Small animal PET with spontaneous inhalation of 15 O-labelled oxygen gases: Longitudinal assessment of cerebral oxygen metabolism in a rat model of neonatal hypoxic-ischaemic encephalopathy

Abstract

Perinatal hypoxic-ischaemic encephalopathy (HIE) is the leading cause of irreversible brain damage resulting in serious neurological dysfunction among neonates. We evaluated the feasibility of positron emission tomography (PET) methodology with ¹⁵O-labelled gases without intravenous or tracheal cannulation for assessing temporal changes in cerebral blood flow ( CBF ) and cerebral metabolic rate for oxygen ( CMRO₂ ) in a neonatal HIE rat model. Sequential PET scans with spontaneous inhalation of ¹⁵O-gases mixed with isoflurane were performed over 14 days after the hypoxic-ischaemic insult in HIE pups and age-matched controls. CBF and CMRO₂ in the injured hemispheres of HIE pups remarkably decreased 2 days after the insult, gradually recovering over 14 days in line with their increase found in healthy controls according to their natural maturation process. The magnitude of hemispheric tissue loss histologically measured after the last PET scan was significantly correlated with the decreases in CBF and CMRO_2. This fully non-invasive imaging strategy may be useful for monitoring damage progression in neonatal HIE and for evaluating potential therapeutic outcomes.

Introduction

Perinatal asphyxia resulting in hypoxic-ischaemic encephalopathy (HIE) is a major cause of premature mortality worldwide, and 25% of survivors suffer from long-term neurological and neurodevelopmental impairments such as cerebral palsy. ¹ The incident occurs in approximately 1.5 per 1000 live births in developed countries. ² The pathophysiology starts with primary energy failure during the hypoxic-ischaemic (HI) event, involving oxidative metabolism failure, cytotoxic oedema and excitotoxin accumulation. The subsequent secondary energy failure results in glutaminergic excitotoxicity, apoptosis and neuroinflammation. ³ The only clinically feasible therapy for HIE neonates is hypothermia starting within 6 h post-incident, but not more than 50% of treated neonates achieve an improved outcome.^{4 –6} The predominantly employed Rice–Vannucci neonatal rodent HIE model^7,8 has provided extensive advances toward developing promising therapies that could enhance the neuroprotective effect of hypothermia.

Given the large variation in brain damage severity in this animal model, ⁹ in vivo follow-up observations on an individual basis are vital for consistent progress monitoring. This interindividual variability is also the case clinically in patients and thus reliable prognostic indicators are highly desired, particularly in the acute phase. In neonates, cerebral oxidative metabolism exhibits a distinct elevation in the early maturational process.^10,11 Oxygen supply derangement to the brain at this stage accordingly causes devastating outcomes including reperfusion-related injury after the initial decrease in cerebral blood flow ( CBF ) post-HI incident.^12,13 The subsequent progressive brain damage or healing process may be detected by a direct physiological parameter for brain tissue viability, i.e. the cerebral metabolic rate for oxygen ( CMRO₂ ). Thus, longitudinal monitoring of CBF and CMRO₂ in neonatal patients with their growth may aid in robustly assessing the disease course and potential treatment outcomes in association with normal developmental changes in brain energy metabolism. Moreover, this experimental approach should be minimally invasive, particularly for vulnerable neonatal rodents.

Studies have utilized the blood oxygenation level-dependent (BOLD) technique^14,15 to quantitatively assess blood oxygenation level in the superior sagittal sinus region in humans using magnetic resonance imaging, from which the oxygen extraction fraction ( OEF ) can be accurately quantified for the whole brain. A combination of BOLD with blood velocity assessment using the phase-contrast technique in the carotid artery region provides CBF and thus CMRO₂ for the whole cerebral region.^16,17 However, regional assessment or parametric imaging of OEF and CMRO₂ remains uncertain because of unknown signal behaviour in the diseased brain due to a varied vasculature abnormality that may violate the essential assumptions required in the theoretical model of Yablonskiy. ¹⁸

Positron emission tomography (PET) with ¹⁵O-labelled gases has been the gold standard tool for decades for quantitative measurements of CBF , CMRO₂ and OEF . Despite the proven clinical value of ¹⁵O-PET, its application to small animals requires exceptionally intensive procedures like tracheotomy and repeated arterial blood sampling^19,20 or the use of ¹⁵O-oxygen–labelled haemoglobin.^21,22 The complexity of the technical procedure and kinetic modelling approaches that require correction for recirculating ¹⁵O-H₂O are additional burdens. Recently, Temma et al. demonstrated a ¹⁵O-PET method that enables spontaneous inhalation of ¹⁵O-labelled gases without needing tracheotomy in mice. ²³ The technique enabled quantitative evaluations of CBF , CMRO₂ and OEF over a one-month time course in a mouse model of chronic cerebral hypoperfusion with bilateral common carotid artery stenosis. Currently, no other methodology allows a non-invasive, repetitive and fully quantitative assessment of cerebral metabolic variables together with tissue perfusion in the 3D domain in experimental settings.

This study depicted temporal changes in CBF , CMRO₂ and OEF after HI insult in rat neonates. We first assembled a novel integrated PET imaging system that enables sequential assessment of CBF , CMRO₂ and OEF in rat neonates with spontaneous inhalation of ¹⁵O-labelled oxygen (¹⁵O-O₂), carbon dioxide (¹⁵O-CO₂) and carbon monoxide (¹⁵O-CO) gases. We also examined the ageing variation in those parametric values in healthy controls at term-equivalent ages to define a comparable time course of normal postnatal brain development across HIE pups.

Materials and methods

Study design

An integrated ¹⁵O-PET system was developed enabling quantitative CBF , CMRO₂ and OEF assessment in small animals by spontaneous inhalation of ¹⁵O-O₂, ¹⁵O-CO₂ and ¹⁵O-CO. Using this system, two sets of experiments were performed; the first experiment was to determine the production rate of metabolized ¹⁵O-H₂O ( k_w ) in arterial blood after ¹⁵O-O₂ inhalation and the second to assess the temporal changes in quantitative CBF , CMRO₂ and OEF values in neonatal rats after HI insult compared with age-matched healthy or sham-operated controls.

Animal subjects and ethical statement

For the first experiment that determined the k_w , thirteen Sprague–Dawley rats consisting of six rats (five males and one female) at 40.3 ± 0.8 days old (d/o) with a body weight of 155.0 ± 30.1 g and seven (all males) at 25.7 ± 1.0 d/o with 65.6 ± 1.0 g were studied. For the second experiment, nine male and nine female Sprague–Dawley rat pups at 9 d/o, whose brain developmental stage is considered equivalent to that of human infants at birth,^24,25 from two litters were randomly divided into HIE, non-operated and sham groups, as summarized in Table 1.

