Tran et al. Journal of Neuroinflammation(2024) 21:121
https://doi.org/10.1186/s12974-024-03053-3

Journal of Neuroinflammation

Open Access

RESEARCH

Early administration of umbilical cord blood
cells following brief high tidal volume
ventilation in preterm sheep: a cautionary tale
Nhi T. Tran1,2*†, Tayla R. Penny1,2†, Kyra YY. Chan1,2, Tanya Tang1,2, Paris C. Papagianis3, Tara Sepehrizadeh4,
Lakshmi Nekkanti1, Valerie A. Zahra1, Yen Pham1, Tamara Yawno1,2,5, Ilias Nitsos1, Sharmony B. Kelly1,2, Alison M. Thiel1,
Michael de Veer4, Dhafer M. Alahmari4,6,7, Michael C. Fahey5, Graham Jenkin1,2, Suzanne L. Miller1,2, Robert Galinsky1,2,
Graeme R. Polglase1,2*† and Courtney A. McDonald1,2*†
Abstract
Background Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access toallogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As
preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated theefficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury.
Methods Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min
ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v.
administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB;n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem
brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further
investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected toin vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant andcells collected for protein and mRNA assessments respectively.

†

Nhi T. Tran and Tayla R Penny are joint first author.

†

Graeme R. Polglase and Courtney A. McDonald are joint seniorauthor.
One Sentence Summary: UCB cells adopt a pro-inflammatory stateand contribute to cerebral white matter injury when administeredduring peak inflammation in ventilated preterms.
*Correspondence:
Nhi T. Tran
nhi.tran@hudson.org.au
Graeme R. Polglase
graeme.polglase@monash.edu
Courtney A. McDonald
courtney.mcdonald@monash.edu
Full list of author information is available at the end of the article

Background
Umbilical cord blood (UCB) cell therapy is emerging as
an exciting and highly promising neuroprotective strategy for neonatal morbidities such as preterm brain injury.
UCB cells are enriched with multiple stem and progenitor cell types – each possessing synergistic pleiotropic
properties that offer cellular and tissue protection [1].
The protective properties of UCB cells are likely to be
mediated by the paracrine secretion of cytokines, chemokines and growth factors [2], resulting in immunomodulatory effects [3]. In addition to their immunomodulatory
properties, UCB cells have advantages over other celltherapy options as UCB cells are readily available from© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The
Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available
in this article, unless otherwise stated in a credit line to the data.

Tran et al. Journal of Neuroinflammation(2024) 21:121

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Results In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial
activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared
to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA
downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed
both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor(BDNF & VEGF) gene and protein expression compared to controls.
Conclusions UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes
white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB
cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells areto become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the
inflammatory state of the preterm infant should be considered when timing UCB cells administration.
Keywords Umbilical cord blood cells, Preterm brain injury, Ventilation, Neuroinflammation, Stem cell therapy,
Preterm ventilation

donors, have a low immunogenicity and greater proliferation profile compared to adult peripheral blood orbone marrow sources of stem cells. As such, multiple
clinical trials in preterm infants have already been conducted reporting a good safety profile [4, 5]. In particular,
autologous UCB cell (5 × 107 cells/kg) administration inpreterm infants ~ 7 h after birth, significantly reduced therequired duration of mechanical ventilation (3.2 days vs.
6.41 days) and oxygen therapy (5.33 days vs. 11.31 days)[6]. This is of particular interest as mechanical ventilationand hyperoxia/hypoxia are both independent risk factors
for respiratory related preterm brain injury [7, 8]. Moreover, preclinical studies have found that earlier administration of UCB cells at 12 h compared to 5 days after
hypoxia-ischemia was more efficacious in reducing whitematter injury [9, 10]. This indicates that delaying UCB
cells administration also delayed the release of peripheral trophic factors entering the brain parenchyma andthereby diminished their neuroprotective potential. EarlyUCB cell administration during peak injury response may
provide superior brain injury prevention – a key consideration in current clinical and preclinical investigations.
Inflammation is a major underlying pathology andantecedent to preterm brain injury. Inflammatory processes develop over time with characteristic increases incytokine and chemokine release into the circulation andwithin the brain, which can have significant downstream
effects that lead to brain injury [11]. The key neuroinflammatory pathways that also contribute to the cerebral
increases in cytokine and chemokine release is glial activation (microglia and astrocytes) and blood-brain barrier (BBB) breakdown [11, 12]. Collectively, increased
neuroinflammatory processes can mediate injury to theimmature oligodendrocytes which is thought to underlie
the characteristic white matter injury in preterm infants[13]. The exact temporal profile of inflammatory processes is difficult to ascertain within the preterm infant
as other confounding underlying pathologies arising inutero, perinatally or postnatally, are likely to be involved.
Nonetheless, if early UCB cell administration is to be
used as a treatment strategy in preterm infants, the effectof UCB cells in a pro-inflammatory environment needs
to be investigated.
Respiratory support is a common postnatal clinicalintervention in preterm infants where even the brief useof respiratory intervention can trigger inflammatory processes [7, 14]. Indeed, within 2 h of protective mechanicalventilation of human preterm infants, levels of multiple
pro-inflammatory cytokines (IL-8, IL-1β, TNF) in arterial
blood are increased [15], which persists with prolonged
durations of ventilation [16]. We have previously demonstrated in preterm lambs that there is peak upregulation of pro-inflammatory cytokine transcripts (IL1B, IL6)
levels in the brain and activation of glial cells within the
cerebral white matter ~ 2 h after high tidal volume ventilation (V T; targeting 10–15 mL/kg for 15 min) [7, 17,18]. Administration of UCB cells during this peak inflammatory response may blunt the systemic inflammatory
response and ultimately, protect the brain from injury.
Here, we first utilised the fetal head-out ventilation
technique in preterm fetal sheep at 125 days of gestation(dGA) [14] to investigate the effects of early UCB cells
administration on systemic inflammatory cytokine levels
and cerebral white matter 24 h following brief high tidal
volume (V T) injurious ventilation. High V T ventilation
was utilised as the injury model as more than 85% of preterm infants inadvertently receive high V T in the delivery
room [19], which has been associated with haemorrhagicand diffuse white matter brain injury [20–22]. As we haveshown that a peak inflammatory response occurs < 2 hafter 15 min of high V T ventilation [7, 17, 18], this study
aims to assess whether UCB cells treatment at 1 h after
15 min of high V T ventilation is able to attenuate thispeak inflammatory response and reduce overall whitematter injury. To further characterise how UCB cells
respond to pro-inflammatory environments, we isolatedTran et al. Journal of Neuroinflammation(2024) 21:121

mononuclear cells (MNCs) from human UCB in vitro
and exposed the cells to pro-inflammatory cytokines.
The cells and supernatant were analysed for inflammatory markers and growth factors to assess the influenceof an inflammatory environment on the functional characteristics of the UCB cells. We hypothesised that in vivo
administration of UCB cells during the early inflammatory period 1 h following brief high V T ventilation
would mitigate the upregulation of inflammation andsubsequent white matter injury in preterm sheep. We
also hypothesised that the MNCs contained within UCB
would be modulated according to their environment
in vitro and a highly inflamed environment may skew
the function by altering their expression of chemokinerelated receptors and release of pro/anti-inflammatory
cytokines and growth factors.

