Tran et al. Journal of Neuroinflammation(2024) 21:142
https://doi.org/10.1186/s12974-024-03137-0

Journal of Neuroinflammation

Open Access

RESEARCH

Intrauterine inflammation and postnatal
intravenous dopamine alter the neurovascular
unit in preterm newborn lambs
Nhi T. Tran1,2, Nadia Hale1, Anawar Aung Win Maung1, Manon Wiersma1,2, David W. Walker1,4, Graeme Polglase1,3,Margie Castillo-Melendez1,2 and Flora Y. Wong1,2,3,4,5*Abstract
Background Intrauterine inflammation is considered a major cause of brain injury in preterm infants, leading tolong-term neurodevelopmental deficits. A potential contributor to this brain injury is dysregulation of neurovascular
coupling. We have shown that intrauterine inflammation induced by intra-amniotic lipopolysaccharide (LPS) in
preterm lambs, and postnatal dopamine administration, disrupts neurovascular coupling and the functional cerebral
haemodynamic responses, potentially leading to impaired brain development. In this study, we aimed to characterise
the structural changes of the neurovascular unit following intrauterine LPS exposure and postnatal dopamine
administration in the brain of preterm lambs using cellular and molecular analyses.
Methods At 119–120 days of gestation (term = 147 days), LPS was administered into the amniotic sac in pregnant
ewes. At 126-7 days of gestation, the LPS-exposed lambs were delivered, ventilated and given either a continuous
intravenous infusion of dopamine at 10 µg/kg/min or isovolumetric vehicle solution for 90 min (LPS, n = 6; LPSDA,n = 6). Control preterm lambs not exposed to LPS were also administered vehicle or dopamine (CTL, n = 9; CTLDA,n = 7). Post-mortem brain tissue was collected 3–4 h after birth for immunohistochemistry and RT-qPCR analysis ofcomponents of the neurovascular unit.
Results LPS exposure increased vascular leakage in the presence of increased vascular density and remodellingwith increased astrocyte “end feet” vessel coverage, together with downregulated mRNA levels of the tightjunction proteins Claudin-1 and Occludin. Dopamine administration decreased vessel density and size, decreased
endothelial glucose transporter, reduced neuronal dendritic coverage, increased cell proliferation within vessel
walls, and increased pericyte vascular coverage particularly within the cortical and deep grey matter. Dopamine also
downregulated VEGFA and Occludin tight junction mRNA, and upregulated dopamine receptor DRD1 and oxidative
protein (NOX1, SOD3) mRNA levels. Dopamine administration following LPS exposure did not exacerbate any effects
induced by LPS.
Conclusion LPS exposure and dopamine administration independently alters the neurovascular unit in the preterm
brain. Alterations to the neurovascular unit may predispose the developing brain to further injury.

*Correspondence:
Flora Y. Wong
flora.wong@monash.edu
Full list of author information is available at the end of the article© 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
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in this article, unless otherwise stated in a credit line to the data.

Tran et al. Journal of Neuroinflammation(2024) 21:142

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Keywords Intrauterine inflammation, Chorioamnionitis, Dopamine, Preterm brain, Neurovascular unit, Neurovascular
coupling

Background
Intrauterine infection or inflammation which manifests
as chorioamnionitis is a major contributor to preterm
cerebral injury leading to adverse neurodevelopmental
outcomes along with long-term disabilities [1–4]. Dysregulation of cerebral blood flow (CBF) occurs alongsideneuropathology in chorioamnionitis [5]. Preterm lambs
and infants with chorioamnionitis have increased basal
CBF and cerebral oxygen delivery [6, 7], likely due to theneuroinflammation. But they can have an even greaterand disproportionate increase in cerebral oxygen consumption and oxygen extraction [7], indicating a baseline
mismatch between the cerebral oxygen delivery and oxygen consumption. We have reported that preterm lambs
exposed to intrauterine inflammation, induced by intraamniotic injection of lipopolysaccharide (LPS), are more
likely to show a negative cerebral functional haemodynamic response (i.e., localised decrease in oxyhaeomoglobin) suggesting that neuronal activity, which increases
the demand for oxygen, is likely to create localized oxygen deficits [8].
Increased local neuronal activity normally leads toincreases of local cerebral perfusion and oxygenation tomeet the increased local metabolic demand – a physiological response known as neurovascular coupling [9].
Tight coupling of cerebral function, metabolism, andblood flow has been demonstrated in numerous physiological, biochemical, and clinical studies [10]. A complex
network of neurons and astrocytes are in close physicalproximity with, and functionally coupled to the basal
lamina, pericytes, smooth muscle cells, and the endothelial cells of cerebral arterioles. The interconnection andinteractions of these cells constitute the “neurovascularunit” (NVU) [11]. This unique vascular coupling systemregulates blood flow, supports brain growth and homeostasis, and coupled with the development of the bloodbrain barrier (BBB), the NVU also enables clearanceof toxic byproducts of brain metabolism and immune
surveillance.
Relatively little research has looked specifically at
the NVU in the developing brain and perinatal period,
despite studies suggesting differences in NVU structure
and/or function between the developing and adult brain.
Importantly, perinatal insults associated with adverse
neurodevelopmental deficits, such as hypoxic-ischemia
or infection such as chorioamnionitis, are typically associated with changes in both CBF and the presence ofneuropathology, suggesting that neurovascular development may be critically affected. Notably, inflammation
has long been known to promote disruption of the BBB

