Multi-Parametric Evaluation of Cerebral Hemodynamics in Neonatal Piglets Using Non-Contrast-Enhanced Magnetic Resonance Imaging Methods

Hypoxic–ischemic encephalopathy (HIE) is a leading cause of neonatal mortality and severe neurological impairment in childhood.¹ The hypoxic–ischemic (HI) event leads to abnormally reduced cerebral blood flow (CBF), and the resulting energy failure can cause cellular death; even if CBF is restored, a multifaceted polymodal secondary energy failure often occurs aggravating brain damage.¹ Therefore, measuring CBF, oxygen extraction fraction (OEF), and cerebral metabolic rate of oxygen (CMRO₂), the three parameters that characterize cerebral oxidative metabolism, is clinically important. Abnormalities in these hemodynamic parameters have been reported in neonates with HIE compared to normal controls.^2-4 However, clinical magnetic resonance imaging (MRI) protocols are not tailored to measure cerebral hemodynamic disturbances in neonates, and it is unclear whether or not HI damage to neurons, glia, or their subcellular compartments directly relates to these hemodynamic changes. This uncertainty is further complicated by the knowledge that these hemodynamic parameters are also affected by other factors such as gestational and postnatal age of the neonate.^{5, 6}

Accurate measurement of CBF and OEF relies on appropriate MRI techniques. Phase-contrast (PC) MRI and arterial spin labeling (ASL) have been successfully applied in human neonates to quantify CBF.^2-6 PC MRI measures the blood flow in the feeding arteries of the brain to provide a global measurement of CBF and has the advantage of short scan time (1–2 minutes).^{3, 5} In contrast, ASL provides regional CBF mapping by magnetically labeling water in the arterial blood as an endogenous tracer.^{2, 4, 6} Brain perfusion mapping in healthy and sick newborns has been conducted by both pulsed ASL (PASL)^{4, 7} and pseudo-continuous ASL (PCASL).^{6, 8, 9} Compared to PASL, PCASL provides higher sensitivity to perfusion signal and has been demonstrated with better image quality in neonatal brains.⁸ However, both PASL and PCASL suffer from low signal-to-noise ratio (SNR) because of the prolonged arterial transit time (ATT) in neonates.⁹ Alternatively, velocity-selective ASL (VSASL) is insensitive to ATT and has demonstrated improved SNR over conventional ASL methods.^10-12

A limited number of MRI-based techniques have been developed to measure OEF in the human neonatal brain without the use of exogenous contrast agents.^{2, 5, 13, 14} Among them, T₂-relaxation-under-phase-contrast (TRUPC) is an MRI technique that can measure OEF in multiple major cerebral veins, such as the superior sagittal sinus (SSS) and internal cerebral veins (ICVs), thereby enabling the assessment of OEF for both cortical and subcortical brain regions.¹⁴ If OEF and CBF are both quantified, CMRO₂ can then be calculated using the Fick principle.⁵

Animal models provide the opportunity to develop new metrics to investigate how CBF, OEF, and CMRO₂ relate to normal brain structure, function, and brain damage under well-controlled conditions. Piglets have similar brain growth patterns and cerebral anatomical structures as human neonates, and the regional distribution of neuropathology in piglet HIE resembles that in human newborns.¹⁵ MRI in piglet models of HI and excitotoxicity, an integral cytotoxic molecular cascade that contributes to neonatal HIE,¹⁶ can also provide salient information relevant to human neonatal brain injuries.^{17, 18} However, so far, neonatal piglets with brain damage have been primarily interrogated using structural imaging, such as T₁-weighted, T₂-weighted imaging, and diffusion imaging techniques.^{17, 18} There is a paucity of MRI sequences to assess oxygen delivery and consumption in clinically relevant neonatal piglet models of HIE.

To facilitate future investigations on neonatal encephalopathy with the piglet model, we sought to test the feasibility of PC, VSASL, and TRUPC in quantifying CBF, OEF, and CMRO₂ in different brain regions using piglets recovered from focal excitotoxic or global HI brain injuries.

Materials and Methods

The Animal Care and Use Committee approved all protocols, which were in compliance with the United States Public Health Service Policy on the Humane Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals. Ten neonatal male piglets were included. Two encephalopathy models were studied: excitotoxic brain injury model and global HI brain injury model.

