measured parameters (i.e., oxy-, deoxy-, and total hemoglobin concentration changes). After the application of the SSR method to the same dataset (b), (d), and (f), moderate improvements are observed in the signal strengths, but significant reductions in noise levels (as shown by the standard error bars) are evident compared to the raw dataset.

4.

Discussion

Previously, we have shown the benefits of high-density imaging arrays in adults. In this feasibility study, we aimed to establish the feasibility of using high-density arrays in infants. A challenge of working with high-density arrays is the bulk of the many fibers. Therefore, we did not start with a full-size imaging pad (e.g., 24 sources $\times 28$ detectors) as used with adults. Rather, we retained the high-density spatial sampling $\left(1\mathrm{cm}\right)$ but worked with a smaller field of view (4 sources $\times 10$ detectors). This amount of fibers was sufficient to cover the visual cortex region and permitted testing of one benefit of high-density arrays: the use of first-nearest neighbor measurements for a superficial signal regression procedure. The $4\times 10$ array used over the visual cortex permits 16 measurements with $1\text{-}\mathrm{cm}$ SD-pair distances and 12 measurements with $2.3\text{-}\mathrm{cm}$ distances. For reference, recent related work by Karen recording infant visual responses used an array with eight measurements at $2.5\text{-}\mathrm{cm}$ distances and two measurements at $3.75\mathrm{cm}$ . Thus, the current $4\times 10$ array has optodes packed over three times more densely (per unit area) than the Karen array. This higher-density arrangement, with the shortest distance being $2.5\times$ shorter and more specific to the scalp, facilitates the regression procedure. Finally, the fibers are arranged in a manner that supports future imaging experiments.

Our results demonstrate the feasibility of using high-density arrays of fibers to record brain activity in infants. The signal levels were sufficient in all nine infants to record from multiple source-detector distances. Of these nine infants, we were able to record with sufficient signal-to-noise ratio to detect visual activations in eight. The regression procedures provided up to a $2.9\times$ improvement in contrast-to-noise ratio of the responses (in $\mathrm{Hb}{\mathrm{O}}_2$ ) to visual activations. Assuming that the competing noise is random, this CNR improvement translates to an approximately $8\times$ reduction in the time needed to obtain similar contrast-to-noise ratios between the two approaches. Since the influences of superficial variations have been largely removed from the measurements, the resulting signals interrogate the brain with greater specificity. While we do not know the shape of the hemodynamic response a priori, we note that qualitatively the general shape of the regressed hemodynamic response is smoother and more similar to the response that would be expected from adults. This method therefore provides increased confidence in the data quality and the characterization of the hemodynamic response compared to traditional single-distance NIRS recordings. Finally, these results provide a basis for advancing future studies with a large-scale imaging pad.

Our results also demonstrate that in quiet resting, healthy term-born infants in the first three days of life, with eyes closed, the visual cortex responds to visual stimulation by an increase in $\mathrm{Hb}{\mathrm{O}}_2$ and HbT and a decrease in HbR. Our data also indicate that the observed response is specific to the visual stimulus and localized to the occipital region. No periodic systemic fluctuation was observed during the extended resting period without visual stimulation. The similarity of the response in the infants indicates the robustness of the measurement and the high reliability of our system within the neonatal population.

Our results are comparable to those from a recent study published by Karen, ³⁰ in which they found a mean $\mathrm{Hb}{\mathrm{O}}_2$ increase of $0.98\mu \mathrm{mol}∕\mathrm{L}$ and a mean HbR change of $-0.17\mu \mathrm{mol}∕\mathrm{L}$ in a group of 20 healthy term infants with a mean age of $5.5\text{days}$ . One difference between the analyses is that the assumed differential path length factors (DPFs) are not the same. Karen applied a DPF the value of 4 from the results by Wyatt, ³¹ which used whole-head time-of-flight measurements taken up to three days postmortem from premature infants who died of intraventricular hemorrhage. In our study, we elected to use the DPF formulas reported by Duncan, ²⁸ as they used both living infants and had a reflectance measurement geometry similar to our own. Uncertainty in the DPF values can lead to errors in the magnitude of the hemodynamic response. Ideally, these DPF values and/or the optical properties of the head would be measured individually for each subject. For instance, the optical properties were recently measured using frequency-domain NIRS (FD-NIRS) in a large group of infants longitudinally.^{32, 33} Though functional responses were not measured in the FD-NIRS study, the results demonstrated the feasibility of measuring the in-situ optical properties, which could, in principal, be combined with the activation measurements detailed here.

An additional potential confound in evaluating the ratio of $\mathrm{Hb}{\mathrm{O}}_2$ to HbR is cross talk between $\mathrm{Hb}{\mathrm{O}}_2$ and HbR due to noise in the spectroscopy. Strangman, Franceschini, and Boas showed that the $780∕830\text{-}\mathrm{nm}$ pairing is susceptible to this problem.³⁴ However, due to the peak in HbR absorption at $760\mathrm{nm}$ , the $750∕830\text{-}\mathrm{nm}$ pairing appears more robust.³⁵ Here we use $750∕850\mathrm{nm}$ , and in the Karen study, they used $730∕830\mathrm{nm}$ , which minimizes this issue.