Table 1.

Age, body weight and number of animal subjects on each PET imaging day in the second experiment. Numbers in parentheses denote values after exclusion from the image data analysis.

PET scan day after HI insult	Day 0	Day 1	Day 2	Day 7	Day 14
Age (days old)	9	10	11	16	23
HIE
Weights (gram)	24.0 ± 1.0(23.9 ± 1.1)	23.1 ± 2.1	26.1 ± 2.7	36.1 ± 4.5(35.3 ± 3.6)	50.6 ± 3.3
Animal number (n)	n = 5 (4)	n = 10	n = 11	n = 10 (7)	n = 5
Scan group 1	n = 5	n = 5	n = 6	n = 5 (2)	–
Scan group 2	–	n = 5	n = 5	n = 5	n = 5
Non-operated
Weights (gram)	24.0 ± 2.9	–	27.8 ± 1.4	35.5 ± 3.8	54.4 ± 6.1
Animal number (n)	n = 4	–	n = 3	n = 4	n = 4
Sham
Weights (gram)	–	–	–	40.2 ± 1.9	–
Animal number (n)	–	–	–	n = 3	–

HI: hypoxic-ischaemic; HIE: hypoxic-ischaemic encephalopathy; PET: positron emission tomography.

All animals were group housed under standard conditions (12-h light/dark cycle, temperature 21 ± 3 °C, humidity 55 ± 15%) with ad libitum access to RM3(E) rodent diet (SDS, UK) and tap water at the Central Animal Laboratory, University of Turku, Finland (UTUCAL). The study was approved by the National Project Authorization Board of Finland (ESAVI/20863/2018) adhering to the 3 R principles in accordance with Finnish National legislation (Act 497/2013, Decree 564/2013) and EU Directive 2010/EU/63 on the protection of animals used for scientific purposes. All reported experiments complied with the ARRIVE guidelines. ²⁶

Integrated ¹⁵O-PET imaging system

A comprehensive PET system was assembled to sequentially assess CBF , CMRO₂ and OEF in small animals as an extension of previous work. ²³ The entire system consisted of an ¹⁵O-oxygen–dedicated small cyclotron (C3D, IBA solutions, Louvain, Belgium), radiosynthesis and purification unit, gas chromatography for quality control, inhalation controller, dedicated PET scanners for each experiment and a radioactive gas evacuation system for effective scavenging of radioactive gases from the animal holder and radioactivity decay.

Radioactive gas production, purification/qualification and inhalation control

¹⁵O was produced using the cyclotron by a ¹⁴N(d,n)¹⁵O nuclear reaction. The target gas was 1.0% O₂ in N₂ and 1.0% CO₂ in N₂, producing ¹⁵O-O₂ and ¹⁵O-CO₂ gases in the target, respectively. Each gas was transported to the hot cell, where ¹⁵O-O₂ and ¹⁵O-CO₂ gases were purified. The ¹⁵O-CO gas was synthesized from the target ¹⁵O-O₂ gas by a charcoal column heated at 950 °C. The radiochemical and chemical purity was assessed each time before inhalation by gas chromatography (Model GC-2014, Shimazu, Kyoto, Japan) with Molecular Sieve and HayeSep Q columns (Figure 1(a)).

Figure 1.

A schematic of the comprehensive PET system newly assembled in this study and consisting of three parts (a–c). (a) Radioactive gas production and purification/qualification system consisting of a cyclotron, target chamber, radioactive gas production/purification unit and inhalation controller. A charcoal column heated at 950 °C (CC950) was utilized for ¹⁵O-CO synthesis. For ¹⁵O-O₂ purification, two columns filled with charcoal at room temperature (CC) and another with MS were used. For the purification of ¹⁵O-CO₂, a column with charcoal heated at 450 °C (CC450) was used. Gas chromatography (GC) was implemented with two columns with HayeSep Q and Molecular sieve (MS) to assess chemical and radiochemical purity. (b) Animal holder dedicated to ¹⁵O-oxygen inhalation PET. A corn-shaped facemask made of a thin fabric sheet is placed over the snout of the animal. Radioactive gases are carried to the facemask for spontaneous inhalation. The animal holder is well sealed and actively scavenged from the front surrounding the radioactive gas supply tube. Fresh air is passively carried from the back into the animal holder. (c) Radioactive gas evacuation system. Approximately 380 l/min of evacuation carries scavenged radioactive gases from the animal holder to the decay ducts installed in the institute basement. Approximately 24 min of transit time in the ducts allows for radioactivity decay by a factor of 4000 before it is released from the chimney to the outside and (d) the ¹⁵O-PET/CT protocol consists of three separate PET scans each with ¹⁵O-CO, ¹⁵O-CO₂ and ¹⁵O-O₂ inhalation conducted in a sequential PET/CT study in the three subject groups of HIE, non-operated and sham. CT: computed tomography; HIE: hypoxic-ischaemic encephalopathy; PET: positron emission tomography; Radio-gas: radioactive gas.

The quality-assured radioactive gases were then mixed with pure oxygen and air so that animals inhaled 20% oxygen during the entire period when animals were in the holder, even while radioactive gases were supplied or when the radioactivity supply rate was changed. The typical flow rate was 300 ml/min and the radioactivity supply rate was 250, 150 and 300 MBq/min, corresponding to ¹⁵O-O₂, ¹⁵O-CO₂ and ¹⁵O-CO, respectively. Using multiple radioactivity detectors and mass flow controllers reliably controlled these parameters. The inhalation gas was mixed with evaporated isoflurane gas at a given concentration (typically 2–2.5%) before it was transferred to the animal holder.

Dedicated ¹⁵O-PET system animal holder

Two sets of animal holder assemblies were constructed to adapt to the HRRT PET scanner (CTI PET Systems, Knoxville, TN, USA) for the first experiment and the β-Cube PET scanner (Molecubes, Ghent, Belgium) for the second experiment. Both holders were sealed to prevent radioactive gas leakage. A corn-shaped facemask made of a fabric sheet (100 μm thickness) gently covered the snout to which the radioactive gases were supplied at a flow rate of 300 ml/min. The air inside the animal holder was evacuated at 7–10 l/min from the front through small holes surrounding the supply tube inside the animal holder. Fresh air was then passively carried to the animal holder from the tail to the front (Figure 1(b)).

Radioactive gas evacuation system

The scavenged radioactive gas from the animal holder was carried to 28 decay ducts (each 9.5 m long and 150 mm diameter, total volume of 4649 l) implemented in the facility building, allowing sufficient radioactivity decay before releasing it from the facility (Figure 1(c)).