Materials and methods
Study design

The first study was an in vivo investigation of UCB
administration at a time of heightened systemic inflammation induced by brief high V T injurious ventilationin an established fetal ovine model to assess the effects
on white matter inflammation and injury. Animal
experimental procedures were approved by the Monash
Medical Centre Animal Ethics Committee A, Monash
University (MMCA/2018/05). All experiments were conducted in accordance with guidelines established by the
National Health and Medical Research Council of Australia. All experiments were performed in accordancewith the ARRIVE guidelines 2.0 (Supplementary Data
File 1) [23].
We then conducted an in vitro study to investigate thecharacteristic alterations to cells isolated from humanderived UCB following exposure to an inflammatoryenvironment induced by cytokine-spiked media. All
human-derived samples were collected with human ethics approval from Monash Health Human Ethics Committee (12387B). Written informed consent was obtained
from all participants prior to collection of UCB.
In vivo sheep preparation

The use of these animals and relevant methodology havepreviously been published [24, 25] and experimental protocols unique to this study are reported in detail below.
Prior to surgery, pregnant ewes at 125 ± 1 day gestation(term ~ 148 days) were randomly assigned to a SHAM(n = 5), VENT (n = 7) or VENT + UCB (n = 7) group. Maleto female ratio for fetuses within each group was SHAM,
3:2; VENT, 5:2; and VENT + UCB, 4:3. As previously
described [24, 25], sterile surgery was performed with amidline laparotomy on pregnant ewes to expose the fetus
head and neck for instrumentation of polyvinyl cathetersin the left carotid artery (for arterial blood sampling) andPage 3 of 17

jugular vein (for UCB administration), and an ultrasonicflow probe (3PS; Transonic Systems, NY, USA) aroundthe right carotid artery to measure carotid blood flow(CBF). To allow ventilation, the fetus’ chest was exteriorised and the fetus was intubated with a cuffed endotracheal tube (ID 4.0-4.5 mm; Smiths Medical, UK) and lungliquid passively drained.
Ventilation for the VENT and VENT + UCB groups was
conducted under sterile conditions using a neonatal positive pressure ventilator (Babylog 8000+, Dräger, Lübeck,Germany) as described previously [24, 25]. Briefly, thefetus was ventilated for 15 min with a high tidal volume(V T) strategy targeting 12–15 mL/kg (normal V T: 5–7
mL/kg). SHAM animals remained intubated and exteriorised for 15 min without mechanical ventilation. CBF
and V T were continuously measured for the 15 min anddigitised using a PowerLab A-D converter and stored on
disk using LabChart7 software (AD Instruments, Bella
Vista, NSW, Australia). CBF could not be recorded fromone lamb from the VENT group due to technical error.
Arterial blood gas was measured at 0 (pre-ventilation), 5,
10 and 15 min of ventilation (ABL80 FLEX, Radiometer
Medical ApS, Denmark).
After 15 min of ventilation, the carotid flow probe wasremoved, and fetuses were extubated and returned tothe uterus. The fetal jugular vein and carotid artery catheters were externalised through the ewe’s right flank. All
incision sites were sutured closed and the ewe and fetus
allowed to recover. Analgesia (buprenorphine; 0.3 mg
i.m.; Temgesic; Reckitt Benckiser, UK and transdermal
fentanyl patch; 75 µg/h; Janssen-Cilag, NSW, Australia)
was administered for post-surgery pain relief. Arterial
blood gas measurements and arterial samples were collected in heparinised tubes regularly throughout the 24 h
study period.
UCB cells treatment

UCB cells were obtained from healthy term lambs (141days gestation of term 148 days) undergoing procedures in a separate study, using UCB collection, storage
and preparation protocols as previously described [9,26]. On the day of cell administration, UCB cell samples
were rapidly thawed, washed and resuspended in media(Gibco, USA), and cell number and viability measured(minimum 75% viability accepted). At 1 h post-ventilation, ~ 80 million allogeneic UCB cells suspended in 3
mL of phosphate-buffered saline (PBS; Gibco, MA, USA)
were administered to VENT + UCB fetuses via the jugularvein catheter, which was then flushed with sterile heparinised saline to ensure all cells were administered [25].
This dosage and administration regime was based on previous studies reporting reduced cerebral inflammation,
white matter injury, oxidative stress and cell death after
hypoxia-ischemia in fetal sheep [9].

Tran et al. Journal of Neuroinflammation(2024) 21:121

Magnetic Resonance Imaging (MRI) and post-mortem
brain collection

Twenty-four hours post-in utero ventilation, ewes wereanaesthetised and the fetus exteriorised via the same
incision site. All lambs were dried, intubated, and stabilised with a standard protective ventilation strategy(Babylog 8000+, Dräger) using volume guarantee set at 7mL/kg and PEEP of 5 cmH2O. Once the lambs were stabilized, the umbilical cord was clamped and the lambsdelivered. Surfactant was administered (100 mg/kg,
CurosurfR, Chiesi Pharma, Italy) and mechanical ventilation was provided targeting 5 mL/kg. Lambs were thentransferred to the 3T MRI system (Siemens Skyra, Erlangen, Germany) at Monash Biomedical Imaging (Clayton,
Australia), with a 15-channel radio frequency transmitter/receiver knee coil. Lambs were placed in supine
position and ventilation maintained using a BabyPAC
portable and MR-compatible ventilator (Pneupac, Smiths
Medical, UK). Due to technical issues, MRI results fromone lamb from the VENT group and two lambs from the
VENT + UCB group were not suitable for analysis; thus
the revised group numbers for this analysis were VENT,n = 6; VENT + UCB, n = 5. MRI acquisition protocols for
T2-weighted image and Diffusion Weighted Imaging(DWI) were conducted as previously published [24, 27,28]. Total acquisition time was around 40 min. Lambs
were then euthanised (sodium pentobarbitone > 100 mg/
kg i.v.; Valabarb Euthanasia Solution; Jurox, NSW, Australia) immediately after scanning was completed. Ewes
were similarly euthanized immediately after delivery of
the lambs.
At post-mortem, the lamb’s brain was removed andthe cerebrum hemisected along the medial longitudinal
fissure. The periventricular and subcortical white matter (PVWM and SCWM respectively) of the left cerebral
hemisphere were dissected and snap-frozen in liquid
nitrogen. The right cerebral hemisphere was cut coronally into 5 mm blocks and immersion fixed in 10% neutral buffered formalin (Amber Scientific, WA, Australia)
for paraffin-embedding.
MRI data analysis

All data were analysed using the FMRIB Software Library(FSL, FMRIB, Oxford, UK [29]). Pre-processing steps
started with extracting brains and creating brain maskusing Brain Extraction Tool. All masks were manuallybrushed and cleaned thoroughly to remove skull andareas other than brain. All DWI data were then corrected
for eddy-current distortion using eddy, and then DTI
parametric maps of fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) created with the Diffusion Toolbox of FMRIB.
RD is the mean of the second and third eigenvalues (L2and L3). Region of interests (ROI) were defined on highPage 4 of 17

resolution T2 images to maximise accuracy. Selected
ROI: the periventricular white matter (PVWM), the frontal white matter (FWM) was identified using The Sheep
Brain Atlas from Michigan State University (Brain Biodiversity Bank, National Science Foundation). High-resolution T2 images of the brains along with the ROIs wereco-registered to DWI images using the Linear Image Registration Tool (FLIRT [29]) as previously published [24,27, 28]. The mean value from 10 adjacent slices within
the given ROI volume was calculated; within-animal ROI
variability was < 16.8%. MRI examination, image processing and analysis of the structural MR images were performed by persons blinded to group allocations.
Immunohistochemistry and quantitative analysis