As in the study of Karen, while six of eight of our infants showed significant increases in $\mathrm{Hb}{\mathrm{O}}_2$ , fewer infants (four) showed significant decreases in HbR. Further, the ratio of the group-averaged contrast over the standard deviation was superior for $\mathrm{Hb}{\mathrm{O}}_2$ (2.0) than for HbR (1.4). Thus, our results reinforce their conclusion that $\mathrm{Hb}{\mathrm{O}}_2$ is a “more reliable indicator of functional activity than [HbR] or BOLD signal.”³⁰ While we found a similar response between healthy adults and infants, discrepancies in the visual activation response between different ages of infants and adults have been reported in previous studies. The pattern found in healthy adults (i.e., increased $\mathrm{Hb}{\mathrm{O}}_2$ and decreased HbR) has been previously reported in infants by both Taga ³⁶ and Karen ³⁰ In contrast, Meek ³⁷ found an increase in both $\mathrm{Hb}{\mathrm{O}}_2$ and HbR. This controversy in neurovascular coupling is also reflected in the literature from BOLD-fMRI studies. Born ¹⁵ found that in sedated or sleeping infants, there was a decrease in BOLD signals in response to visual stimulation, whereas in adults, there was an increase in BOLD signals. Yamada ²⁰ and Martin ³⁸ both showed an interesting dichotomy: younger infants showed the typical adult pattern of BOLD signal increase, whereas older infants showed a BOLD signal decrease in the visual cortex, while the oldest infants returned to a positive BOLD response. The timing of these changes varied between the two studies, with the first transition occurring at $8\text{weeks}$ of age in the infant cohort from Yamada, and at $16\text{weeks}$ in those from Martin; the latter transition was observed at $40\text{months}$ in Martin with negative BOLD responses peaking at $1\text{to}2\text{years}$ of age. A summary of the study methodologies and results is presented in Table 3 .

Table 3

Summary of the literature on hemodynamic responses in infants. Papers from the same investigators are grouped into single rows.

Another consideration when analyzing the literature on infant neurovascular coupling is that many BOLD-fMRI studies are often conducted with the infants under sedation to prevent movement. Multiple studies have found effects on the BOLD response due to anesthesia, including nonlinear and nonmonotonic dose-response curves.³⁹ Cannestra found that pentobarbital in adults caused negative BOLD responses to visual stimulation.⁴⁰ Thus, interpretation of findings from sedated infants using BOLD-fMRI is complex. In contrast, NIRS studies can be conducted on both awake (as in Taga ³⁶) and resting/sleeping (as in Karen ³⁰ and the current study) infants. It is unclear whether sleep state can also affect the neurovascular response. While electrophysiology studies of visual evoked potentials have shown differences in the latency and amplitude of the electrical response between sleeping and awake infants,^{41, 42} no clear evidence is available to show an effect on the hemodynamic response. While the cited NIRS results seem to report similar visual response patterns regardless of the level of consciousness, to our knowledge, no randomized controlled NIRS study has investigated the effect of behavioral states and sedation on the visual activation pattern.

It has been hypothesized that maturation or development of the neurovascular coupling may modulate the activation patterns observed in infants.^{20, 26, 30, 38} Increasing white matter myelination at $8\text{weeks}$ of age with an increase in metabolic demand was proposed by Yamada to explain the negative BOLD response seen in older infants.²⁰ In studies that recruited a higher percentage of infants that had been born preterm, a higher percentage of infants were seen with an inverted functional response to visual stimulation later in life,^{20, 26} suggesting that prematurity may also affect subsequent cerebral metabolism and should be controlled. To resolve these ambiguities, it is crucial for studies to be tightly controlled in both gestational age range and clinical course. For this reason, we restricted ourselves to studying healthy term-born infants within only the first three days of life. While there are changes in baseline cerebral hemodynamics during this time window, this is a tight timing constraint relative to the existing literature. Our findings show a functional response pattern similar to that of adults. By qualitative visual inspection, it does appear that the response is slower in the infants than the adults; however, a more detailed study with great numbers of infants and adults would be needed to quantitatively evaluate the potential timing difference.

While the proposed superficial signal regression method improves the reliability of infant data, further optimization is possible. The superficial noise signal is produced from the most closely spaced SD pairs. These SD pairs are separated by $1\mathrm{cm}$ , giving the signal an approximately $3\text{-}\mathrm{mm}$ penetration depth. We have found that in some infants the $1\text{-}\mathrm{cm}$ spacing is sufficient to sample brain signals. In the case where the functional brain signal is mixed in with the physiological noise signal, the regression may reduce the intensity of the brain signal in addition to reducing the noise. As a result, the contrast is degraded. Alternative solutions, such as independent component analysis (ICA), might allow for better removal of noise sources that are present in all channels.⁴³ In addition, extension of the imaging cap to a larger field of view will provide more robust imaging data. In adults, where larger fields have been used, the spatial localization of the response has provided further improvements in contrast to noise ratio. The potential for these proposed improvements in data analysis and instrumentation, along with further optimization and expansion of the stimulation paradigm, suggest that while there is yet work to be done, it is reasonable to foresee a robust protocol emerging for routine clinical use.

Resolving the controversy over the true neurovascular coupling in infants has been challenging. Our results show that in newborn healthy term infants in the first three days of life during a visual stimulus, there is an increase in $\mathrm{Hb}{\mathrm{O}}_2$ and a decrease in HbR. This pattern of visual response is similar to our adult data, as well as some recently published findings in infants, suggesting that the visual activation response for infants at term is similar to the mature response. Since the age, behavioral/conscious state, and visual stimulation paradigm all may alter the neurovascular response, we aimed to control these variables in our study. We believe that the use of NIRS provides a crucial insight into the hemodynamics that is not found with BOLD-fMRI, and further, that the high-density NIRS technique with superficial signal regression provides a more reliable measurement of brain hemodynamics than typically afforded by traditional NIRS systems. Future research can use the flexible techniques proposed here to analyze longitudinal variations that may occur in the neurovascular coupling of infants. Establishing a firm understanding of the normal hemodynamic response in healthy infants is a critical first step toward evaluating abnormal responses in high-risk infants to aid clinicians in the early recognition of cerebral dysfunction.