Animal preparation and experimental procedures

First experiment (assessment of metabolized ¹⁵O-H₂O)

Each animal assigned for the first experiment was anaesthetized with isoflurane (1.5–3%), and the left common carotid artery was cannulated with a polyethylene tube (SP-31, outer diameter: 0.8 mm, inner diameter: 0.5 mm, Natsume Seisakusho Co., Ltd, Tokyo, Japan). The cannula was secured by tight ligation around the cannulated vessel with 5-0 silk sutures (Ethicon, NJ, USA) and flushed with heparinized 0.9% NaCl to prevent blood clotting.

The animal was then moved to the animal holder and placed in the HRRT PET scanner. After ensuring the stabilisation of the animal under isoflurane anaesthesia, a 6 min attenuation correction scan was conducted. Next, a 6 min dynamic PET scan with a 5 s frame duration was started 15 s before initiation of ¹⁵O-O₂ inhalation. Arterial blood samples (0.1 ml each) were manually obtained 4–5 times at 30 s intervals after 30 s of ¹⁵O-O₂ inhalation from the catheter placed in the carotid artery. The blood samples were immediately divided into two samples. One sample was quickly centrifuged and the plasma radioactivity concentration was measured using a BeWell-Q3 well counter (Molecular Imaging Labo, Suita City, Japan), and the other sample was used to determine the whole blood radioactivity concentration.

Second experiment (sequential ¹⁵O-PET imaging)

HIE was induced in 9 d/o rats as described previously.^7,8 Briefly, under isoflurane anaesthesia (1.5–3%), the left common carotid artery was exposed, ligated (6-0 silk sutures: Ethicon, NJ, USA) and then completely cut with electrocautery. After resting with their dam for 60 min, pups were exposed to 120 min of hypoxia in a chamber containing 8% oxygen and 92% nitrogen (P360 ProOx, BioSpherix, NY, USA) at 36–37 °C rectal temperature. Pups were then returned to their dam again for recovery for over 60 min and kept in standard housing condition after sequential imaging studies. Sham pups underwent a similar surgical procedure without carotid artery ligation and hypoxic insult. Non-operated pups experienced no interventions.

PET and computed tomography (CT) scans were sequentially performed on eleven HIE, four non-operated and three sham pups using β-Cube and X-Cube small animal scanners (Molecubes). During all scans, the animal was placed on a heating pad to maintain its body temperature, and respiration rate was continuously monitored using a sensor pad placed underneath the animal’s chest.

After ensuring the stabilisation of the animal under isoflurane anaesthesia, a series of list-mode PET scans were acquired (Figure 1(d)). Two 14 min scans were started at the time of initiating continuous inhalation of both ¹⁵O-O₂ and ¹⁵O-CO₂ over 8 min. A separate 10 min scan was acquired from the time of initiating continuous inhalation of ¹⁵O-CO over 3 min on one HIE pup on days 1, 7 and 14 before the ¹⁵O-O₂ and ¹⁵O-CO₂ scans to assess cerebral blood flow ( CBV ). Thereafter, the holder containing the animal was moved to the CT scanner for attenuation correction. Ten-to-fifteen-minute breaks separated PET scans to change cyclotron settings and allow radioactivity decay in the animal’s body. Since the β-Cube PET scanner has a limited data transfer rate of 8 Mcps in the electric circuit, the radioactivity supply rate was adjusted not to exceed this limitation during each inhalation. After PET/CT scans, the animal was kept on a heating pad to recover from anaesthesia and returned to its regular housing.

These sequential scans were repeated four times, first within 6 h (day 0) and then at 24 h (day 1), 48 h (day 2) and 7 days (day 7) post-insult for HIE pups in scan group 1, and on days 1, 2, 7 and 14 post-insult for HIE pups in scan group 2. Age-matched non-operated pups were sequentially scanned four times at ages equivalent to those of HIE pups on days 0, 2, 7 and 14. Sham pups were scanned only on day 7 after the surgical intervention. The PET imaging time course with postoperative days and ages in the three subject groups is presented in Table 1. Image data analysis excluded animals that indicated poor radioactivity inhalation dose in the body during scanning or inconstant body position between ¹⁵O-O₂ and ¹⁵O-CO₂ scans.

Data processing

In the first experiment, PET images were reconstructed to generate dynamic images with 12 × 5 s, 8 × 15 s and 6 × 30 s frames for 6 min using the 3D OSEM method with depth-of-interaction compensation. ²⁷ Whole blood time-activity curves were obtained from the left ventricular (LV) region in the reconstructed images.

Well-counter counts for the plasma and whole blood samples were normalized by acquisition time duration(s) and weights for each sample, followed by correction for radioactivity decay to the PET initiation time. They were further corrected for the cross-calibration factor determined using a 5 cm diameter and 10 cm long cylindrical phantom so that the well counter-based activity concentration for whole blood was equivalent to that of the LV values in the PET image, as we previously reported. ²⁸ Plasma counting rates were corrected for the plasma/whole blood radioactivity concentration ratio for ¹⁵O-H₂O. ²⁹ The arterial blood concentration of ¹⁵O-O₂ [Bq/ml] was then obtained by subtracting the ¹⁵O-H₂O concentrations from the whole blood radioactivity concentrations for each sample.

In the second experiment, PET images were reconstructed to generate dynamic images with a 1 min frame duration for the entire period of 14 min using 3D OSEM with depth-of-interaction compensation for ¹⁵O-O₂ and ¹⁵O-CO₂ and 10 min for ¹⁵O-CO scans. Radioactivity decay correction was set uncorrected in the second experiment to avoid degradation of the pixel count precision in decay-corrected images for short-lived ¹⁵O radioisotopes, which occurs specifically in the β-Cube scanner.

Data analysis

Determination of the production rate of metabolized ¹⁵O-H₂O in arterial blood

For the first experiment, a volume of interest (VOI) was selected on the LV cavity region in the dynamic PET images obtained after 30 s continuous inhalation of ¹⁵O-O₂ using Carimas 2.1 (Turku PET Centre, Turku, Finland). The total blood arterial input function (AIF), ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ , was determined by interpolating the LV time–activity curve (TAC), as previously validated. ²⁸ We evaluated the agreement between this and the well counter-derived whole blood radioactivity concentration curves in each animal experiment.