Coronal sections (8 μm) at the level of the frontal, parietal, temporal, and occipital lobes (4 slides/animal/
antibody) were stained with rabbit anti-ionised calcium
binding adapter molecule-1 (Iba-1; 1:1500, Wako Pure
Chemical Industries, Osaka, Japan) to identify microglia;
rabbit anti-sheep serum (1:1000, Sigma-Aldrich, USA)to identify vascular extravasation of protein; mouse antioligodendrocyte transcription factor-2 (Olig2; 1:1000,
Merck, Darmstadt, Germany) for oligodendrocytes; rat
anti-myelin basic protein (MBP; 1:200; Merck, Darmstadt, Germany) for mature myelin and oligodendrocytes;
and rabbit anti-glial fibrillary acidic protein (GFAP; 1:400,
Sigma-Aldrich, USA) to identify astrocytes as described
previously [30]. Briefly, sections were pre-treated withcitrate buffer (pH 6.0) in a microwave oven. Sections
were incubated with secondary biotinylated IgG antibody raised against the corresponding animal secondary(1:200; Vector Laboratories, CA, USA) and reacted using
the Vectastain Elite ABC Kit (Vector Laboratories, CA,
USA). A ‘Terminal deoxynucleotidyl transferase dUTP
nick end labelling’ (TUNEL) assay (ApopTag® Peroxidase
In Situ Apoptosis Detection Kit, Millipore, USA) was
also conducted according to manufacturer’s instructions.
Negative controls (primary antibody omitted) demonstrated no positive staining.
Analyses were conducted at equivalent sites within the
PVWM and SCWM of sections from the frontal, parietal,
temporal, and occipital lobes of each lamb. Slides werecoded and the assessors blinded to the groups. Analysis
was conducted using either ImageScope (Aperio Technologies; Leica Biosystems, Germany) or ImageJ software(National Institutes of Health, USA) as described previously [30]. Cell density (cells/mm2) of Iba-1+ microglia(resting, ameboid and total), GFAP+ astrocytes, TUNEL+
cells, Olig2+ oligodendrocytes, MBP+ cells and number of blood vessel profiles with protein extravasation
were manually quantified in 3 non-overlapping fields in
the PVWM and 6 non-overlapping fields in the SCWM
from 2 separate gyri in each section (area = 0.14 mm2).

Tran et al. Journal of Neuroinflammation(2024) 21:121

The fractional area coverage (%) of Iba-1, GFAP and MBP
immunoreactivity was assessed within the same fields.
For Iba-1 immunostaining, the total number of microglial aggregations and the fractional area coverage of theseaggregations within the PVWM and SCWM were also
assessed. Data from all fields were averaged for eachbrain region then averaged across subjects in each experimental group.
Magpix cytokine analysis

Arterial plasma was collected following ventilation (0[pre-ventilation], 15 min [end of ventilation] and 1, 3,
6, 12 and 24 h [post-ventilation recovery]) and used toquantify levels of IFNγ, IL-1β, IL-10, TNFα, IL-8 and IL-6
using a Milliplex MAP bovine cytokine magnetic beadpanel assay kits according to manufacturer’s instructions (cat#: CYT1-91 K; MerckMillipore, USA). In brief,
96-well plates were coated with samples, assay buffer,serum matrix and antibody-immobilised beads. Plates
were incubated overnight at 4 °C, washed and incubatedwith detection antibodies for 1 h. Streptavidin-phycoerythrin was added to the plates for 30 min. Sheath fluid
was added to the plates and cytokine concentrations were
quantified using a Bio-Plex MAGPIX® Multiplex reader
with xPOTENT® software (Bio-Rad, CA, USA).
In vitro UCB mononuclear cell isolation

For in vitro studies, human term UCB samples wereobtained from women with uncomplicated pregnancies undergoing elective caesarean section at term (> 37
weeks gestation). Women gave written, informed consent for the collection of their UCB. After clampingof the cord and delivery of the placenta, UCB was collected from the umbilical vein using blood collection
bags containing anticoagulant (Macopharma, Tourcoing, France). On average, ~ 100 mL of UCB was collected. UCB was stored at room temperature for up to
48 h until processing and cell isolation. To obtain themononuclear layer of cells, UCB was transferred to
50 mL falcon tubes and diluted 1:1 with PBS. This wascentrifuged at 3100 rpm for 12 min, with no brake. The
mononuclear cells were separated and washed in 20 mL
PBS and centrifuged at 800 g for 5 min to isolate a cellpellet. Red blood cell lysis buffer (ammonium chloride,potassium bicarbonate and EDTA dissolved in double
distilled water; Sigma-Aldrich, USA) was added for
3–5 min to lyse excess red blood cells. The reaction was
stopped with excess media (16.5% fetal bovine serumand DMEM/F12; Gibco, USA), followed by centrifugation at 400 g for 5 min and the supernatant was thenaspirated. Cell viability was determined using trypan
blue exclusion dye (Gibco), and cells were counted witha hemocytometer. The mononuclear cells were theneither used for magnetic bead separation of individual

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cell types or cryopreserved for later use. For cryopreservation, UCB mononuclear cells were frozen at a densityof 20 × 106 cells/mL; in 40% complete media (DMEM/
F12, 16.5% FBS, 1% antibiotics), 50% fetal bovine serum(FBS; Gibco) and 10% dimethyl sulfoxide (DMSO;
Sigma Aldrich). Cells were then transferred to freezer
tubes and left in a freezing container (MrFrosty, Thermo
Fisher Scientific) overnight at -80°C, following which
they were transferred to liquid nitrogen. To thaw, sample tubes were quickly removed from liquid nitrogenand placed directly into a 37°C water-bath until thawed.
Samples were washed to remove DMSO, and cell counts
and viability were determined.
In vitro stimulation of cells

For stimulation of cells, three separate donors wereused for each cell type and each stimulation experiment was performed in triplicate. Briefly, cells were rapidly thawed and washed with media (DMEM/F12, 10%
FBS and 1% Antibiotic-Antimycotic, Gibco), then centrifuged at 480 g for 10 min, the supernatant was aspirated and cells were resuspended in media. Cells werecounted using a hemocytometer and viability assessedusing trypan blue exclusion dye.
There were four conditions: media alone, media
with TNFα, media with IFNγ, and media with TNFα
& IFNγ combined. 48-well plates were set up, the four
conditions were plated in triplicate for supernatantcollection. For RNA, the four conditions were plated
in duplicate. 1 × 106 cells were seeded for RNA wells.
After the cells were initially seeded, the plates wereincubated at 37°C with 21% O2 and 5% CO2 overnight
to allow the cells to recover from thawing after cryopreservation. After 24 h of recovery, supernatant wasremoved and the cells were exposed to media only, 10
ng/mL of TNFα, 10 ng/mL of IFNγ or both 10 ng/mLof TNFα and 10 ng/mL of IFNγ. Media was added toincrease the final volume in the wells to 500 uL. The
plates were incubated for 16 h at 37°C with 21% O2 and
5% CO2. Supernatant was collected for protein analysis
and cells were harvested for either flow cytometry or
RNA isolation.
Luminex protein analysis

Neat cell supernatant was placed into 96-well plates andanalyzed with a Magnetic Luminex protein array system (Human Premixed Multi-Analyte Kit; RD Systems,
Minneapolis, MN, USA) for the presence of Angiopoietin-1 (Angpt1), SDF-1, IFN-γ, IL-6, VEGFA, BDNF,
GDNF, IL-10 and TNFα. This assay is designed for thesimultaneous detection of multiple human biomarkers
in cell culture supernatants and the assay was performed
strictly according to the manufacturer’s instruction (Catalogue Number LXSAHM).