and neurovasculature in both the mature and immature
brain [11–13]. Given the adverse long-term consequenceof chorioamnionitis, insights into the neurovasculareffects of intrauterine inflammation in the preterm brain,
particularly on the NVU, will be important for understanding the events that lead to brain injury, dysfunction,and repair, as well as identification of therapeutic targets.
In addition, we have previously reported that infusionof the inotrope dopamine, often required to treat hypotensive preterm infants following infection/inflammation, increases the baseline cerebral oxygenation but also
further increases the incidence of negative functional
haemodynamic responses in preterm lambs exposed to
intrauterine inflammation [8, 14]. To fully understand theimplications of these effects on neurovascular coupling,histopathological analysis of the NVU would provide
mechanistic insights at the cellular and molecular levels.
In the present study, we investigated the effects on thecellular and molecular components of the NVU in preterm newborn lambs previously exposed to intra-amniotic LPS. We also determined the effects of postnatal
dopamine infusion on the NVU with and without exposure to this intrauterine inflammation. We hypothesizedthat exposure to intrauterine inflammation alters the
cerebral vasculature in the brain of preterm lambs so thatneurovascular coupling is obtunded, and that dopamine
treatment further exacerbates these changes.

Methods
Experimental proceduresThis study utilized post-mortem preterm lamb brain tissue collected after in vivo physiological studies as previously published [8]. The use of animals was approved
by Monash Medical Centre Animal Ethics Committee(MMCA-2016/17 and 2016/23) and was conducted inaccordance with the Australian Code of Practice for thecare and use of Animals for Scientific Purposes established by the National Health and Medical ResearchCouncil of Australia.
Time-mated pregnant ewes (Merino-Border Leicester
cross) used in this study were supplied by the Monash
Animal Research Platform. At 119–120 days of gestation (dGA; Term is ~ 147 dGA), pregnant ewes received
an ultrasound-guided intra-amniotic injection of lipopolysaccharide (LPS; E Coli 055: B5, 10 mg/mL) or saline(control) to induce intra-uterine inflammation and elicit
the multi-system complications of chorioamnionitis
observed in human infants as reported previously [15].
At 126–7 dGA, ewes were anaesthetised by intravenous
injection of sodium thiopental (20 mg/kg; Jurox, NSW,

Tran et al. Journal of Neuroinflammation(2024) 21:142

Australia), followed by tracheal intubation, positive
pressure ventilation and inhaled anaesthesia of isofluorane 1.5–3.5% in oxygen air, and the lambs (n = 28) weredelivered by caesarean section. Each lamb was weighed,intubated and ventilated in volume-guaranteed mode at~ 5–7 ml/kg (Babylog8000Plus; Draeger; Germany) whileanaesthesia was maintained. Prophylactic surfactant wasadministered via the endotracheal tube (100 mg/kg Curosurf; ChiesiPharma; Italy). After delivery of the lamb, theewe was killed by intravenous injection of pentobarbitone (~ 100 mg/kg; pentobarbitone sodium 325 mg/ml,
Virbac, Milperra, NSW, Australia).
A pulse oximeter probe was placed on the lamb’s rightforelimb or tail to measure SpO2 (Radical 4, Masimo
Frenchs Forest NSW, Australia). Polyvinyl catheters containing heparinised saline were placed into the umbilicalvein and artery, or the jugular vein and carotid artery, toallow continuous measurement of mean arterial bloodpressure (MABP) and heart rate (HR) (DTX Plus Transducer; Becton Dickinson), for withdrawing blood forregular blood-gas analysis (ABL30; Radiometer, Copenhagen, Denmark) and for administration of 10% glucose(at 3 ml/kg/h) as fluid maintenance. Ventilatory settings
and fractional inspired oxygen were adjusted to maintain
SpO2 at > 90% and PaCO2 at 45–55 mmHg.
Thirteen lambs across the Control and LPS groups
were randomly allocated to receive a continuous intravenous infusion of dopamine at 10 µg/kg/min (mid-clinicaldose used in hypotensive preterm infants [16]) in heparinised saline for 90 min. The groups were: control, (CTL;n = 9), LPS, (LPS; n = 6), control and dopamine (CTLDA;n = 7) or LPS and dopamine (LPSDA; n = 6). All lambs wereventilated for 3–4 h and then were killed by intravenous
injection of pentobarbitone at 100 mg/kg.
Post-mortem tissue preparation