The following model formulation was employed to estimate the metabolized ¹⁵O-H₂O curve, ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ , from the total blood AIF, ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ : ²⁵

$${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)={\mathit{k}}_{\mathit{w}}\text{\hspace{0.17em}·\hspace{0.17em}}{\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)\otimes {\mathit{e}}^{-{\mathit{k}}_{\mathit{w}}\text{·}\mathit{t}}$$

(1a)

and then the ¹⁵O-O₂ AIF, ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ , as shown below:

$${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)={\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)-{\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$$

(1b)

where ${\mathit{k}}_{\mathit{w}}$ [min⁻¹] is the production rate of the metabolized ¹⁵O-H₂O in arterial blood after ¹⁵O-O₂ inhalation. Next, we determined ${\mathit{k}}_{\mathit{w}}$ for each PET scan so that the simulated ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ in equation (1a) reproduced the well counter-derived metabolized ¹⁵O-H₂O curve by means of non-linear least square fitting (NLLSF). ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ and ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ in the second experiment were then calculated from PET-derived ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ with the given $\mathit{k}$ _w following equations (1a) and (1b).

Calculation of parametric values

Parametric values of CBV , CBF , OEF and CMRO₂ were calculated for two VOIs selected in the brain. CBV s were first determined from a ¹⁵O-CO image for six examinations for both ipsi- and contralateral VOIs by normalising the average radioactivity concentrations in the cerebral VOIs by that in the LV VOI. The averaged CBV value was used in the following calculation.

OEF and CBF ( f ) were both determined by simultaneous NLLSF of paired tissue time activity curves (tTACs) obtained with ¹⁵O-O₂ and ¹⁵O-CO₂ inhalation to the following equations. ²⁹

For the ¹⁵O-O₂ inhalation PET data:

$$\begin{array}{c}{\mathit{C}}_{\mathit{i}}\left(\mathit{t}\right)=(1-{\mathit{V}}_{\mathit{o}})\cdot \mathit{OEF}\cdot \mathit{f}\cdot {\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)\otimes {\mathit{e}}^{-\frac{\mathit{f}}{\mathit{p}}\cdot \mathit{t}}\\ \hspace{1em}+(1-{\mathit{V}}_{\mathit{w}})\cdot \mathit{f}\cdot {\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)\otimes {\mathit{e}}^{-\frac{\mathit{f}}{\mathit{p}}\cdot \mathit{t}}\\ +{\mathit{V}}_{\mathit{o}}\cdot {\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)+{\mathit{V}}_{\mathit{w}}\cdot {\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)\mathrm{}\end{array}$$

(2a)

and for the ¹⁵O-CO₂ inhalation PET data:

$${\mathit{C}}_{\mathit{i}}\left(\mathit{t}\right)=\mathit{f}\cdot {\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)\otimes {\mathit{e}}^{-\frac{\mathit{f}}{\mathit{p}}\cdot \mathit{t}}+{\mathit{V}}_{\mathit{w}}\cdot {\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$$

(2b)

where the fractional volume of the arterial blood for ¹⁵O-O₂ ( ${\mathit{V}}_{\mathit{o}}[\mathit{ml}/\mathit{ml}]$ ) and that for ¹⁵O-H₂O ( ${\mathit{V}}_{\mathit{w}}[\mathit{ml}/\mathit{ml}]$ ) were determined from CBV $[\mathit{ml}/\mathit{ml}]$ assuming a fractional venous blood volume ( F_vein ) of 0.835 ³⁰ as:

$$\mathrm{}\mathrm{}\mathrm{}\mathrm{}\mathrm{}{\mathit{V}}_{\mathit{o}}=\mathit{CBV}\cdot \{\left(1-{\mathit{F}}_{\mathit{vein}}\right)+(1-\mathit{OEF})\cdot {\mathit{F}}_{\mathit{vein}}\}$$

(3a)

$${\mathit{V}}_{\mathit{w}}=\mathit{CBV}\cdot (1-{\mathit{F}}_{\mathit{vein}})$$

(3b)

and p represents the tissue-to-blood partition coefficient of water (0.91 g/ml). The first-pass extraction fraction for ¹⁵O-water is limited in high flow regions, such as the rat brain, because of the high blood flow in small animals. Thus, this study defines CBF as a flux of ¹⁵O-water from the blood to the tissue space, as defined for CMRO₂ assessment using ¹⁵O-oxygen.

The AIF for ¹⁵O-O₂ ( ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ ) and metabolized ¹⁵O-H₂O ( ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ ) were estimated following equations (1a) and (1b), using the production rate of k_w determined in experiment 1. The AIF for the ¹⁵O-CO₂ inhalation experiment ( ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ ) was determined from the LV TAC as ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)={\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ .

The CMRO₂ values were calculated as the product of CBF ( f ), OEF and the oxygen content in arterial blood, [O₂] _a , as: ³⁰

$${\mathit{CMRO}}_2={\left[{\mathit{O}}_2\right]}_{\mathit{a}}\times \mathit{CBF}\times \mathit{OEF}$$

(4)

where [O₂] _a is calculated as a product of the oxygen volume confined per unit gram of haemoglobin (1.39 [ml/g]), the haemoglobin concentration in the blood [g/ml] and the fractional saturation of oxygen in arterial blood (e.g. 98%).

Time course of parametric values

Time courses of absolute CBF (ml/min/g), CMRO₂ (ml/min/g) and OEF in ipsi- and contralateral brain VOIs were evaluated in the HIE, non-operated and sham groups over 14 days after the day of HI insult (day 0) or postnatal day 9. Age, group and hemispheric region dependency in CBF , CMRO₂ and OEF were evaluated at each time point and between animal groups.

Histological analysis

After the last PET scan on day 14, HIE and non-operated pups (n = 5 and n = 4, respectively) were transcardially fixed with 4% paraformaldehyde under deep anaesthesia with 4% isoflurane. The brains were removed, processed and embedded in paraffin sections. Coronal slices were obtained in 2 mm intervals from the frontal pole using a microtome (Leica RM2265, Nussloch, Germany) and stained with haematoxylin-eosin (H&E) for morphology. Microglial activation was examined using Iba1. Antigen retrieval was performed in citrate buffer (pH 6.0, BioSite BSC-OKHURL) for 20 min using a pressure cooker (Decloaking chamber, Biocare Medical NxGen). Sections were incubated with endogenous enzyme block (hydrogen peroxide) and pre-protein block (BrightDiluent, normal antibody diluent, WellMed BD09-125) to avoid non-specific binding. A primary antibody of Iba1 (1:2000, 019-19741, Wako Ltd., Japan) was applied for 60 min, followed by secondary antibody (BrightVision, 1 step detection system goat anti-rabbit HRP, WellMed DPVR110HRP) for 30 min and detection reagents (BrightDAB, WellMed BS04-110) for 10 min at room temperature.