Tran et al. Journal of Neuroinflammation(2024) 21:121

mRNA expression

mRNA expression of genes of interest from frozen tissue
are outlined in Table S1. RNA extraction, cDNA preparation and analysis were conducted as described previously[31]. Briefly, mRNA was extracted from the PVWM and
SCWM (RNeasy Midi RNA Extraction kit; Qiagen, VIC,
Australia) and reverse-transcribed into cDNA according to the manufacturer’s instructions (SuperScript® III
First-Strand Synthesis System for RT-qPCR kit; Invitrogen). Genes of interest were measured by quantitative
PCR using the Fluidigm Biomark HD system (Fluidigm
Corporation, CA, USA). Samples were run in triplicates,
averaged and normalised to endogenous housekeeping gene ribosomal protein S18 (RPS18) expression
then expressed relative to SHAM group using the 2–∆∆Ct
method.
mRNA expression within cultured cells of genes ofinterest is outlined in Table S2. Cells were harvested for
RNA isolation after the 16 h of stimulation. In a modification of the protocol included in the Bioline RNA kit,
the cells were first centrifuged after collection, the supernatant was aspirated off and the cells were suspended in
2 µL TCEP and 100 µL RNA Lysis Buffer RL. This solution was then frozen at -80°C until ready for use. After
thawing, RNA was isolated according to manufacturer’s
instructions (Bioline, Meridian Bioscience, USA). The
concentration of the isolated RNA was measured on a
Nanophotometer (Implen). RNA was reverse-transcribed
into cDNA (SuperScript III reverse transcriptase, Invitrogen; Life Technologies, USA) according to manufacturer’s
instructions. Relative mRNA expression was measured
by quantitative real-time PCR using Applied Biosystems
7900HT Fast Real-Time PCR system. The expression of
all genes was normalized to β-actin for each sample using
the 2–∆∆Ct method.
Statistical analysis

Statistical analyses were conducted using GraphPad
Prism (version 10.1.1; GraphPad Software, CA, USA).
Data were tested for normality by Shapiro-Wilk test andstatistical analysis conducted accordingly. Data violating the assumption of sphericity were corrected with
the Greenhouse-Geisser method. An alpha of P < 0.05
was adopted to establish statistical significance. All data
(Flow cytometry, DTI parameters, proteins, RT-qPCR
and immunohistochemistry analyses) were comparedusing a one-way ANOVA, and significance followed bypost-hoc testing using Tukey’s multiple comparisons. For
non-parametric data, Kruskal-Wallis test was performed
and a Dunn’s post-hoc analysis used. Blood gas, CBF and
ELISA data were compared using two-way repeated measures ANOVA. The independent variables assessed weregroup (PGROUP) and time of measurement (PTIME). Where
there was a significant interaction between independent

Page 6 of 17

variables (PGROUP X TIME), a post-hoc analysis with Tukey’s
multiple comparisons test was undertaken. DTI data arepresented as box plots with 5–95% confidence intervalsof median, with maximum–minimum error bars. Allother data are presented as mean ± SD.

Results
Head-out ventilation in preterm fetal sheep and UCB cells
administration

Singleton fetuses at 125 ± 1 days of gestational age(dGA) from all three groups had similar body and brain
weights (Fig. 1A, B). Ventilation with an intact umbilical cord achieved a V T of ~ 8.40 mL/kg by the end of the
15 min ventilation strategy in all ventilated fetuses, withno difference in delivered V T between groups (VENT:
8.32 ± 1.67; VENT + UCB: 7.77 ± 2.36 mL/kg; P = 0.624)(Fig. 1C). Higher V T could not be achieved in line withthe low compliance and high airway resistance of theimmature lungs characteristic of preterm infants [32].
Carotid blood flow (CBF) gradually decreased during
the high V T ventilation in both ventilation groups dropping to 40–42% of baseline by 15 min (Fig. 1D). Fetal pH,
PaCO2, and PaO2 were not altered during high V T ventilation (Fig. 1E-H). At 15 min of ventilation comparedto the SHAM group, SaO2 was significantly higher in the
VENT group and trended higher in the VENT + UCB
group (P = 0.035 and 0.077 respectively; Fig. 1H).
To test our hypothesis that early UCB treatment would
attenuate the peak upregulation of cerebral pro-inflammatory mediators that occurs after > 1 h following highV T ventilation [7, 17, 18], UCB cells administration was
conducted 1 h post-ventilation – a clinically feasible timing for an intervention. One hour after the initiation ofventilation, VENT + UCB fetuses were administered allogeneic 24.5 ± 5.0 million UCB cells/kg (~ 80 million cells)
intravenously obtained from healthy term lambs. UCB
administration did not alter pH, PaO2 and SaO2 during
the 24 h recovery period, but VENT + UCB fetuses had
a higher PaCO2 levels compared to the VENT group at 1
and 12 h (Fig. 1D-G).
UCB cell administration following brief high VT ventilationinduces white matter injury

The effects of brief high V T ventilation and UCB cells on
white matter were assessed using magnetic resonanceimaging-diffusion tensor imaging (MRI-DTI) (Fig. 2A)
and immunohistochemical staining (Fig. 2B) in multiple
white matter regions of interest.
In the periventricular white matter (PVWM), mean
fractional anisotropy was not different between groups(Fig. 3C). For measurements of axial, radial and meandiffusivity within the PVWM, brief high V T ventilation alone did not result in changes to DTI measurements compared to SHAM, however the administration

Tran et al. Journal of Neuroinflammation(2024) 21:121

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Fig. 1 Head-out ventilation in preterm fetal sheep and UCB cells administration. (A) Schematic overview of in vivo experimental paradigm in fetal sheep
at 125 ± 1 dGA. All fetuses underwent sterile instrumentation. VENT and VENT + UCB fetuses underwent 15 min of high tidal volume (V T ) ventilation. All
fetuses were then returned in utero for 24 h of recovery. VENT + UCB fetuses were administered umbilical cord blood (UCB) cells 1 h post-ventilation. All
fetuses were delivered at 24 h for MRI. (B) Body weight and brain weight were not different between groups. (C) V T increased over time in VENT and
VENT + UCB fetuses to the same degree. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. (D) Carotid blood flow (CBF) decreased during 15 min of high V Tventilation. Two-Way repeated measures ANOVA. #P < 0.05 SHAM vs. VENT; +P < 0.05 SHAM vs. VENT + UCB. SHAM; n = 5, VENT; n = 6 and VENT + UCB; n = 7.
(E-H) Arterial blood gas parameters during 15 min of ventilation (grey) and recovery period. (E) pH, (F) arterial partial pressure of carbon dioxide (PaCO2),(G) arterial partial pressure of oxygen (PaO2), and (H) arterial oxygen saturation level (SaO2) at timepoints relative to ventilation. Two-Way repeatedmeasures ANOVA. #P < 0.05 SHAM vs. VENT; *P < 0.05 VENT vs. VENT + UCB. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. All data in (B-H) are mean ± SD

of UCB cells significantly increased axial, radial andmean diffusivity in the VENT + UCB group compared
to VENT (P = 0.009, 0.037, 0.013 respectively) (Fig. 3DF). The increases in axial, radial and mean diffusivity inthe VENT + UCB group suggests a degree of alteration
to the structures within the major white matter tract,demyelination or increased edema [33]. All mean DTI

measurements were similar across groups within thefrontal white matter (FWM) (Fig. 3C-F).
White matter was assessed using immunostaining of
Olig2+, a marker for all oligodendrocyte lineage cells,and MBP+, a marker for mature myelin, in the PVWM
and subcortical white matter (SCWM) (Fig. 2B,G&H).
Ventilation alone did not alter the white matter oligodendrocyte cell density compared to SHAM. The