Brains were transcardially perfused in situ with isotonic
saline, then removed, weighed and divided longitudinallyat the mid-sagittal plane into the 2 cerebral hemispheres.
The right hemisphere was dissected for sampling of cortical grey matter (cortex), white matter (WM; combined
sampling from the subcortical and periventricular whitematter) and striatum (combined sampling from the putamen and caudate nucleus); samples were snap frozen inliquid nitrogen and stored at -80 °C for mRNA analyses. The left hemisphere was sectioned coronally into
10-mm-blocks for immersion fixation in chilled 4% paraformaldehyde (0.1 M; pH 7.4) for 3 days before paraffin
embedding. Coronal brain sections (8 μm) were cut at thelevel posterior to the ansate sulcus which corresponds tothe somatosensory cortex (the sheep brain atlas section
number 600, Michigan sheep atlas; [17]). Two coronalbrain sections that were 80 μm apart were used for each

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immunohistochemical marker and results were averaged
between the sections for each lamb.
Single-label immunohistochemistry

Immunohistochemical labelling was conducted using primary antibodies: rabbit anti-NeuN (1:200; Abcam, UK;
CAT#: 177487) for mature neurons; rabbit anti-microtubule-associated protein 2 (MAP2; 1:250; Sigma-Aldrich,
USA; CAT#:M3696) for neuronal dendrites; rabbit antisheep serum (1:700; Sigma-Aldrich, USA; CAT#:S4265)
for serum protein extravasation to detect vascular leakage; rabbit anti-matrix metallopeptidase 9 (MMP9; 1:200;
Thermo Fisher Scientific, USA; CAT#:RB-1539-P) as amarker for inflammation and basement membrane integrity; rabbit anti-glucose transporter 1 (GLUT1; 1:250;
Abcam, UK; CAT#:ab14683) to identify mature endothelial cells and; rabbit anti-Ki67 (1:100; Thermo Fisher Scientific, USA; CAT#:MA5-14520) for proliferating cells.
Briefly, tissue sections were de-paraffinized, then pretreated for antigen retrieval with citrate buffer (10 mM
Tri-sodium citrate in dH2O, pH 6.0; Sigma Aldrich), PBS
washes, endogenous peroxidase blocking, and blockedwith serum free protein block (Code X090, Dako, USA).
Primary antibodies were incubated overnight at 4°C in
Dako antibody diluent (Code S3022, Dako, USA). Sections were incubated in secondary goat biotinylated antirabbit IgG for 2 h (1:200; Vector Laboratories, UK; CAT#:
BA-100) then incubated with avidin-biotin complex(ABC Elite kit; 1:1:200 in PBS; Vectastain®, Vector Laboratories, UK) and visualized with 3,3’-diamniobenzidine
solution (DAB; MP Biomedicals, USA). Ki67 and sheep
serum antibody labelling were counter-stained with haemotoxylin. Sections were digitally scanned (Image Scope,
Aperio Technologies Inc., Germany) and analysed at 40x
magnification.
Double-label immunohistochemistry

To identify astrocytes associated with blood vessels,sections were incubated in mouse monoclonal antiGFAP (1:500; Sigma, USA; CAT#:G3893-.2ML) andrabbit anti-laminin (1:200; Novus Biologicals, USA;
CAT#:NB300-144). To identify pericytes, sections wereincubated in mouse monoclonal α-smooth muscle actin(α-SMA, 1:50; Sigma, USA; CAT#:A5228) and rabbit
polyclonal anti-desmin (1:50; Sigma, USA; CAT#:D8281).
Tissue sections were de-paraffinized, pre-treated for
antigen retrieval, then washed with sodium borohydride(10 mg/ml) in 0.1 M PBS to reduce autofluorescence.
Sections were then blocked with serum free protein
block and primary antibodies incubated overnight at 4 °C
in Dako antibody diluent. Immunoreactivity was visualized using the appropriate secondary antibodies foreach primary antibody with Alexa Fluor®488 goat antirabbit (1:500, Invitrogen, USA) and Alexa Fluor®594 goatTran et al. Journal of Neuroinflammation(2024) 21:142

anti-mouse (1:500, Invitrogen, USA). Sections were coverslipped using an aqueous mounting media (code S3023;
Dako, California, USA), viewed and images obtained witha Nikon C1 Digital Eclipse Modular Confocal Microscope System (Nikon Instruments Inc. Japan) at 20x magnification for analyses.
Immunohistochemistry quantification

All analyses were undertaken by investigators blinded
to the treatment groups. Regions of interest included
the cortical grey matter (cortex), subcortical white matter (SCWM), periventricular white matter (PVWM) andcaudate. Within the cortex, 2 non-overlapping fields ofview (FOV; 0.04 mm2) were placed within each of the 6
gyri (see Supplementary Fig. 1) (except for MAP2, see
below) and statistical analysis (one-way ANOVA) was
undertaken within each group to assess whether there
was an effect of gyri. No significant effect of gyri wasfound for any of the parameters and thus data for all gyri
were averaged for each animal. For SCWM, 4 FOVs wereplaced within each of the first 4 gyri medial to the baseof the gyri. A significant effect of gyri was also not found
for the SCWM and thus data for gyri were averaged foreach animal. For the PVWM and caudate, 4 FOVs in eachregion were used for analysis.
NeuN and vascular Ki67 were quantified using immunopositive cell densities. For NeuN, manual cell counts
Table 1 Genes of interest
Biological Process
Dopamine-related