Image Processing

The T₂-weighted images were first co-registered to the VSASL images using rigid body transformation with SPM12 (Statistical Parametric Mapping; Wellcome Department of Imaging Neuroscience, Institute of Neurology, London, UK). Data were then analyzed with Matlab 2019b (The MathWorks, Natick, MA, USA) and ImageJ (Rasband W., National Institutes of Health, USA).

VSASL-based CBF maps were calculated using the subtraction of label from control images (∆S) divided by the equilibrium magnetization of tissue (SI_PD) as:

Here the brain blood partition coefficient, λ, was 0.9 mL/g; α_label is the labeling efficiency and α_BGS is the correction factor for background suppression, taken as 0.57 and 0.86, respectively, as used in a previous study on adult human brain.¹¹ The duration of the flow dephasing module, TE_prep, was 20 msec, and T_2,tissue was assumed to be 90 msec. The tissue T₁ of newborn was assumed to be about 1600 msec.²⁰ Since the mean Hct values of piglets were 22.9% (Table 1), the blood T₁ at 3 T was calculated to be 2105 msec based on a Hct-T₁ conversion model.²¹ Hence T_1,eff was taken as 1853 msec, as averaged between tissue T₁ and blood T_1.¹¹ Finally, CBF units were converted from mL/g/s to mL/100 g/minute through a factor of 6000.

TABLE 1. Physiological and Hemodynamic Parameters of Each Piglet

					TRUPC		PC		VSASL
Piglet #	Treatment	Time after injury (hour)	Hct (%)	EtCO2 (mmHg)	OEF (ICV) (%)	OEF (SSS) (%)	CBF (mL/minute/100 g)	CMRO2 (μmol/minute/100 g)	rCBF	CBF (mL/minute/100 g)	CMRO2 (μmol/minute/100 g)
1	Sham	168	21.6	34.5	48.1	61.2	27.3	75.9	N/A	N/A	N/A
2	Sham	23	22.5	38.0	52.8	55.4	19.9	51.9	1.5	35.7	93.3
3	QA (240 nmol)	96	24.1	36.0	46.3	63.4	21.5	68.9	1.3	21.9	70.3
4	QA (720 nmol)	96	24.3	34.0	N/A	68.5	10.7	37.4	N/A	22.2	77.4
5	QA (720 nmol)	91	24.3	36.0	52.7	59.1	36.3	109.4	1.4	22.2	67.1
6	QA (720 nmol)	20	16.8	46.5	49.1	48.5	55.4	94.8	0.9	43.3	74.2
7	QA (960 nmol)	25	23.4	77.5	21.2	21.3	279.3	292.3	0.7	64.4	67.4
8	QA (960 nmol)	24	20.4	42.0	37.9	43.1	45.0	83.1	0.8	36.8	68.0
9	HI	45	23.4	43.0	41.4	49.1	N/A	N/A	0.8	28.5	68.8
10	HI	65	28.4	39.0	40.5	45.5	41.1	111.5	0.9	36.4	98.7
Mean			22.9	38.8	46.1	54.9	32.1	79.1	1.1	30.9	77.2
SD			3.2	4.3	5.6	8.8	14.9	26.2	0.3	8.3	12.2
CoV (SD/mean, %)			14.0	11.1	12.2	16.0	46.4	33.1	28.3	26.9	15.8

• Piglet #7 was considered as an outlier due to almost twice-of-average EtCO₂, and excluded from the statistical analyses.
• QA = quinolinic acid injection; HI = global hypoxia-ischemia; Hct = hematocrit; EtCO₂ = end-tidal carbon dioxide; TRUPC = T₂-relaxation-under-phase-contrast; PC = phase contrast; VSASL = velocity-selective arterial spin labeling; OEF = oxygen extraction fraction; SSS = superior sagittal sinus; ICV = internal cerebral vein; CBF = global cerebral blood flow for PC or whole-brain averaged CBF for VSASL; rCBF is the relative CBF of putamen and caudate of right side to the left side; CMRO₂ = cerebral metabolic rate of oxygen of the whole brain; CoV = coefficient of variation; N/A = data not acquired.

A neuropathologist and neuroanatomist with 36 years of experience in human and large animal brain (monkey, dog, cat, piglet) neuroanatomy and 20 years of experience in MRI (L.J.M.) and an MRI physicist with 10 years of MRI experience (D.L.) annotated the brain structures on the VSASL-based CBF maps. Regions of interest (ROI) for the whole brain were manually drawn from the final CBF maps (D.L.) to calculate the average CBF. For all piglet brains, ROIs were manually drawn in consensus to encompass the left and separately right putamen and caudate. Relative CBF (rCBF) was defined as the ratio of mean CBF of the right ROI to the left ROI.