Tran et al. Journal of Neuroinflammation(2024) 21:121

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Fig. 2 White matter disturbances present in VENT + UCB lambs but not VENT only lambs. (A) Representative MRI images of ROIs (yellow circles) in theperiventricular white matter (PVWM) and frontal white matter (FWM) for DTI analysis. Figure is reproduced from [28]. (B) Immunohistochemical analyses
conducted in the PVWM and subcortical white matter (SCWM) across the major cerebral lobes (frontal, parietal, temporal and occipital). (C-F) MRI-DTI
analysis of C) mean fractional anisotropy (FA), (D) axial diffusivity (AD), (E) radial diffusivity (RD), and (F) mean diffusivity (MD) measurements; units = arbitrary unit (AU). Box plots showing interquartile range and min-max, the line marks the median and + marks the mean; SHAM; n = 5, VENT; n = 6 andVENT + UCB; n = 5. One-Way ANOVA, Tukey’s post-hoc comparisons; *P < 0.05, **P < 0.01. (G) Olig2+ immunostaining marks all oligodendrocyte lineage
while MBP + immunostaining marks mature myelin. (H) Representative images of Olig2 and MBP immunoreactivity in the PVWM. Scale bar = 50 μm.
Quantitative analysis of (I) Olig2+ and (J) MBP+ cellular population (cell density) and (K) MBP+ fractional area coverage. One-Way ANOVA, Tukey’s post-hoc
comparisons. All data (I-K) are mean ± SD and SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7 and *P < 0.05, **P < 0.01

Tran et al. Journal of Neuroinflammation(2024) 21:121

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Fig. 3 Increased vasculature damage within white matter in VENT + UCB group. (A) Quantitative analysis of TUNEL+ cell population (cell density) in the
PVWM and SCWM. One-Way ANOVA. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7 (B) Gene expression levels of cell death related genes (P53 [P53]and caspase3 [CASP3]) and tight junction related genes (occludin [OCLN] and claudin1 [CLDN1]) expressed as a fold change relative to SHAM. SHAM;n = 5, VENT; n = 7 and VENT + UCB; n = 7. One-way ANOVA. Tukey’s post-hoc comparisons; *P < 0.05, **P < 0.01. (C) Quantitative analysis of the number ofvessels with serum extravasation. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. One-Way ANOVA, Tukey’s post-hoc comparisons; *P < 0.05. All data aremean ± SD. (D) Representative images of sheep serum-positive staining in the PVWM and SCWM (arrow indicates intact vessel, arrowhead indicates vessel
profile with protein extravasation). Scale bar = 50 μm

administration of UCB cells however significantly
reduced oligodendrocyte number in the PVWM and
SCWM in the VENT + UCB group compared to VENT
(P = 0.026 and 0.030 respectively; Fig. 2I). Assessment of
mature myelin (MBP; Fig. 3G&H) revealed no changes
in cell density or fractional area coverage within the
PVWM and SCWM; though there was a near-significant
decrease in the number of MBP+ cells in the PVWM inthe VENT + UCB group compared to SHAM (P = 0.051;
Fig. 2J&K). Together, the administration of UCB cells
following brief high V T ventilation reduced the numberof oligodendrocytes, with a trending decrease in axonal
myelination.
This loss of oligodendrocyte populations within thewhite matter was however not due to apoptosis as indicated by the lack of increased TUNEL+ staining within
the same areas (Fig. 3A). A lack of apoptosis is consistent
with the decreased mRNA levels of CASP3, the gene that
encodes caspase3, in the VENT + UCB group comparedto SHAM (P = 0.007; Fig. 3B). Moreover, the decrease in
CASP3 mRNA also occurred in the VENT group whencompared to SHAM (P = 0.001; Fig. 3B). This indicatesthat the downregulation of caspase3 activity is a result of
the brief high V T ventilation within the white matter tractand is not mediated by UCB cells.
Blood vessel integrity was assessed by counting thenumber of blood vessels with serum extravasation.

VENT + UCB had a greater number of vessels withserum extravasation compared to SHAM in the SCWM
(P = 0.024; Fig. 3C&D). In the PVWM, the number of vessels with serum extravasation was significantly higher
in the VENT + UCB compared to VENT group, and atrend for a significant difference compared to SHAM
(P = 0.040 and 0.073 respectively; Fig. 3C&D). Increased
blood vessel extravasation suggests blood-brain-barrier(BBB) disruption in the VENT + UCB group, which isconsistent with the decreased mRNA levels of OCLN and
CLDN1, key tight junction proteins, in the PVWM and
SCWM compared to the SHAM group (Fig. 3B). Interesting to note is that OCLN and CLDN1 mRNA levelsin the PVWM were also decreased in the VENT groupcompared to SHAM (P = 0.012 and 0.038 respectively;
Fig. 3B). At the transcriptional level, the downregulationof OCLN enhances intracellular free Ca2+ concentration
thereby increasing cellular permeability [34]. Claudin-1
has a complex BBB destabilising role and its downregulation contributes to BBB leak [35]. The data suggest thatwhile brief high V T ventilation alone is not causing overt
white matter changes at 24 h, there is indication of cellular dysregulation likely involving the integrity of the
BBB. The early administration of UCB cells following
brief high V T ventilation disturbed white matter integrity,
caused a reduction in the oligodendrocyte populationand increased permeability of the BBB.

Tran et al. Journal of Neuroinflammation(2024) 21:121

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UCB cells following brief high VT ventilation induces
systemic and cerebral inflammation

ventilation-induced injury. As such, we investigated thepotential mechanisms underlying the response of UCB
derived MNCs in a pro-inflammatory environment invitro; we also investigated mRNA and protein effects on
individual cell types (HSCs, EPCs and monocytes) which
are reported in Table S3 & S4 respectively. A pro-inflammatory environment was simulated by spiking media
with the pro-inflammatory cytokines IFNγ and TNFα
(10ng/mL; Fig. 5A), both known to be differentiallyupregulated in the brain and systemically with preterm
birth and to play a role in white matter injury [38].

Inflammation is a key driver of preterm-related whitematter injury. To characterise systemic inflammation
caused by brief high V T ventilation and UCB cells, wemeasured the concentration of cytokines within arterial plasma periodically over the 24 h experimental
period (Fig. 4A-F). Plasma IFNγ and IL-10 levels differed
between groups over time with levels markedly increasedin the VENT + UCB group compared to both VENT and
SHAM (Fig. 4A,C). Plasma IL-1β, TNFα, IL-8, and IL-6
levels were not different between groups (Fig. 3B, D-F).
At 24 h, mRNA expression of pro-inflammatory cytokine IL6 was increased within the subcortical white matter (SCWM) in the VENT + UCB group but not VENT
only group (Pvs.SHAM=0.037; Fig. 4G). mRNA expression of IL1B and TNF was unchanged in VENT and
VENT + UCB group (Fig. 4G).
Immunohistochemical staining of Iba-1+ and GFAP+
was analysed to assess neuroinflammation/neuroglial
reactivity within the cerebral white matter. Iba-1 stains
microglia, the resident immune cells found within thebrain. Morphological assessment delineates whether
microglia are in a resting (ramified) or activated (ameboid) state [36] (Fig. 4H). Brief high V T ventilation alone
did not affect Iba-1+ staining in the PVWM or SCWM
(Fig. 4I-M). The administration of UCB treatment after
brief high V T ventilation increased the number of total
Iba-1+ cells compared to the VENT only group (P = 0.009;
Fig. 4I) which was due to an increase in resting/ramified
microglia (P = 0.008; Fig. 4J) and not the number of activated/ameboid microglia (Fig. 4K). Consistent with theincrease in cell density was the increase in Iba-1+ fractional area coverage in the VENT + UCB group compared
to VENT in the SCWM (P = 0.010; Fig. 4L). In addition, the VENT + UCB group had higher percentages of
SCWM area covered by aggregations compared to the
SHAM group (P = 0.027; Fig. 4M). Neither ventilation nor
UCB treatment altered GFAP+ cell density and fractional
area coverage staining (Fig.S1).
Overall, our data indicate a very minimal pro-inflammatory response in the VENT group but amplified
inflammation, both systemically and within the brain, inthe VENT + UCB group.
Investigating the effect of inflammation on UCB derived
mononuclear cells in vitro