Vasculature-related

Oxidative stressHousekeeping genes

Gene Name
Dopamine receptor D1
Dopamine receptor D2
Monoamineoxidase A
Monoamineoxidase B
Dopaminetransporter
Angiopoietin 1
Angiopoietin 2
Claudin-1
Occludin
Vascular endothelial growth
factor A
Myeloperoxidase
NADPH oxidase 1
Superoxide
dismutase 2,
extracellular
Nitric oxide
synthase 3
Ribosomal Protein L32

ID
DRD1

Taqman Code
Bt03223051_s1

DRD2

Oa04895884_m1

MAOA

Oa04709308_m1

MAOB
SLC6A3

Oa03251801_
mH
Oa04675060_m1

ANGPT1ANGPT2
CLDN1
OCLN
VEGFA

Oa04876148_m1
Oa04857533_m1
Oa03217991_m1
Oa04728972_m1
Oa04653812_m1

MPO
NOX1
SOD3

Oa04654413_g1
Oa04709255_g1
Oa04858164_m1

NOS3

Oa04907031_
MH
Oa04893129_g1

RPL32

Page 4 of 16

were conducted in FOVs placed within cortical layers IV
and V. Pyknotic cells were excluded from neuronal cellcounts. For Ki67, the vascular immunopositive cells was
expressed as the number of positive cells divided by theperimeter of vessels identified within FOVs.
For MAP2, GFAP and laminin, area coverage within
FOVs was quantified using ImageJ software (FIJI,
National Institutes of Health, USA), where a set threshold
was optimised for detection of positive labelling in FOVs
for each antibody respectively. For MAP2 analysis, area
coverage was quantified within cortical layers IV, V andVI, in order to capture both basal and apical dendrites,
using 4 FOVs in each of the 6 gyri. No differences were
found across gyri and cortical layers within each group;
thus the area coverage for each of the 24 FOVs were averaged. For GFAP and laminin analysis, laminin-positive
area coverage was analysed by viewing only the green
fluorescence channel as a measure of vascular density;and GFAP-positive area coverage was analysed by viewing only the red fluorescence channel to assess astrogliosis. For quantitative assessments of area coverage within
vessels (MMP9, GLUT1) in each FOV, blood vessel walls
were traced in which area coverage analyses were applied
as described above and expressed as percentage of thevessel area.
Double label analysis of GFAP-positive immunolabelling and laminin-positive blood vessel walls were measured to quantify astrocyte attachment to blood vessels
and; desmin-positive and αSMA-positive blood vessel
walls were measured to assess vascular pericyte density.
Both analysis were expressed as a percentage of colocalization within blood vessels at x20 magnification, as previously described [18].
Analysis of sheep serum was conducted as previously
described [19]. Briefly, blood vessels with sheep serum
extravasation were manually identified across the total
cross-sectional area of each region of interest and examined for positive immunoreactivity in the surrounding parenchyma. The number and proportion of vessels
exhibiting serum extravasation for each brain region wereaveraged across the lambs in each experimental group.
To assess vascular morphology, vessel perimeter was
analysed by measuring the external circumference of
only cross-sectional vessels as identified using lamininpositive immunolabelling. A minimum of ~ 10 vessels per
FOV was measured, and the average perimeter per vessel
per brain region per animal was calculated.
RT-qPCR

Gene expression for 14 genes relating to vasculature
integrity, dopamine and oxidative stress were assessed(Table 1). RNA extraction, cDNA preparation and analysis was conducted as described previously [19]. Briefly,
RNA was extracted from frozen brain tissue (60–80 mg)

Tran et al. Journal of Neuroinflammation(2024) 21:142

Page 5 of 16

using an RNA extraction kit (RNeasy Midi Kit, Qiagen,Germany). RNA yield was determined by spectrophotometry (Nanodrop, Analytical Technologies, Biolab)
then transcribed to cDNA, and pre-amplified to 50 ng/µl (SuperScript® III First-Strand Synthesis System for
RT-PCR kit; Invitrogen) with a mixture of pooled 20X
TaqMan gene expression assay probes and 2X Taqman
PreAmp Master Mix (Life Technologies, ThermoFisher,
USA). Gene expression was analysed using a Fluidigm
Dynamic array Biomark HD system (Fluidigm, USA).
Genes were determined by relative expression calculated
by change in cycle threshold (ΔCt) between each geneof interest and endogenous housekeeping gene RPS32.
Levels of mRNA expression relative to geometrical average of house-keeping genes were determined using the
2−ΔΔCT method [20] and expressed relative to the CTL
group. Due to insufficient sampling, mRNA analysis of
LPS lambs in the caudate could not be conducted.
Statistical analysis

All statistical analyses were conducted using GraphPad
Prism (version 10.2.1; GraphPad Software, CA, UnitedStates). Data were assessed for normality using the Shapiro-Wilk Test. Physiological parameters of animals at
the start and end of experiment were compared using
paired t-tests. For animal characteristics, RT-qPCR (greyand white matter) and immunohistochemical analyses, data sets were assessed for the main effects of LPS(PLPS), dopamine (PDA) and interactions between LPS
and dopamine (PINT) by two-way ANOVA. Where a significant interaction was observed, post-hoc analysis was

performed using Tukey’s multiple comparison test. For
statistical analysis of RT-qPCR data in the caudate, a oneway ANOVA was conducted and significant F values were
followed up with Tukey’s multiple comparison test. All
mRNA expression from RT-qPCR analysis are expressed
as relative change (2−ΔΔCT) and log10-transformed tomaintain normal distribution for statistical analysis. Data
are presented as mean ± SD and an alpha of 0.05 was considered statistical significance for all analyses.