The PC data were processed following previously reported methods.⁵ Briefly, the PC sequence produces three images: an anatomic image, a complex difference (CD) image showing the vessels, and a flow velocity map. For each ICA, an ROI was manually drawn by an MRI physicist with 9 years of MRI experience (D.J.) in the CD image by closely tracing the boundary of the artery. This ROI was then applied to the flow velocity map, and the flow rate (in mL/minute) of the artery was quantified by spatially integrating the velocities within the ROI. The PC based CBF (mL/100 g/minute) was calculated by dividing the sum of the flow rates of the two ICAs by the brain mass (in 100 g), which was estimated from the volumes of ROIs of the whole brain manually drawn from the high-resolution T₂-weighted images (D.L.), assuming a brain tissue density of 1.06 g/mL.⁵ Note that we did not include the vertebral arteries or the basilar artery in the PC CBF calculation because they were not visible in the TOF angiogram and are very small even in juvenile pigs.²²

The TRUPC images were reconstructed and corrected for eddy-current induced phase errors¹⁴ using in-house Matlab scripts. Two preliminary ROIs were drawn (D.J.) in the CD images to encompass the SSS and ICV, respectively. Within each ROI, the eight pixels with the highest intensities were selected as the final mask. Mono-exponential fitting of the signal intensities inside the final mask was conducted where each pixel was allowed to have an individual initial signal but was forced to have the same T₂. Venous oxygenation (Y_v) values were converted from the blood T₂ using a published calibration model,²³ based on the measured hematocrit level of each piglet. OEF was then calculated using:

assuming an arterial blood oxygenation, Y_a, of 100%. As a metric of the reliability of the OEF value, the width of the 95% confidence interval of 1/T₂ (ΔR₂) was estimated from the mono-exponential fitting process. OEF values with ΔR₂ ≥ 10 Hz suggested poor data quality and were excluded.

CMRO₂ was then calculated as:

where C_ery = 21 μmol O₂/mL blood cells²⁴ and Hct was the measured hematocrit of each piglet. Here the global CMRO₂ in units of μmol/100 g/minute was derived both from the whole-brain average of VSASL-based CBF maps and the PC-based CBF values separately, with OEF obtained from SSS.

Results

Ten neonatal piglets (2–4 days old, 1–2.2 kg) underwent excitotoxic brain injury from QA (N = 6), global HI injury (N = 2), or sham procedure (N = 2). Sham piglets had highly stable physiological parameters, including arterial pH ~7.4, arterial partial pressure of carbon dioxide (PaCO₂) ~41 mmHg, arterial partial pressure of oxygen (PaO₂) ~140 mmHg, and base excess approximately −3. The physiologic severity of the HI protocol induced similar blood gas physiology at 7 minutes of asphyxia with arterial pH 6.84 and 6.92, PaCO₂ 115 mmHg and 108 mmHg, PaO₂ 18 mmHg and 20 mmHg, and base excess −14.3 and −10.9 in the two piglets. Both piglets were resuscitated with the restoration of pH to greater than 7.30, normocarbia, and normoxia, and extubated 3 hours after the HI injury.