Pro-inflammatory stimuli preconditioning has beenshown to increase the immunomodulatory functions ofother stem cells such as mesenchymal stromal cells [37],but it is unclear as to how a pro-inflammatory environment effects UCB derived MNCs. Our in vivo investigations indicate that the UCB cells may have adopted apro-inflammatory state when administered early after

Pro-inflammatory stimulation upregulated mRNAand protein expression of pro- and anti-inflammatory
cytokines and chemokines

IL-1β is a major pro-inflammatory cytokine associatedwith preterm white matter injury [39]. mRNA expression of IL1B in MNCs was upregulated following TNFα
stimulation (P = 0.015; Fig. 5C). We also found that IL18
mRNA expression was increased following stimulation with TNFα, though this did not reach significance(P = 0.07; Fig. 5C). We did not observe any changes inmRNA or protein expression of IL-6 in response to proinflammatory stimuli (Fig. 5B and C).
TNFα is a pro-inflammatory cytokine which is known
to be upregulated in the setting of brain injury, contributing to injury pathogenesis. TNF mRNA expression was upregulated in MNCs when stimulated withthe combination of TNFα and IFNγ (P = 0.027; Fig. 5C).
This suggests that the UCB MNCs are responding to the
inflammatory environment and subsequently producing
pro-inflammatory cytokines, further perpetuating the
inflammatory response.
Interestingly, we also observed an increase in IL-10
expression. IL-10 is a potent anti-inflammatory cytokine
predominately produced by leukocytes, a cell population found within UCB MNCs. Stimulation with TNFαand the combination of TNFα and IFNγ resulted in an
increase in IL-10 expression in MNCs (P = 0.001 and
P = 0.007 respectively; Fig. 5D).
In addition to inflammatory cytokines, we also investigated the effect of pro-inflammatory stimuli on theexpression of key chemokines and their receptors. Stromal cell-derived factor 1 (SDF-1) is a chemokine thatplays key roles in stem cell migration as well as tissue
repair and regeneration. The balance between SDF-1 andits receptor chemokine receptor type 4 (CXCR4) is essential for trafficking stem cells to the site of injury, and stem
cells expressing CXCR4 will migrate to sites of injury
where SDF-1 expression is increased [40]. SDF-1 protein
expression was significantly increased following stimulation with TNFα and the combination of TNFα and IFNγ
(P = 0.0001 and P < 0.0001, respectively; Fig. 5B). We did
not observe any changes in mRNA levels of CXCR4 orTran et al. Journal of Neuroinflammation(2024) 21:121

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Fig. 4 Inflammation present in the VENT + UCB group but not VENT group. Plasma cytokine levels (A) IFNγ, (B) IL-1β, (C) IL-10, (D) TNFα, (E) IL-8 and (F)IL-6 over 24 h following 15 min of brief high V T ventilation (grey bar). SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. Two-Way repeated measures ANOVA.
(G) Gene expression levels of inflammatory cytokines (interleukin 1 beta [IL1B], interleukin 6 [IL6] and tumour necrosis factor [TNF]) expressed as a foldchange relative to SHAM. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. One-way ANOVA. Tukey’s post-hoc comparisons; *P < 0.05. (H) Representative
images of Iba-1 positive immunohistochemical staining in the SCWM. Insert demonstrates significant microglial aggregation. When resting, microglia
display ramified morphology but as microglia are activated, they adopt an ameboid morphology. Scale bar = 50 μm. Quantitative analysis in the PVWM
and SCWM of (I) the total number of Iba-1+ cells, (J) the number of resting/ramified microglia, (K) the number of ameboid microglia, (L) the fractional area
coverage of Iba-1+ staining, and (M) the percentage of white matter occupied by aggregation. SHAM; n = 5, VENT; n = 7 and VENT + UCB; n = 7. One-Way
ANOVA. Tukey’s post-hoc comparisons; *P < 0.05, **P < 0.01. All data are mean ± SD

Tran et al. Journal of Neuroinflammation(2024) 21:121

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Fig. 5 Pro-inflammatory stimulation of UCB MNCs resulted in differential expression of key cytokines, caspases and growth factors. (A) Schematic overview of in vitro experimental paradigm where UCB MNCs were stimulated with the pro-inflammatory factors TNFα & IFNγ for 16 h. (B) Conditioned media
was collected to measure protein content of inflammatory-related proteins and growth factors. Cells were also harvested to measure mRNA expression
levels of (C) inflammatory cytokines, (D) caspases and growth factors expressed as fold change relative to control. One-Way ANOVA, Dunnett’s post-hoc
comparisons. All data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Control (grey circles), n = 3; TNF (purple squares), n = 3;
IFN (green triangles), n = 3; TNF + IFN (orange diamond), n = 3, with each performed in triplicate

C-C chemokine receptor types (CCR1, CCR2, CCR4 &CCR5; Table S3).
One key role of caspases is to cleave inactive precursor
pro-IL-1β and pro-IL-18 to form mature IL-1β and IL-18
which is then secreted from the cell via the NLR family
pyrin domain containing 3 (NLRP3) inflammasome. We
observed a significant increase in CASP4 mRNA levels
following stimulation with IFNγ and the combination of
TNFα and IFNγ (P = 0.010 and P = 0.007 respectively), but
no changes to CASP1 or CASP5 (Fig. 5D).
Pro-inflammatory stimulation upregulated protein
expression of growth factors

UCB MNCs can secrete growth factors that support andpromote cellular growth and differentiation. In this study,
pro-inflammatory stimulation with TNFα and combined
TNFα and IFNγ resulted in increased protein expression of VEGF (P = 0.0006 and P = 0.0002, respectively),
BDNF (P = 0.0010 and P = 0.0014, respectively) and
GDNF (P = 0.0003 and P = 0.0004, respectively; Fig. 5B).
Interestingly, pro-inflammatory stimulation did not alter
growth factor mRNA expression in MNCs (Fig. 5D). The
increased protein expression in the absence of mRNA

expression may be due to the temporal delay in protein
synthesis following transient upregulation of mRNA
expression. This suggests a temporary neuroprotective role of UCB derived MNCs via a release of beneficial growth factors, that is being driven by exposure to a
TNFα mediated inflammatory environment.

Discussion
Preterm newborns often receive respiratory support after
birth irrespective of their underlying pathologies, andwhilst life-saving, respiratory support can contribute tobrain injury [7]. We hypothesised that administration of
UCB cells 1 h after 15 min of high V T ventilation in thepremature fetal sheep would reduce ventilation induced
cerebral white matter inflammation and injury. Despite
the effect of brief high V T ventilation alone on cerebral
white matter being relatively mild, unexpectedly, we
found that early administration of UCB cells after ventilation increased diffusivity of white matter, increased
cerebral inflammation, decreased BBB integrity anddecreased oligodendrocyte cell population in the cerebral
white matter at 24 h. To further investigate the mechanisms underlying these adverse effects, we assessed theTran et al. Journal of Neuroinflammation(2024) 21:121