Results
Lamb characteristics

Lamb characteristics (126–127 dGA) of the four groups
are presented in Table 2. The lambs in all 4 groups wereof similar weight. The male to female ratios were balanced but due to the small numbers for each group, sexdifferences could not be assessed between groups.
Arterial blood gases, pH, and other metabolites wereassessed at the beginning of experiment following delivery and resuscitation of the lamb, and again at the end ofexperiment after ~ 3–4 h of gentle ventilation (Table 2).
At the beginning of the experiment, there were no differences in pH, PaCO2, PaO2, HCO3−, and base excess
between groups. pH, PaCO2, and PaO2, remained atsimilar levels by the end of experiment and remained
similar across groups. At the end of experiment, lambs
which received dopamine had increased HCO3− andbase excess, with the increase in HCO3− greatest in
LPS-exposed lambs (LPS vs. LPSDA: P = 0.008). Moreover, in CTL, CTLDA and LPS lambs, HCO3− concentrations had decreased by the end of experiment compared

Table 2 Lamb characteristics and blood gas measurements at the beginning (after delivery and stabilisation with ventilation) and endof experimentGroupsn
Gender (male/total)
Weight (kg)Parameter
pH
PaCO2 (mmHg)
PaO2 (mmHg)
HCO3− (mmol.L− 1)
Base excess (mmol.L− 1)
Haemoglobin (g.dL− 1)
Parameter
pH
PaCO2 (mmHg)
PaO2 (mmHg)
HCO3− (mmol.L− 1)
Base excess (mmol.L− 1)
Haemoglobin (g.dL− 1)

CTL
CTLDA
9
7
4/9
3/7
3.16 ± 0.31
3.01 ± 0.46
Beginning of Experiment7.30 ± 0.11
7.31 ± 0.12
51.89 ± 13.51
55.26 ± 13.80
96.58 ± 90.63
98.20 ± 38.60
25.16 ± 3.75
26.70 ± 3.00
-1.11 ± 3.68
-0.29 ± 3.99
11.76 ± 1.79
13.33 ± 1.92
End of Experiment7.34 ± 0.12
7.37 ± 0.10
43.58 ± 12.82
42.60 ± 9.04
59.74 ± 20.89
82.00 ± 31.27
21.19 ± 5.11^
23.69 ± 2.25^^-4.80 ± 6.29
-1.10 ± 3.57
11.73 ± 2.19
13.49 ± 1.71

LPS
6
3/6
3.44 ± 0.39

LPSDA
6
3/6
2.96 ± 0.45

2-WAY ANOVA
PLPS
PDA

PINT

0.445

0.057

0.296

7.32 ± 0.04
47.67 ± 8.09
88.67 ± 46.68
24.20 ± 1.29
-2.13 ± 2.06
9.62 ± 1.41

7.28 ± 0.09
61.10 ± 11.36
123.52 ± 30.64
27.14 ± 1.16
0.23 ± 2.89
12.17 ± 1.74

0.860
0.865
0.722
0.822
0.849
0.030*

0.826
0.088
0.459
0.062
0.232
0.008**

0.506
0.296
0.499
0.545
0.560
0.498

7.28 ± 0.08
40.83 ± 8.47
97.67 ± 19.10
18.32 ± 4.34
-7.43 ± 5.20
8.90 ± 1.73

7.31 ± 0.18
57.28 ± 27.70
88.30 ± 33.40
25.90 ± 1.36##-0.73 ± 4.22
12.80 ± 1.60#

0.214
0.344
0.052
0.821
0.566
0.034*

0.501
0.223
0.554
0.002**
0.014*
0.002**

0.987
0.172
0.155
0.089
0.449
0.182

Data expressed as mean ± SD and analysed by 2-Way ANOVA; post hoc tests followed up with Tukey’s multiple comparisons test. Significant main effects are in bold
and indicated as P*<0.05, P**<0.01; significant post-hoc comparison between LPS vs. LPSDA are indicated as P#<0.05, P#<0.01. Blood gas measurements comparisons
between beginning and end of experiment in each group were analysed using paired t-tests and paired significant differences are indicated as P^<0.05, P^^<0.01Tran et al. Journal of Neuroinflammation(2024) 21:142

to the beginning of the experiment (P = 0.015, P = 0.008,
P = 0.053 respectively). Haemoglobin concentrations weresimilar between the start and end of experiment withingroups, but there was a difference between groups withLPS exposure lowering haemoglobin concentrations,whereas lambs allocated to the dopamine treatment had
increased haemoglobin concentrations irrespective of thetime of sampling (Table 2).
Data expressed as mean ± SD and analysed by 2-Way
ANOVA; post hoc tests followed up with Tukey’s multiple comparisons test. Significant main effects are inbold and indicated as P*<0.05, P**<0.01; significant posthoc comparison between LPS vs. LPSDA are indicated as
P#<0.05, P#<0.01. Blood gas measurements comparisons
between beginning and end of experiment in each groupwere analysed using paired t-tests and paired significant
differences are indicated as P^<0.05, P^^<0.01.
Vascular leakage and vascular remodeling are increased by
LPS-exposure