The area (mm²) of each hemisphere was measured on H&E-stained sections using CaseViewer 2.4 (3DHISTECH Ltd., Budapest, Hungary). The hemispheric volume of each brain was calculated by summing the hemispheric area of each brain slice and multiplying the sum of the section thickness. The mean ipsi-/contralateral hemispheric-volume ratios were compared between HIE and non-operated groups. Additionally, the ipsi-/contralateral hemispheric-volume ratio in each HIE animal was compared with the ipsi-/contralateral ratios of CBF and CMRO₂ values obtained on day 14.

Statistics

Results are presented as mean ± SD. The student’s paired or unpaired t-test was applied to compare two variables as appropriate. Pearson's analysis was used to test for correlation with GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). P-values <0.05 were considered statistically significant. Image data analysis was blinded to avoid any subjectivity.

Results

Five of the thirteen rats that were assigned to the first experiment were excluded from the data analysis because of unsuccessful arterial blood sampling or extremely low ¹⁵O-O₂ inhalation dose during the PET scan. Otherwise, all well counter-based whole-blood radioactivity concentrations agreed with the PET image-derived LV TAC in all cases and were further validated by an earlier study using the same experimental setup. ²⁸ Fitting the predicted metabolized ¹⁵O-water TAC to the manually separated ¹⁵O-water TAC resulted in average k_w values of 0.33 ± 0.06, 0.33 ± 0.07 and 0.33 ± 0.07 ${\min }^{-1}$ , corresponding to younger (n = 4, 25.5 ± 1.0 d/o, 63.0 ± 0.8 g), older (n = 4, 40.0 ± 0.0 d/o, 166.5 ± 20.6 g) and all animals (n = 8, 32.8 ± 56.9 d/o, 114.8 ± 56.9 g), respectively. No significant difference was found in k_w values between the two age groups. From these results, we defined a k_w value of 0.33 min⁻¹ in the calculation of CBF , CMRO₂ and OEF for all animals in the second experiment.

The whole-body distribution of ¹⁵O-gases in a rat scanned on days 1, 7 and 14 post-HI insult is shown in Figure 2 as sagittal PET/CT images accumulated for a period of 0–10 min, 0–5 min and 0–8 min after initiation of ¹⁵O-CO, ¹⁵O-O₂ and ¹⁵O-CO₂ gas inhalation, respectively. Radioactivity was visible on the surface of the snout inside the facemask (arrow a), in the gas supply tube (arrow b) and on the inner surface of the animal holder (arrow c). These radioactivity spots were negligibly small and did not significantly impact image data analysis. Radioactivity in the brain relative to the heart was lower with ¹⁵O-O₂ than that with ¹⁵O-CO₂. Radioactivity in the lung region was greater in ¹⁵O-O₂ images than ¹⁵O-CO₂ images.

Figure 2.

Representative sagittal whole-body PET/CT images of a HIE rat placed in the animal holder on days 1, 7 and 14 post-HI insult. Summed images over 0–10 min after ¹⁵O-CO, 0–5 min after ¹⁵O-O₂ and 0–8 min after ¹⁵O-CO₂ inhalation were displayed. White arrows (a–c) indicate the effects of radioactivity accumulated in the snout (a), emitted from the radioactive gas supply tube (b) and on the inner surface of the animal holder (c). CT: computed tomography; HIE: hypoxic-ischaemic encephalopathy; PET: positron emission tomography.

Figure 3 shows ¹⁵O-gas accumulation in a HIE brain scanned on day 14 (Figure 3(a) to (c)) and LV TAC and tTACs (Figure 3(d) to (f)) for the selected VOIs defined in the ¹⁵O-CO image (Figure 3(a)). The decreased radioactivity in the ipsilateral hemisphere is clearly visible in both ¹⁵O-O₂ and ¹⁵O-CO₂ PET images (white arrows), while ¹⁵O-CO images showed no interhemispheric difference at any time point after HI insult. LV radioactivity concentrations were nearly equal between the ¹⁵O-O₂ and ¹⁵O-CO₂ inhalation scans although the supply rate of ¹⁵O-CO₂ radioactivity was lower (200 MBq/ml) than that of ¹⁵O-O₂ (500 MBq/ml). The peak values of TACs in both the ipsi- and contralateral brain regions were lower during ¹⁵O-O₂ inhalation compared with ¹⁵O-CO₂, as seen in Figure 3(b) and (c), respectively.

Figure 3.

Representative transaxial, coronal and sagittal PET/CT images of the brain of a HIE rat on day 14 post-insult. PET images are summed over 0–10 min after ¹⁵O-CO (a), 0–5 min after ¹⁵O-O₂ (b) and 0–8 min after ¹⁵O-CO₂ (c) inhalation. The lesion is indicated by the white arrows in the left hemisphere in both ¹⁵O-O₂ (b) and ¹⁵O-CO₂ images (c). VOI locations in the LV, the lesion in the ligated-side (ipsilateral) hemisphere and its symmetrical position (contralateral) are also indicated on the ¹⁵O-CO images (a). The measured tTACs in ipsilateral (square) and contralateral (diamond) brain regions, and LV TAC (circle) from the same animal successively administered ¹⁵O-CO (d), ¹⁵O-O₂ (e) and ¹⁵O-CO₂ (f) are also presented. Note that LV TAC for ¹⁵O-CO is presented as one-tenth value (d). CT: computed tomography; HIE: hypoxic-ischaemic encephalopathy; LV, left ventricle; PET: positron emission tomography; TAC: time-activity curve; tTAC: tissue TAC; VOI: volume of interest.