immunomodulatory state of MNCs derived from human
UCB when introduced to a pro-inflammatory environment. We discovered that UCB cells incubated in media
spiked with pro-inflammatory cytokines (TNFα andIFNγ) adopted a predominantly pro-inflammatory state
as characterized by upregulation of pro-inflammatory
genes and production of pro- and anti-inflammatory
cytokines. Nevertheless, UCB cells also upregulated specific growth factors suggesting potential neuroprotectiveroles of cells, even in the setting of profound inflammation. This study highlights the need to assess the timingof administration of UCB cells to preterm infants, especially in cases where infants are receiving respiratory support in periods of high systemic inflammation to avoid
undesired side effects such as white matter pathology.
We previously demonstrated in the same lambs that15 min of brief high V T ventilation led to significant lunginjury indicated by persisting leukocyte infiltration andstructural changes [25]. Interestingly, we found that early
UCB cell treatment did not reduce or exacerbate this ventilation-induced lung injury [25]. Here however, we founddifferent responses in the preterm brain. UCB cells given
during the period of peak inflammation exacerbatedwhite matter injury when ventilation alone was benign.
We suspect that returning the lambs in utero following
ventilation causes the lungs to become liquid filled once
again, reinstating the right-to-left shunting and minimal
pulmonary perfusion. As the pulmonary circulation isbuffered, the intravenous administration of UCB cells canonly target other accessible organs such as the brain. We
and others have reported extensively that UCB treatment
alone does not induce systemic nor cerebral injury whenadministered > 12 h after the initial insult in preterm andterm asphyxiated lambs [9, 26]. Therefore, the white matter injury observed in this study is likely to be due to theinteraction between the systemic consequences of ventilation and the direct effect on UCB cells.
Inflammation is a key mediator of preterm brain injury[41], particularly when infants are exposed to systemic
inflammation [42]. In this study, 15 min of high V T ventilation induced negligible increases in systemic inflammation across the 24 h period studied, probably due tothe relatively short duration of the ventilation intervention. However, the intravenous administration of UCB
cells following ventilation increased systemic cytokines
which persisted during the 24 h. We theorise that thetransient high V T ventilation created an acute inflammatory environment and the introduction of the UCB
cells to this environment inappropriately triggered thecells’ innate pro-inflammatory response mechanisms tocause downstream neuropathological consequences. A
key mechanism by which the brief high V T ventilation
may have increased the susceptibility of the white matterto further damage is alterations to the BBB. Ventilation

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induces BBB permeability within minutes in pretermlambs [43], with evidence of brain diffusion and metabolic disturbances by 1 h following high V T ventilation[27]. Indeed, we observed evidence of altered endothelial
integrity within the white matter as indicated by a downregulation of tight junction proteins, but did not observe
histological evidence of increased BBB permeability in
VENT lambs. The lack of disturbed BBB integrity at 24 h
likely reflects a recovery from the transient permeability induced by ventilation. Complete BBB permeability
recovery has been shown to occur at 24 h after 15 min of
cerebral ischemia in adult rats [44]; and thus the 24 h ofnormalised cerebral reperfusion in the absence of continued ventilation likely explains the intact BBB comparedto the other preterm lamb studies reported BBB permeability following ventilation [27, 43]. A disturbance tothe BBB integrity may have rendered the immature brain
temporally vulnerable to infiltration of systemic inflammatory cytokines, leukocytes and trophic factors that
promoted localized cerebral inflammation from the UCB
cells.
Pro-inflammatory preconditioning of cell therapies,
such as mesenchymal stromal cells (MSCs), has beenshown to increase their immunosuppressive and reparative capacity [37]. Little research has been conducted
to assess the effect of pro-inflammatory stimulation on
UCB MNCs and the individual cells within this population. UCB MNCs are comprised of many stem and progenitor cells, including MSCs, hematopoietic stem cells(HSC), endothelial progenitor cells (EPC) and monocytes, and thus it is important to understand how eachof these cell types respond to inflammatory stimulationindividually, and together. Here, we demonstrate that
MNCs derived from human UCB increased gene expression of pro-inflammatory cytokines and chemokines,
with corresponding increases in protein when stimulatedwith pro-inflammatory stimuli in vitro. This reflects theincrease of pro-inflammatory cytokines seen in vivo, further confirming that when placed into a suboptimal environment UCB cells will mount an inflammatory response.
Adding to the complexity, MNCs also upregulated antiinflammatory cytokine and growth factor release following pro-inflammatory stimulation suggesting potential
reparative responses. Specifically, there was an increasein IL-10 expression, which is consistent with the in vivo
study where we observed a transient increase in IL-10
protein expression following UCB administration. There
was also an increase in expression of the growth factors
VEGF, GDNF and BDNF, which highlights a neuroprotective role of UCB cells in the setting of inflammation.
These growth factors are known to modulate injury in
response to neonatal hypoxia ischemia via anti-inflammatory, anti-apoptotic and pro-angiogenic mechanisms[45–47]. UCB is a heterogeneous population of cells andTran et al. Journal of Neuroinflammation(2024) 21:121

thus we further investigated which cell populations may
be responsible for driving inflammation. We individually
measured gene and protein expression of HSCs, EPCs
and monocytes, when exposed to a pro-inflammatoryenvironment, however we were unable to extrapolate any
discernable pattern to identify which predominate cell
population was driving the responses observed within
the total MNC population (Table S3 & S4).
On the basis of our findings in preterm sheep, we suggest that caution should be taken in future studies if UCB
cells are to be used in preterm infants as an early therapeutic intervention strategy for preterm brain injury. The
growing interest in, feasibility and safety profile of UCB
cells for preterm infants encourages their early administration to optimise neuroprotection [4, 9, 48, 49], however in these studies “early” treatment is still > 12 h post
initial insult. Notwithstanding the other morbidities thatare likely to occur in preterm infants, the majority ofpreterm infants require respiratory support for varying
periods of time. If UCB cells are to be administered as atreatment strategy for morbidities such as hypoxic-ischemic encephalopathy or intraventricular haemorrhage,
the timing of administration in relation to the pathogenesis of inflammatory processes, especially following ventilatory interventions, must be considered. This is criticalas up to 96% of infants < 32 weeks GA receive respiratory
support at birth [50] which can occur for a duration ofdays to weeks [51]. The neuroinflammatory processes
following prolonged ventilation most likely differs to abrief high V T ventilation method; for example, we haveshown persisting increased levels of systemic cytokines
during 24 h of in utero ventilation with V T of 3 mL/kg
in fetal sheep with evidence of heightened microglial andastrocytic responses within the brainstem [52]. Whether
UCB cells demonstrate a similar pro-inflammatory statein cases of prolonged ventilation or ventilation with otherco-morbidities requires further interrogation.
The use of UCB for ventilation-induced brain and lunginjury should not be dismissed however, as the timing foradministration may be key to its efficacy. Both preclinicaland clinical studies utilising UCB cell therapy as a treatment for perinatal-related complications have demonstrated particular efficacy when administration occurs
after 6–24 h after birth/injury [6, 9]. This timing caneven be delayed to 3–10 days after preterm birth, with
UCB cells and UCB-derived cells reducing intraventricular haemorrhage rates and bronchopulmonary dysplasia(BPD) severity [53, 54]. It is also important to note thatin our previous study by Li et al., demonstrating beneficial effects of allogeneic UCB cells, UCB cells preparation
was the same but cells were administered 12 h after the
insult [9]. The optimal therapeutic window for administration may therefore be after peak inflammatory mechanisms have occurred whereby UCB cells would play a

Page 14 of 17

more restorative role rather than as a preventative therapeutic strategy.
A major limitation of our study is the absence of measurable white matter injury or systemic inflammation following exposure to mechanical ventilation that we haveshown previously [7, 17, 18, 55]. This suggests a subclinical manifestation of the injury, creating a subtle proinflammatory environment that was adequate to induce apro-inflammatory response of UCB cells. Future studies
should consider a more injurious model to fully explore
the effects of UCB cells in the setting of established brain
inflammation/injury. A further limitation was our inability to ascertain the exact mechanisms by which the UCB
cells exacerbated white matter injury following acutemechanical ventilation. Due to the experimental paradigm, we were unable to investigate how the UCB cellularconstituents reacted to the cytokines at a temporal andspatial level, future investigations using metabolomics
and proteomics platforms may reveal these exact mechanisms. We did not label the UCB cells in this study andcould not trace if they entered the brain parenchyma,
though we know that UCB cells are present in low numbers in the brain after intravenous administration in preterm [9] and near-term [26] lambs and that their effects
are predominantly due to trophic actions. We haveidentified multiple mechanisms by which UCB cells canadopt an injurious state – whether these mechanisms are
unique to ventilation-induced injury or are also involvedin other perinatal pathologies needs further investigation. In this study we have demonstrated an increase inneuroinflammation and a decrease in white matter integrity at 24 h post-injury, however due to the short-term
nature of this study we do not know if these changes will
persist or if they are transient and thus may resolve overtime. To fully ascertain this, future studies should include
later timepoints to determine how injury progresses after
ventilation and UCB administration. Multiple iterationsof ventilation-induced brain injury need to be assessedto ascertain the exact interaction between ventilation and
UCB cells; e.g., prolonged ventilation and UCB administration during ventilation or after ventilation cessation.