Brain regions were assessed for vascular leakage as indicated by vessels surrounded by a halo of extravasatedsheep serum (Fig. 1A&C). In the SCWM, PVWM andcaudate regions, LPS-exposure increased the number ofvessels with serum extravasation, with significant posthoc differences between the groups not administereddopamine within the PVWM and Caudate (P = 0.019 and0.017 respectively; Fig. 1A&C). There was no effect of
LPS or dopamine on vascular leakage in the cortex.

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Intra-amniotic LPS administration alone increased
basement membrane breakdown and vascular remodeling within the SCWM, as indicated by the increase inarea coverage of MMP9 immunolabelling in the control
LPS lambs (P = 0.014; Fig. 1B&D). This effect was notobserved in the dopamine-exposed lambs.
Vasculature, astrocytic morphology and vessel coverage
are altered by LPS-exposure and dopamine administration

Assessment of the extracellular matrix protein laminin that lines endothelial cells was used as a surrogate measurement for vascular density and vessel size
(Fig. 2A&B). LPS-exposure increased laminin expression,and therefore the calculation of vascular density, within
the PVWM and caudate (Fig. 2A). In contrast, dopamine administration reduced the vascular density in the
SCWM (Fig. 2A), and also decreased the overall vessel
size within the SCWM (Fig. 2B).
Dopamine increased astrocyte coverage in the cortex,
with the greatest effect occurring in the lambs that had
been previously exposed to LPS (P = 0.021; Fig. 2C). No
other effects on astrocyte coverage were observed.
Astrocytic “end feet” coverage on vessels was assessedby colocalization of GFAP- and laminin-immunoreactivity (Fig. 2D&E). LPS-exposure alone resulted inincreased astrocytic vessel coverage in the SCWM and
PVWM (P = 0.041 and P = 0.030 respectively). In contrast,
dopamine in LPS-exposed lambs decreased astrocyte

Fig. 1 Sheep serum and MMP9 positive immunohistochemistry(A) Quantification of vessels with sheep serum immunopositive leakage and (B) quantification of MMP9 area coverage within vessels. Data are mean ± SD.
Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.005. (C) Representative images of sheep serum extravasation from blood vessels into the brain parenchyma(black arrowhead), and sheep serum contained within the blood vessels (white arrowhead) in subcortical white matter (SCWM), periventricular whitematter (PVWM) and caudate; and (D) representative images of MMP9 positive immunohistochemistry in the subcortical white matter (SCWM), periventricular white matter (PVWM). Scale bar represents 100 μm

Tran et al. Journal of Neuroinflammation(2024) 21:142

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Fig. 2 Laminin and GFAP positive immunohistochemistry(A) Quantification of laminin positive area coverage and (B) average vessel perimeter size as assessed using laminin positive immunolabelling. (C) Quantification of GFAP positive area coverage and (D) quantification of percentage of colocalization of GFAP with laminin positive immunolabelling. Data aremean ± SD. Two-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.005. (E) Representative images of laminin (green) and GFAP (red) positive immunofluorescentlabellingand colocalization indicating astrocytic “end feet” covering of vessels in the cortical grey matter (cortex) and periventricular white matter (PVWM).
Scale bar represents 50 μm

Tran et al. Journal of Neuroinflammation(2024) 21:142

Page 8 of 16

vessel coverage in the SCWM (P = 0.0005; LPS vs. LPSDA;
Fig. 2D&E).

brain regions examined irrespective of LPS exposure (all
PDA<0.05; Fig. 3A&B).

Dopamine increases pericyte vascular coverage

Dopamine increases proliferative cells within vessel walls

Pericyte vascular coverage, considered a major factor in
regulating capillary blood flow, was assessed using colocalization of vascular desmin and αSMA. Dopamine
administration increased pericyte vascular coverage in all

Within the cortex, SCWM and caudate, dopamine
administration increased vascular Ki67-immunopositive cells (Fig. 4A&C). In the SCWM specifically, the
LPS lambs were most affected by dopamine-mediatedFig. 3 Desmin and αSMA positive immunohistochemistry(A) Quantification of percentage of colocalization of desmin with αSMA positive immunolabelling. Data are mean ± SD. Two-way ANOVA. (E) Representative images of desmin (green) and αSMA (red) positive immunofluorescent labellingand colocalization indicating pericyte coverage of vessels in thecortical grey periventricular white matter (PVWM). Scale bar represents 50 μm

Tran et al. Journal of Neuroinflammation(2024) 21:142

increase in vascular Ki67-immunopositive cells (P = 0.026;
Fig. 4A&C). In the PVWM of control lambs however,dopamine had an opposite effect and decreased Ki67immunopositive cells within vessels (P = 0.031; Fig. 4).
Dopamine decreases endothelial glucose transporter