A representative result from the NLLSF analysis for fitting CBF and OEF simultaneously to two tTACs of ¹⁵O-O₂ and ¹⁵O-CO₂ is shown in Figure 4. Three AIFs were determined during ¹⁵O-O₂ inhalation PET imaging from total blood ( ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ ) and metabolized ¹⁵O-H₂O ( ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right))$ and ¹⁵O-O₂ ( ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ ), as estimated using equations (1a) and (1b), respectively (Figure 4(a)). ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ continued to increase during the ¹⁵O-O₂ inhalation period of 8 min. However, ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ increased only until 3 min, plateauing at approximately 60% of the peak of ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ , while ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ increased until the end of ¹⁵O-O₂ inhalation. Figure 4(b) demonstrates that the total tTAC ( ${\mathit{C}}_{\mathit{t}}\left(\mathit{t}\right)$ ) as given in equation (2a) reproduced well the measured tTAC. The additional three tTACs shown in this figure correspond to each of three terms shown in equation (2a), i.e. the response for ¹⁵O-O₂ AIF ( ${\mathit{C}}_{\mathit{o}}\left(\mathit{t}\right)$ ), that for metabolized ¹⁵O-H₂O AIF ( ${\mathit{C}}_{\mathit{w}}\left(\mathit{t}\right)$ ) and the vascular components V _o  ·  A _o ( t ) + V_w  ·  A_w ( t ). This figure also shows that while the total tTAC ( C_t(t) ) continued to increase during the ¹⁵O-O₂ inhalation period of 8 min, ${\mathit{C}}_{\mathit{o}}\left(\mathit{t}\right)$ plateaued at approximately 3 min until the end of ¹⁵O-O₂ inhalation. In contrast, ${\mathit{C}}_{\mathit{w}}\left(\mathit{t}\right)$ exceeded the level of that for ${\mathit{C}}_{\mathit{o}}\left(\mathit{t}\right)$ around 4 min after ¹⁵O-O₂ initiation. Figure 4(c) shows the AIF for ¹⁵O-CO₂ inhalation, in which the total blood activity is equal to the AIF for ¹⁵O-H₂O (A_t(t) = A_w(t)), whereas Figure 4(d) presents the fit results for the ¹⁵O-CO₂ inhalation scan given the formulation in equation (2b). The contribution of vascular radioactivity is negligibly small because of the small vascular component and large CBF values, as estimated in equation (3b). Averaged CBVs obtained from the ¹⁵O-CO scan were 0.049 ± 0.023 and 0.053 ± 0.026 ml/ml corresponding to the ipsi- and contralateral regions, respectively. Because of the small value, V_w was neglected and the calculated V_o was given as determined.

Figure 4.

A typical example of AIFs (a) and the results from simultaneous fitting to tTACs (b) for ¹⁵O-O₂, AIFs (c) and results from simultaneous fitting to ¹⁵O-CO₂ tTACs (d) obtained from a HIE rat on day 14 after the insult (same case shown in Figure 3). (a) Three input functions are given for the ¹⁵O-O₂ fitting analysis, i.e. the total blood AIF ( ${\mathit{A}}_{\mathit{t}}\left(\mathit{t}\right)$ ) (solid line), AIF for ¹⁵O-O₂ ( ${\mathit{A}}_{\mathit{o}}\left(\mathit{t}\right)$ ) (dashed line) as calculated by equation (1b) and AIF for metabolized ¹⁵O-H₂O ( ${\mathit{A}}_{\mathit{w}}\left(\mathit{t}\right)$ ) (dash-dotted line) as calculated by equation (1a) with the production rate of ¹⁵O-H₂O, k_w , determined from the first experiment. The observed tTAC (closed circles) for the ¹⁵O-O₂ scan was well reproduced by NLLSF (solid line) that consisted of the three components shown in (c), as estimated as a response to the ¹⁵O-O₂ AIF (dashed line), a response to the AIF for ¹⁵O-CO₂ (dash-dotted line) and the contribution of the blood radioactivity (dotted line), corresponding to the first, second and third terms of equation (2a), respectively. Of note is that the AIF for ¹⁵O-O₂ reached to a plateau at approximately 3 min after the initiation of continuous inhalation of ¹⁵O-O₂, while the contribution of metabolized ¹⁵O-H₂O continued to increase, exceeding the contribution of ¹⁵O-O₂ and reaching approximately 60% of the total radioactivity concentration at the end of ¹⁵O-O₂ inhalation. In contrast, the AIF for ¹⁵O-CO₂ inhalation consists of only a single component of ¹⁵O-H₂O that is equal to the whole blood TAC, i.e. ${\mathit{C}}_{\mathit{t}}\left(\mathit{t}\right)$ . AIF: arterial input function; HIE: hypoxic-ischaemic encephalopathy; NLLSF: non-linear least square fitting; TAC: time-activity curve; tTAC: tissue TAC.

The temporal profile of mean quantitative hemispheric CBF , CMRO₂ and OEF values (Figure 5(a) to (c), respectively), and mean ipsi-/contralateral ratios of CBF , CMRO₂ and OEF (Figure 5(d) to (f), respectively), as a function of days post-HI insult or equivalent days after birth (d/o) were determined in HIE, non-operated and sham subject groups (Figure 5). Non-operated pups showed significant increases from 9 to 23 d/o in both CBF (p < 0.01) and CMRO₂ (p < 0.05) by approximately 2.5-fold and 2-fold increases, respectively (Figure 5(a) and (b)). CBF in the ipsilateral brains of HIE pups was significantly reduced at the lowest level on day 2 (p < 0.0001, compared to day 1) and then increased on day 7 (p < 0.01) and day 14 (p < 0.01) compared to day 2. CMRO₂ values in the ipsilateral hemispheres of HIE animals also reached the lowest levels on day 2 (p < 0.01, compared to day 1) and significantly increased on day 14 from day 2 (p < 0.05). Pearson’s test showed a correlation between age and CBF (p = 0.0005) or CMRO₂ (p = 0.0003) in non-operated pups. The ipsilateral hemispheres of HIE pups indicated a linear relationship between CBF (p = 0.0007) or CMRO₂ (p = 0.0125) and postoperative days from day 2 to day 14. OEF showed no time dependent or interhemispheric differences in any animal group except for a significant increase on day 2 in the ipsilateral hemisphere compared to contralateral hemisphere (p < 0.0001) in HIE pups (Figure 5(c)). The ratios of CBF and CMRO₂ in HIE pups reached the lowest on day 2 and CMRO₂ ratios showed no temporal changes thereafter (Figure 5(d) and (e)). The ratio of OEF in HIE animals was significantly increased on day 2 (p < 0.01, compared to day 1) and then decreased on day 7 (p < 0.05) (Figure 5(f)).

Figure 5.

The mean temporal values of (a) CBF (ml/min/g), (b) CMRO₂ (ml/min/g) and (c) OEF in VOIs selected on ipsilateral (closed circle) and contralateral (open circle) hemispheres on days 0, 1, 2, 7 and 14 post-insult in HIE, at the corresponding age of 9, 11, 16 and 23 d/o in non-operated (ipsilateral: closed square; contralateral: open square) and on day 7 after the surgical operation in sham pups (ipsilateral: closed diamond; contralateral: open diamond). The ipsi- to contralateral ratios of CBF (d), CMRO₂ (e) and OEF (f) of the three subject groups on each imaging day (HIE: circle-dot; non-operated: square-dot; sham: diamond-dot) are also presented. Values are expressed as mean ± SD and those in a pair of each imaging time point or each hemisphere are compared by student’s paired t-test. Asterisks shown above closed circles indicate significant differences between each hemisphere. Ipsilateral HIE brains presented significantly lower values than contralateral hemispheres at all time points in CBF (a) and except for day 0 in CMRO₂ (b). OEF in ipsilateral HIE brains showed a significant increase on day 2 compared to contralateral hemispheres (c). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS; not significant. CBF: cerebral blood flow; CMRO₂: cerebral metabolic rate for oxygen; d/o: day old; OEF: oxygen extraction fraction; HIE: hypoxic-ischaemic encephalopathy; VOI: volume of interest.