Quantitative regional CBF maps obtained by three-dimensional VSASL from a sham piglet are shown in Fig. 1, with corresponding T₂-weighted images. The ROIs of different structures in this sham piglet's brain are delineated in Fig. S1 in the Supplemental Material. The piglet neocortex showed non-uniform cerebral perfusion with higher CBF in the primary sensory cortical areas (auditory, visual, somatosensory, and olfactory: 41.6 ± 5.2, 39.5 ± 5.7, 33.0 ± 8.5, and 67.7 ± 47.7 mL/100 g/minute, respectively) (Fig. 1, slices 5, 6, 10, 13–16). Primary motor cortex had a CBF of 37.5 ± 6.3 mL/100 g/minute (Fig. 1, slice #7). In contrast, the lateral convexity of the frontal cortex showed lower perfusion (20.7 ± 4.5 mL/100 g/minute) (Fig. 1, slice #1–4). The hippocampus had very low apparent perfusion (12.9 ± 5.1 mL/100 g/minute) (Fig. 1, slice #12). In subcortical forebrain gray matter, the caudate nucleus and putamen (33.2 ± 5.3, 31.7 ± 4.1 mL/100 g/minute) were divisible from the central thalamus and globus pallidus by their higher perfusion (Fig. 1, slice #9). Thalamus was also notable because some parts showed low cerebral perfusion while other parts, such as the visual relay lateral geniculate nucleus (LGN) and auditory relay medial geniculate nucleus (MGN), had comparably higher CBF (49.5 ± 6.5, 49.7 ± 6.9 mL/100 g/minute) (Fig. 1, slice #12). The brainstem tectal collicular nuclei visual and auditory relay centers correspondingly showed high CBF (51.2 ± 39.6 mL/100 g/minute) (Fig. 1, slice #14, 15). Moreover, brainstem structures such as the pons and middle cerebellar peduncle, which are known to provide afferents to cerebellum, were delineated by high perfusion (75.8 ± 21.6 mL/100 g/minute) (Fig. 1, slice #14) in a pattern consistent with high perfusion in the cerebellar vermis (49.7 ± 14.5 mL/100 g/minute) (Fig. 1, slice #17).

Different brain levels seen by H&E staining and SOD2 immunohistochemistry are shown next to the CBF maps (Fig. 2). In a descriptive evaluation of SOD2 immunolocalization, different brain regions appeared distinct because they were divisible by varying intensities of mitochondrial positivity. Figure 2a illustrates this finding with a comparison of high SOD2 immunoreactivity in the LGN (Fig. 2a, right) to the much lower SOD2 staining in the nearby hippocampus (Fig. 2a, left) within the same tissue section. In brainstem, the superior and inferior colliculi were identifiable in the H&E section and in the CBF maps (Fig. 2b). Cerebellar cortex showed exquisite distinctions in intracellular mitochondrial loads even within the same cell types but in different cortical subregions. In the lateral cerebellar hemisphere, the neocerebellum, the Purkinje cell bodies were generally SOD2 negative at this age (Fig. 2c, left). In the cerebellar vermis, the medial unpaired component of the cerebellar cortex, Purkinje cells had contrastingly high mitochondrial enrichment (Fig. 2c, right).

Figure 3 shows two different piglets with 960 nmol QA injection in the right putamen that were scanned ~24 hours after the injury. The T₂-weighted images show the unilateral injury as witnessed by the higher signal intensity (Fig. 3a,c, red arrows). Figure 4 shows the TRUPC data of a sham piglet. The physiological values (Hct, EtCO₂) and MRI-based hemodynamic parameters (CBF, OEF, and CMRO₂) of each piglet are listed in Table 1, along with their group-averaged values. One piglet with 960 nmol QA injection was considered as an outlier due to almost twice-of-average EtCO₂ and was excluded from the statistical analyses. The Hct and EtCO₂ of the remaining nine piglets were 22.9 ± 3.2% and 38.8 ± 4.3 mmHg with CoV of 14.0% and 11.1%, respectively. With the PC-based method, the global CBF was 32.1 ± 14.9 mL/100 g/minute with a CoV of 46.4%. Using the VSASL-based approach, the CBF averaged from the whole-brain CBF map was 30.9 ± 8.3 mL/100 g/minute with a CoV of 26.9%. The OEF measured at the SSS was 54.9 ± 8.8% with a CoV of 16.0%. In one piglet, the OEF at ICV was excluded due to ΔR₂ ≥ 10 Hz. Across the remaining eight piglets, OEF at ICV was 46.1 ± 5.6% with a CoV of 12.2% and was significantly lower than the OEF at SSS (P < 0.05). With the OEF obtained from SSS, the global CMRO₂ derived from PC-measured CBF was 79.1 ± 26.2 μmol/100 g/minute with a CoV of 33.1%, and the global CMRO₂ derived based on VSASL-measured CBF was 77.2 ± 12.2 μmol/100 g/minute with a CoV of 15.8%.

We found a significant positive correlation between PC and VSASL based global CBF values (N = 7, r = 0.82, P < 0.05, Fig. 5a). Negative correlations between OEF at SSS and both VSASL global CBF (N = 8, r = −0.88, P < 0.05, Fig. 5b) and PC global CBF (N = 8, r = −0.79, P < 0.05, Fig. 5c) were observed. EtCO₂ was negatively correlated with OEF at SSS (N = 9, r = −0.84, P < 0.05) but positively correlated with PC global CBF (N = 8, r = 0.81, P < 0.05). There was no correlation between EtCO₂ and PC-based CMRO₂ (N = 8, r = 0.55, P = 0.16) (Fig. 5d–f).