Microvascular GLUT1 expression provides a marker for
BBB maturity and function [21]. Dopamine decreased
GLUT1 coverage in the SCWM in both control and
LPS lambs (PDA=0.013), and also in the cortex and caudate in the LPS groups (P = 0.002 and 0.025 respectively)(Fig. 4B&D). In the PVWM, LPS-exposure decreased
GLUT1 area coverage (Fig. 4B&D). Within the cortex,
LPS-exposure increased GLUT1 area coverage, but thisincrease was not observed in the LPS-treated lambs afteradministration of dopamine (P = 0.043).
Dopamine decreases coverage of neuronal dendrites

Mature neuronal population and neuronal dendritic
integrity within grey matter regions were assessed with
NeuN and MAP2 immunolabelling respectively (Fig. 5).
There was a significant interaction of LPS and DA on
NeuN immunopositive neurons in the cortex and caudate, although post-hoc analysis did not identify significant differences between groups (Fig. 5A&C). Dopamine
administration decreased neuronal dendrite coverage
within both the cortex and caudate (both PDA<0.05;
Fig. 5B&C). However, in the caudate specifically, thedecrease in neuronal dendrite coverage with dopamine
was greater in LPS-exposed lambs (P = 0.045; Fig. 5B&C).

Page 9 of 16

Effects of LPS and dopamine treatment on mRNA levelsof genes relating to vasculature integrity, dopamine
receptors/transporters, and markers of oxidative stress

Vascular integrity LPS exposure decreased angiopoietin ANGPT1 (activator) mRNA levels in the cortex andwhite matter but did not affect expression of ANGPT2(agonist/antagonist) (Fig. 6A&B). LPS exposure also
decreased expression of genes associated with formation of vascular tight junctions - CLDN1 and OCLN – inthe white matter of lambs not administered dopamine(P = 0.028 and < 0.0001 respectively; Fig. 6B). Dopamine
decreased VEGFA mRNA expression in the cortex in bothcontrol and LPS groups (P = 0.027 and 0.009 respectively;
Fig. 6A), and also in the control groups in the white matter(P = 0.012; Fig. 6B). Within the white matter, dopamineadministered lambs had decreased OCLN mRNA levels
compared to CTL lambs (CTL vs. CTLDA, P = 0.004 andCTL vs. LPSDA, P = 0.030; Fig. 6B). In the caudate, dopamine showed a trend for upregulating ANGPT2 mRNAlevels in CTLDA lambs compared to CTL lambs (p = 0.051;
Fig. 6C).
Dopamine signalling LPS exposure decreased dopamine receptors DRD1 and DRD2 mRNA levels in thecortex and white matter (Fig. 6A&B). In the white matter
specifically, the LPS-mediated decrease in DRD1 mRNAlevels noted above was significantly less following dopamine administration, with mRNA levels higher in LPSDA
lambs compared to LPS only (P = 0.020; Fig. 6B). In thecaudate, dopamine administration significantly increased
the DRD1 mRNA levels compared to CTL (P = 0.0002)

Fig. 4 Ki67 and GLUT1 positive immunohistochemistry(A) Quantification of Ki67 immunopositive cells within vessel walls and (B) quantification of GLUT1 area coverage within vessel walls. Data are mean ± SD.
Two-way ANOVA, *P < 0.05, **P < 0.01. (C) Representative images of Ki67 immunopositive cells (black arrowhead) within the blood vessels in the subcortical white matter (SCWM) and caudate, and (D) representative images of GLUT1 positive immunohistochemistry in the cortical grey matter (cortex) andperiventricular white matter (PVWM). Scale bar represents 100 μm

Tran et al. Journal of Neuroinflammation(2024) 21:142

Page 10 of 16

Fig. 5 NeuN and MAP2 positive immunohistochemistry(A) NeuN immunopositive cell density indicating mature neuronal population and (B) MAP2 immunopositive area coverage indicating coverage of neuronal dendrites. Data are mean ± SD. Two-way ANOVA, *P < 0.05. (C) Representative images of NeuN- and MAP2-positive cells in the cortical grey matter(cortex; Layer V) and caudate. Scale bar represents 50 μm

with a trend for increase also occurring in LPSDA lambs(P = 0.051; Fig. 6C). The mRNA levels of dopamine transporter SLC6A3 was decreased in dopamine administeredlambs compared to non-dopamine administered lambs inthe cortex (Fig. 6A). mRNA expressions of monoamineoxidases A and B, the key enzymes that metabolises dopamine, were unaffected by dopamine but were downregulated in LPS lambs compared to CTL lambs in the whitematter (P = 0.001 and 0.018 respectively; Fig. 6B).
Oxidative stress LPS decreased the mRNA levels of
NOX1 (Fig. 6A) in the cortex and white matter. In thewhite matter, LPS decreased SOD3 mRNA levels in lambs
not administered dopamine (P = 0.011; Fig. 6B). Dopa-

mine increased both NOX1 and SOD3 mRNA levels in
the caudate, with NOX1 expression also increased in the
LPSDA compared to CTL lambs (Fig. 6C).