Conclusion
Overall, we demonstrate that a single systemic dose ofallogeneic UCB cells 1 h after acute high V T ventilationin preterm lambs induced damage to the integrity ofwhite matter tracts, compromised vascular integrity andincreased neuroinflammation at 24 h. Our results suggest that brief ventilation sensitises the preterm brain
to injury, and the early introduction of UCB cells intoa vulnerable or pro-inflammatory microenvironment
may exacerbate inflammatory responses that can lead towhite matter injury. Future clinical studies using UCB
cells should consider how ventilation, and other common

Tran et al. Journal of Neuroinflammation(2024) 21:121

interventions at birth, may alter the inflammatory environment, and thus how UCB cells will subsequently
respond to this environment. As such, caution for earlyadministration in preterm infants needs to be observed.
Abbreviations°C	Degrees Celsius
AD	Axial diffusivity
ANOVA	Analysis of varianceAU	Arbitrary units
BBB	Blood-brain-barrier
BDNF	Brain-derived neurotrophic factor
CASP-	CaspaseCat#	Catalog number
CCR-	C-C chemokine receptor typecDNA	Complementary Deoxyribonucleic acid
CLDN1	Claudin 1
CXCR-	Chemokine receptor typedGA	Days gestational age
DMEM	Dulbecco’s Modified Eagle Medium
DMSO	Dimethylsulfoxide
DTI	Diffusion tensor imaging
DWI	Diffusion weighted imaging
EDTA	Ethylenediaminetetraacetic acidFA	Fractional anisotropy
FBS	Fetal bovine serum
FiO2	Fraction of inspired oxygeng	G-forceg	Grams
GDNF	Glial cell line-derived neurotrophic factor
GFAP	Glial fibrillary acidic proteinh	HourIba-1	Ionised calcium binding adapter molecule-1
IFN	Interferon
IGF	Insulin-like growth factor
IgG	Immunoglobulin GIL	Interleukin
kg	Kilogram
MBP	Myelin basic protein
MD	Mean diffusivitymg	Microgramsmin	Minutes
mL	Millilitermm	Millimetersmm	Milometers
MNCs	Mononuclear cells
MRI	Magnetic resonance imaging
mRNA	Messenger ribonucleic acidn	Numberng	Nanograms
NLRP3	NLR family pyrin domain containing 3
OCLN	Occludin
Olig2	Oligodendrocyte transcription factor-2
P	
p-value
P53	Tumor protein 53
PaCO2	Partial pressure of carbon dioxide
PaO2	Partial pressure of oxygen
PBS	Phosphate-buffered saline
PCR	Polymerase Chain Reaction
PEEP	Peak end-expiratory pressurepg	Picogram
PIP	Peak inspiratory pressure
PVWM	Periventricular white matter
RD	Radial diffusivityRNA	Ribonucleic acid
ROI	Region of interests
Rpm	Rotations per minute
RT-PCR	Reverse transcription polymerase chain reaction
SaO2	Oxygen saturation
SCWM	Subcortical white matter

Page 15 of 17

SD	Standard deviation
SDF-1	Stromal cell-derived factor 1
TCEP	Tris(2-carboxyethyl)phosphine
TNF	Tumor necrosis factor
TUNEL	Terminal deoxynucleotidyl transferase dUTP nick end labelling
UCB	Umbilical cord blood
VEGF	Vascular endothelial growth factor
VENT	Ventilation
V T	Tidal volumeµL	Microliterµm	Micrometre

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03053-3.
Supplementary Material 1: Fig. S1: No changes to GFAP immunoreactivity in the white matter
Supplementary Material 2: Table S1: Details of genes investigated using
RT-qPCR by Fluidigm on frozen tissue
Supplementary Material 3: Table S2: Primer sequences for in vitro gene
expression analysis
Supplementary Material 4: Table S3: mRNA expression in in vitro study
Supplementary Material 5: Table S4: Protein expression in in vitro study
Supplementary Material 6: Data file S1: ARRIVE Checklist for animal
research
Acknowledgements
The authors acknowledge the assistance of Jade Barbuto, and the facilities
and technical assistance of Monash Biomedical Imaging, Monash Histology
Platform, and MHTP Medical Genomics Facility.
Author contributions
MCF, GJ, SLM, GRP, CAM conceptualized and designed the study. MdV, GRP,
CAM designed the methodology. SLM, GRP, CAM acquired funding. GRP, RG,
CAM administrated and supervised the project. NTT, KYYC, TT, PCP, TS, LN,
VAZ, YP, TY, IN, SBK, AMT, DMA, RG, GRP, CAM undertook the experiments. NTT,
TRP, KYYC, TT, TS, DMA, GRP, RG, CAM undertook formal analysis of the data.
NTT, TRP undertook data visualization and writing of original draft. All authors
critically reviewed the manuscript and approved the final manuscript as
submitted and agree to be accountable for all aspects of the work.
Funding
This study was supported by the Cerebral Palsy Alliance Australia Early Career
Fellowship 1110195 (CAM); National Health and Medical Research Council
Senior Research Fellowship 1105526 (GRP); National Health and Medical
Research Council Senior Research Fellowship APP2016688 (SLM); Inner Wheel
Australia (SLM); Victorian Government’s Operational Infrastructure Support
Program (SLM).
Data availability
Data is provided within the manuscript or supplementary information
files. The datasets used during the current study are available from thecorresponding author upon reasonable request.

Declarations
Ethics approval and consent to participate
All procedures were approved by the Hudson Institute of Medical Research
Animal Ethics committee and were conducted in accordance with the
National Health and Medical Research Council Code of Practice for the Careand Use of Animals for Scientific Purposes (Eighth Edition). All human-derived
samples were collected with human ethics approval from Monash Health
Human Ethics Committee.

Tran et al. Journal of Neuroinflammation(2024) 21:121

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
The Ritchie Centre, Hudson Institute of Medical Research, Clayton, VIC,
Australia
2
Department of Obstetrics and Gynaecology, Monash University, Clayton,
VIC, Australia
3
Department of Pharmacology, Biomedicine Discovery Institute, Monash
University, Clayton, VIC, Australia
4
Monash Biomedical Imaging, Monash University, Clayton, VIC, Australia
5
Department of Paediatrics, Monash University, Clayton, VIC, Australia
6
Monash Biomedicine Discovery Institute and Department of Medical
Imaging and Radiation Sciences, Monash University, Clayton, VIC,
Australia
7
Department of Diagnostic Imaging, Kind Saud Medical City, Riyadh,
Saudi Arabia
Received: 2 January 2024 / Accepted: 23 February 2024

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