A comparison of ¹⁵O-O₂ PET/CT images and histology results from a HIE rat on day 14 and an age-matched non-operated rat is shown in Figure 6. Infarct regions measured by H&E staining in the HIE brain coincided with areas showing extremely decreased accumulation of ¹⁵O-O₂ in PET images (Figure 6(a))_. Additionally, increased microglial activation was displayed by Iba1 outside the margins of the infarct cores, where small reductions in ¹⁵O-O₂ accumulation were observed. The mean ipsi-/contralateral hemispheric-volume ratio of HIE pups showed a 62.6% reduction compared with non-operated pups (p < 0.0001) (Figure 6(b)). The hemispheric volume ratios of HIE pups indicated a linear relationship with the ratios of hemispheric CBF (p = 0.0112) and CMRO₂ (p = 0.0091) values measured in each brain VOI (Figure 6(c) and (d), respectively).

Figure 6.

(a) Representative PET/CT images of the transaxial view and each coronal slice at the striatum (1) (3) and hippocampus level (2) (4) of the brains of a HIE pup on day 14 and an age-matched non-operated pup are shown as summed images over 0–5 min after ¹⁵O-O₂ inhalation. Coronal images stained with H&E and Iba1 of the same animals at equivalent slicing levels are also displayed. Note that the area of the histological tissue loss in H&E images as indicated by grey arrows in the HIE animal is consistent with the defect region in ¹⁵O-O₂ PET images as indicated by white arrows. Increased microglial activation is also visible around the infarct tissues in Iba1 images as indicated by grey arrows. (b) Ratios of the ipsilateral to contralateral hemispheric volume are presented as mean ± SD, displaying a significantly decreased mean ratio in HIE (****p < 0.0001) compared to non-operated pups (n = 5 and n = 4, respectively). (c, d) The ipsi-/contralateral ratio of CBF and CMRO₂ on day 14 in each HIE pup showed significant correlations with the ratio of the ipsi-/contralateral hemispheric volume in histology measured on the same day (p = 0.0122 and p = 0.0091, respectively). CBF: cerebral blood flow; CMRO₂: cerebral metabolic rate for oxygen; CT: computed tomography; H&E: haematoxylin-eosin; HIE: hypoxic-ischaemic encephalopathy; PET: positron emission tomography.

Discussion

Overview of the findings

The comprehensive ¹⁵O-oxygen PET system for small animals developed in this study provided quantitative CBF , CMRO₂ and OEF values non-invasively by spontaneous inhalation of ¹⁵O-O₂ and ¹⁵O-CO₂ without tracheotomy or intravascular cannulations required using an intravenous ¹⁵O-H₂O administration approach. The sophisticated radioactive gas supply and scavenging systems implemented for two PET scanners enabled good quality dynamic PET images. The physiological model-based estimation of metabolized ¹⁵O-H₂O in arterial blood was essential as it avoids labour-intensive procedures and minimizes uncertainties in A_w ( t ) and A_o ( t ) values when assessing individual blood samples with small volumes. Simultaneous NLLSF to a pair of dynamic PET images for ¹⁵O-O₂ and ¹⁵O-CO₂ enabled reliable estimation of parametric values. This system allowed repetitive measurements even on consecutive days initiated immediately after the HI insult in vulnerable infant rats.

The technique detected time-dependent changes in CBF , CMRO₂ and OEF associated with postnatal age and post-HI injury state for different brain regions and subject categories. Important findings in this study are the following: (a) remarkable reductions in CBF and CMRO₂ on day 2 after HI insult; (b) continuous recoveries after day 2 in the affected hemisphere in parallel to the increases in the contralateral hemisphere and (c) similar increase rates in CBF and CMRO₂ with age-matched controls (Figure 5). The non-operated rats displayed a relatively large increase from 9 to 23 d/o, which was similar to a previous report demonstrating that whole brain CBF and CMRO₂ , measured by H₂ clearance and the arteriovenous difference in O₂ content, dramatically increased in infant rats from 10 to 20 d/o. ³¹ Our finding is also consistent with a clinical PET study that indicated 3–4 times lower oxygen consumption in term infants than adults as a reflection of brain maturation. ³² This difference is considered attributed to the escalating cerebral energy demand compensating for the increased Na+ pump activity in neuronal cells^33,34 for structural and functional maturational processes at the ages examined in this study.

We confirmed that both LV TAC for ¹⁵O-oxygen scan in the first experiment agreed with those obtained by the well counter, which is consistent with an earlier finding ²⁸ despite the smaller average animal size in this study. This may be attributed to the contribution of the myocardium surrounding the LV chamber, which has a high flow value, resulting in a similar tTAC in the myocardial wall as the LV blood TAC. We also expect a similar phenomenon in the second experiment, in which a high-resolution β-Cube scanner was employed. However, this remains a hypothesis requiring further verification.

In HIE animals, CBF in the ipsilateral hemispheres showed a transient increasing tendency on day 1 post-insult _, which may be consistent with a previous report, indicating that hyperperfusion occurring 24 h after HI intervention measured by the laser speckle method could be associated with the more severe tissue damage outcome. ¹³ The subsequently marked reductions in both CBF and CMRO₂ on day 2 may be attributed to the neuronal cell death and apoptosis reportedly maximized at 24–72 h after neonatal HI insult.^8,35,36 The pathophysiological background of the continuous increases in CBF and CMRO₂ in the ipsilateral VOI after day 2 may also be an important finding. Of note is that the ipsi-/contralateral ratio of CMRO₂ in HIE brains showed no significant increase after day 2. Thus, one may hypothesize that the surviving neuronal cells in the injured region keep growing while the damaged tissues with no energy consumption persisted.

Histological tissue losses measured after the last PET scan showed spatial agreement with severely decreased CBF and CMRO₂ areas (Figure 6). In contrast, CMRO₂ reduction was milder in the area where increased microglial activation was detected by Iba1 surrounding the infarct core region. Post-ischaemic inflammation reportedly modulates the exacerbation of neural damage in neonatal HIE, further suggesting a unique immune response in the immature brain.^37,38 The application of repetitive imaging techniques of ¹⁵O-gas inhalation PET along with neuroinflammation-targeting PET using 18 kDa translocator protein (TSPO) would be useful for evaluating the age- and time-dependent effects of microglia-mediated inflammatory response on delayed neuronal damage expansion.