For the analysis of neuron counts to MRI, two piglets (one sham and one HI) without neuropathology cell counts were excluded. A third piglet (with QA 720 nmol) was excluded due to imaging artifact in the deep brain CBF map and unreliable OEF at ICV (ΔR₂ ≥ 10 Hz). In the remaining six piglets, rCBF of putamen and caudate and OEF obtained from ICV showed no significant correlations with the ratio of degenerating-to-total neurons (rCBF: r = −0.54, P = 0.30, Fig. 6a; OEF: r = −0.77, P = 0.10, Fig. 6d), the ratio of normal-to-total neurons (rCBF: r = 0.31, P = 0.56, Fig. 6b; OEF: r = 0.66, P = 0.17, Fig. 6e), or the ratio of injured-to-total neurons (rCBF: r = −0.37, P = 0.50, Fig. 6c; OEF: r = −0.49, P = 0.36, Fig. 6f).

Discussion

Our study demonstrated the feasibility of multi-parametric evaluation of cerebral hemodynamics in neonatal piglets with excitotoxic and HI brain injury using non-invasive MRI techniques. CBF was evaluated by PC MRI and VSASL, and OEF was evaluated by TRUPC MRI. VSASL provided a high-resolution neuroanatomical interrogation of regional CBF that appears to be related to gray matter mitochondrial enrichment. When averaged across the whole brain, the VSASL-based CBF values of each piglet showed a significant correlation with PC-based global CBF. EtCO₂ was negatively correlated with OEF at SSS. In the striatal areas injured by QA, decreased perfusion signal was revealed in regional CBF maps. We found no significant correlation between neuron counts and rCBF in putamen and caudate or OEF at ICV.

The annotated maps of CBF are salient because they show regional CBF variations in newborn piglet brain within the context of known neuroanatomy. We observed distinct CBF differences in cerebral cortex that match known functional domains. For example, higher perfusion was seen in the primary sensory cortices (olfactory, somatosensory, visual, auditory) compared to the dorsolateral prefrontal cortex. This finding is consistent with advanced maturation in sensory brain regions but not in areas with cognitive and executive functions at this age.²⁶ However, the cingulate cortex also had generally higher CBF than other cortical regions and has executive functions too, but it also has autonomic and limbic functions involved in processing aversive stimuli. The very low perfusion in hippocampus in neonatal piglets reinforces this conclusion. Furthermore, we observed starkly segregated, high perfusion regions within functionally active neural systems and their connectomes. For example, the piglet central visual system, which is an energetically demanding network,²⁷ had high perfusion throughout the brainstem (tectum), diencephalon (LGN), and neocortex (occipital cortex). Thus, mapping CBF in specific brain networks could advance neonatal neurocritical care medicine because metabolism-connectivity could be partially responsible for the neonatal brain's regional vulnerability to HIE.²⁸ Our observation of relatively higher perfusion to thalami, basal ganglia, brainstem, cerebellum, and sensorimotor cortex than other regions agree with results from applying long or multiple-PLD three-dimensional PCASL in human neonate brain.^{6, 9}

The contrast of perfusion in various brain structures was supported by the localization of mitochondria by SOD2 immunohistochemistry. Visual differences in regional CBF suggest that different structures have varying oxidative phosphorylation needs and that regional mitochondrial SOD2 differences might distinguish these structures. In prior work,^{28, 29} cytochrome c oxidase enzyme histochemistry was used to map in situ metabolic activity in neonatal piglet brain by disclosing the catalytic activity of complex IV. We could not apply this technique in the current study due to limitations in tissue processing. Notwithstanding, the SOD2 pattern generally matches the mitochondrial activity reported by Martin et al.²⁹ The perfusion pattern we observed also resembles the reported cerebral glucose metabolism of human newborns using ¹⁸F-fluorodeoxyglucose PET,²⁶ reflecting the metabolic activity at the early stages of brain development. The forebrain perfusion was consistent with olfaction being a dominant primary sense in pig.³⁰

Measuring OEF is traditionally performed by PET imaging with ¹⁵O-labeled radiotracers.³¹ However, the invasive nature of the procedure and exposure to ionizing radiation are major obstacles to its applications in neonates.³¹ Optical methods such as near-infrared spectroscopy (NIRS) have been widely used to monitor neonatal cerebral oxygenation but have difficulty in probing the deep brain structures due to the limited penetration depth of light.³² In the past decade, several MRI-based techniques have been developed to measure OEF on a global level in neonates.^{2, 5, 13} However, regional OEF measurement is more desirable because many diseases, such as HIE and stroke, mainly affect specific brain regions.^{19, 33} In this work, we used the TRUPC MRI technique to quantify OEF in two major cerebral veins: the SSS which drains the majority of cerebral cortical veins, the falx cerebri, meninges, diploic veins, and emissary veins; and the ICV which is formed by the unification of the thalamostriatal, septal, and choroidal veins and drains deep brain regions including thalami and basal ganglia. Among them, OEF at SSS has been shown to be correlated well with whole-brain averaged OEF measured by ¹⁵O-PET.³⁴ We found that EtCO₂ correlated positively with PC-based CBF but negatively with OEF at SSS, and there existed a negative correlation between OEF at SSS and CBF (both PC-based and ASL-based). These findings are consistent with previous reports on human adults^{35, 36} and were thought to be related to the well-known vasodilatory effect of blood CO₂ content and the balance between blood supply and oxygen extraction. We observed a lower OEF at ICV than OEF at SSS, which was consistent with previous reports on human neonates.¹⁴ The lower OEF at ICV may be explained by the higher CBF in deep brain structures like thalami and basal ganglia observed in this study and previous literature.^{6, 9}

In our exploratory study of neuropathology in a small sample size, both the rCBF of putamen and caudate and OEF at ICV exhibited negative trends with the ratio of degenerating-to-total neurons; however, none of the associations were statistically significant. Unlike in stroke, piglet models of encephalopathy have selective and regional vulnerability with asynchronous cellular injury and degeneration. Damaged neural cells are not at the same stage of injury at any given time in vivo. To address this, we examined multiple recovery durations to capture different time points after brain injury to better study the neuropathology. We also specifically counted degenerating neurons that were undergoing irreversible, end-stage cell death.¹⁷ It should be noted that our neuronal cell counting was conducted in microscope fields that represented a very small portion of the large putamen and caudate ROI that was used to quantify rCBF. Moreover, the ICV-derived OEF included large brain regions that are drained by ICV but were not examined by microscopic neuropathology, such as the thalami. Therefore, given the small sample size (N = 6), it was not surprising that we did not observe significant associations.

Though we did not observe significant relationships between neuron counts and rCBF or OEF, other reports indicate that such a relationship does exist. Using an HI rat model induced by unilateral common carotid artery ligation and subsequent hypoxia, Vannucci et al found reduced CBF in multiple regions of the ipsilateral cerebral hemisphere and that the extent and spatial distribution of CBF reduction correlated with those of ischemic neuronal necrosis observed in a similar rat model.³⁷ Kurth et al showed that piglets with longer HI duration tended to have more severe neuronal damage and also a higher cerebral oxygen saturation measured by NIRS, which indicated a lower OEF in these piglets.³⁸ In human neonates, it has also been demonstrated that neonates with HIE had lower OEF than normal controls,² and neonates with severe HIE had even lower OEF than those with moderate HIE.³ Although the relationships between CBF and/or OEF and neuropathology that we observed were statistically insignificant among a small sample size, our findings may support the rationale for larger studies investigating whether MRI can detect depressed tissue oxygen metabolism with neuronal cell death in neonatal brain.

Medications that induce general anesthesia are known to alter CBF, OEF, and CMRO₂. Because all piglets in our study received the same anesthetic regimen, we expect similar anesthesia-related effects on these physiologic parameters in all piglets. Additionally, the inhaled oxygen concentration was closely monitored and maintained at 50% for all piglets during their MRIs scans. We did not study differences in cerebral oxygen supply and demand because that was beyond the scope of our study.

Conclusion

We measured hemodynamic parameters (CBF, OEF, and CMRO₂) in neonatal piglet brain using non-contrast MRI techniques. VSASL-based CBF maps revealed well-defined regional CBF patterns that corresponded to known functional domains and matched to brain regions with varying mitochondrial enrichments as seen by direct localization of these organelles. CBF and OEF relationships to neuropathology were inconclusive. Our results suggest that these methods may be useful for monitoring perfusion and oxygenation of brain development and neuropathological conditions in human neonates.