Methodological advantages

¹⁵O-O₂ gas is the sole tracer that can trace the kinetics of oxygen molecules and oxygen metabolism. It was shown in both the first and second experiments that ¹⁵O-O₂ is metabolized quickly in neonatal rats so that the metabolized ¹⁵O-H₂O replaces ¹⁵O-O₂ in arterial blood shortly after starting ¹⁵O-O₂ inhalation, as typically seen in Figure 4(a). Since the ¹⁵O-H₂O extraction rate to the brain in the capillary bed is over two-fold higher than that of ¹⁵O-O₂, the contribution of metabolized ¹⁵O-H₂O to the brain (the second term in equation (2a)) exceeds the level of that of the original ¹⁵O-O₂ during the continuous inhalation of ¹⁵O-O₂ (Figure 4(b)), at 3–5 min after ¹⁵O-O₂ inhalation initiation. To compensate for the contribution of metabolized ¹⁵O-H₂O, the additional administration of ¹⁵O-CO₂ (equivalent to intravenous ¹⁵O-H₂O) was applied as many PET methods that utilized ¹⁵O-O₂ inhalation protocol.

Simultaneous NLLSF is considered to be a reliable approach for estimating both CMRO₂ and CBF defined in equations (2a) and (2b), respectively. Using a single rate constant, k_w , that takes into account the kinetics of ¹⁵O-H₂O in parallel to that of ¹⁵O-O₂ plays an important role in this study in terms of stabilizing NLLSF estimation. This approach detected small but significant increases in CMRO₂ during cognitive stimulation that increased CBF to a greater amount; ²⁹ however, another approach failed to detect an increase in CMRO₂ where the metabolized ¹⁵O-H₂O was neglected. ³⁹

Limitations

There are several limitations to this study. Firstly, the animal model used exhibits hemispheric HI injury generated by unilateral ligation of the common carotid artery with subsequent global hypoxic insult. Considering the impact of interhemispheric collateral circulation, the use of ipsi-/contralateral ratios of CBF and CMRO₂ may have been biased, especially in the acute phase. Secondly, anaesthesia is a potentially confounding factor in measuring cerebral haemodynamics since isoflurane can derange CBF autoregulation. ⁴⁰ Furthermore, isoflurane may possess neuroprotective properties, and thus its exposure time may be correlated with decreased brain infarction in the Rice–Vannucci model. ⁴¹ Another anaesthetic agent may be a suitable replacement, especially for the study of cerebral haemodynamics or ischaemic disease. It should also be mentioned that limited vital monitoring was performed because of the difficulty in placing highly sensitive equipment in the limited space of the animal holder to detect stable signals from small pups. Moreover, the physiological condition is unlikely constant during the entire PET imaging protocol because of the long scan period, and systematic errors may be attributed to transient changes in CBF and/or CMRO₂ .

A standardized CBV value determined from limited animal scans of a single rat was used to calculate OEF and CMRO₂ . The contribution of CBV to OEF and CMRO₂ was small and around 1% in the present data both in the control and HIE VOIs, partly attributed to the observed higher CBF and CMRO₂ values than those in humans. However, CBV could be increased, for example, in chronic ischaemic lesions, ⁴² during haemodynamically altered conditions, ⁴³ and even in neurodegenerative diseases. ⁴⁴ Kinetic fitting of V_o and V_w with CMRO₂ and CBF as proposed by previously ⁴⁵ is highly desired as an alternative not only to remove the ¹⁵O-CO scan but also to improve the accuracy by removing the need for assuming a fixed arterial-to-venous blood volume ratio.

A k_w of 0.33 ± 0.07 min⁻¹ was obtained in the first experiment for 24 and 40 d/o rats, whereas it was approximately twice in an earlier study on 7–8-week-old rats. ⁴⁵ One reason for this discrepancy could be the different tracer administration procedures, i.e. earlier study used intravenous injection of ¹⁵O-O₂–labelled oxyhaemoglobin instead of gaseous ¹⁵O-oxygen. Further systematic investigations are warranted.

We applied the average k_w value obtained from the first experiment to all analyses for the second experiment. To estimate the effects of ±20% variation in k_w , the CBF and CMRO₂ values were calculated by changing k_w by ±20% using the tTAC obtained from the day 14 study, which was the worst situation because of the highest CBF and CMRO₂ values. The change in k_w by +20% caused changes in CBF by −3.7% and −7.5%, whereas a change by −20% resulted in +0.0% and +2.9%, corresponding to the ipsilateral and contralateral hemispheres, respectively. Similarly, changes in k_w by +20% and −20% caused changes in CMRO₂ by +8.4% and +3.1% and −4.1% and −1.0%, respectively, in the ipsilateral and contralateral hemispheres. The range of changes in CBF and CMRO₂ were not large compared with the range of the time courses shown in Figure 5. However, a more systematic error propagation study is planned for future work.

Future implications

The present ¹⁵O-oxygen PET system can be further improved if the entire examination duration is shortened. A PET scanner with better counting rate performance is essential for employing a single PET scan protocol during the dual inhalation of ¹⁵O-O₂ followed by ¹⁵O-CO₂ with a short interval, as demonstrated previously.^46,47 This protocol can reduce the total anaesthetisation time and minimize possible physiological changes during PET scans. The intrinsic vascular radioactivity correction could also be compensated without the need for an additional ¹⁵O-CO scan. Continued research in this study would be of great value to evaluate possible pharmacological interventions and the early prognostic value of ¹⁵O-PET for neonatal HIE with a larger sample size. Such technical innovations would also facilitate the broader use of ¹⁵O-PET in preclinical and clinical applications, including in neonatal patients.

It is also important to establish a technique that can determine the appropriate k_w value for the given subject groups without the need for blood sampling. The indirect calorimeter would allow assessments of k_w through whole-body oxygen metabolism by measuring O₂ consumption and CO₂ production, as demonstrated earlier. ⁴⁵ More sophisticated equipment, such as a ventilated chamber for small animals,^48,49 may also be a possibility.

Conclusions

We demonstrated that the present ¹⁵O-gas inhalation PET system quantitatively assesses temporal changes in CBF and CMRO₂ associated with acute cerebral perfusion derangement and tissue damage evolving 48 h after HI insult in rat neonates. The technique also depicts subsequent pathological progression over 14 days interrelated with escalating cerebral energy metabolism along with the maturation process. This completely non-invasive imaging strategy may be of value for developing early therapeutic interventions and their response monitoring in neonatal HIE with highly individualized in vivo follow-up.