Discussion
Intrauterine inflammation is a common precursor topreterm brain injury, and has often been associated with
impaired brain development and long-term neurodevelopmental abnormalities [22]. Infants exposed to infection and inflammation often suffer from cardiovascular
dysfunction including hypotension, and are therefore
treated with dopamine infusion to restore blood pressure. To our knowledge, this is the first study on the combined and separate impacts of intrauterine inflammation

Tran et al. Journal of Neuroinflammation(2024) 21:142

Page 11 of 16

Fig. 6 Fold change of mRNA levels
mRNA expression of genes relating to vasculature integrity, dopamine, and oxidative stress measured within the (A) cortex, (B) combined white matterand (C) caudate. mRNA expression expressed relative to CTL. Data are mean ± SD. Cortex and white matter data analysed using two-way ANOVA, Tukey’s
post-hoc analyses. Note mRNA levels in the caudate of LPS lambs were not conducted and data analysed using a one-way ANOVA, Tukey’s post-hoc
analyses. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Tran et al. Journal of Neuroinflammation(2024) 21:142

and dopamine infusion on the preterm NVU, including
histological assessments of the NVU, and the transcriptional changes of genes associated with the impact ofinflammation and dopamine. Overall, the effects of LPS
and dopamine on the NVU were either independent or
opposite in direction, and we found no compoundingor synergistic effects of LPS and dopamine. Exposure to
intrauterine inflammation was associated with increased
vascular leakage in the presence of increased vessel density, increased astrocytic “end-feet” vascular coverage,
basement membrane breakdown and vascular remodeling mainly within the white matter, with downregulated
gene expression for tight junctions also in the whitematter. On the other hand, dopamine administration
decreased vessel density and size, downregulated VEGF
gene expression, and increased pericyte vascular coverage, but it also decreased astrocytic “end-feet” vascular
coverage despite an overall higher astrocytic coverage.
In addition, dopamine decreased glucose transporterand barrier function with downregulation of the OCLN
tight junction gene, and decreased neuronal dendrite
coverage. As expected, dopamine upregulated genes for
dopamine receptors, and for markers of oxidative stressin the caudate. Taken together, this study demonstrates
that components of the preterm NVU are disrupted byintrauterine inflammation and are also sensitive to thepresence of increased levels of dopamine.
The effects of LPS exposure on the preterm NVU

LPS and vessels/BBB Disturbances of the NVU physiology and morphology have been identified as one of theleading causes of neurodevelopmental disorders [23].
We have previously demonstrated in preterm lambsthat exposure to intrauterine inflammation results in anegative cerebral functional haemodynamic response
following somatosensory stimulation [8]. This negative
haemodynamic response suggested impaired neurovascular coupling, possibly reflecting cellular changes at thelevel of the NVU. Our findings of an increase in cerebral
vascular leakage after LPS exposure associated with anincreased vessel density and astrocytic “end-feet” vascular
coverage suggests disrupted structure of the NVU, possibly leading to functional deficits. Studies in adult rats haveshown that disrupted endothelial cells in the pial arteries disturb the stimulus-evoked vasodilation associatedwith neurovascular coupling [24]. The increases in MMP9
expression associated with the LPS treatment is suggestive of vascular remodeling, and the decreased expressionof the tight junction proteins (CLDN1, OCLN) further
support vascular and BBB disruption in the white matter[25, 26]. Such inflammation-induced BBB permeability
and leakage has previously been associated with preterm
white matter brain injury in the sheep fetus [27–29].

Page 12 of 16

LPS and astrocytes Astrocytes are a key component of
the NVU, and they modulate the vasculature interface andthe BBB [30]. In our study, LPS exposure increased astrocytic end-feet vessel coverage in the white matter, but not
the overall astrocytic coverage. This result is in contrast
to a previous study conducted in fetal sheep at a younger
gestation (~ 104–108 dGA) where LPS was administeredas a chronic LPS infusion and boluses over 5 days, resulting in decreased astrocytic end-feet vessel coverage,
increased astrocytic coverage, and reduced microvascularvessel density [31]. The differences in astrocyte and vasculature morphology may reflect the study’s younger gestation, the chronic LPS regimen and/or the timing of brain
assessment. For example, the disruption to the NVU may
arise from both direct cytokine actions and secondary
cytokine production in the fetal brain by activated glial
cell such as astrocytes [32]. This cytokine induction differs
in cases of repeated and acute LPS challenges [33]. We
cannot ascertain whether the increased astrocytic endfeet coverage contributes to increased BBB leakage; orthe BBB breakdown and inflammation induces increased
astrocytic end-feet coverage. We speculate that, firstly, theincreased astrocytic end-feet coverage post-LPS exposure
is dysfunctional or ineffective, thus causing increased BBB
leakage [34]. Alternatively, the increased astrocytic endfeet coverage is a response to the BBB leakage following
LPS exposure [35]. Regardless, the increased astrocytic
end-feet vessel coverage highlights injury to the NVU andlikely its dysfunction.
LPS and dopamine genes We found that LPS exposure
downregulated gene expression for dopamine receptors D1 and D2, and monoamine oxidase A and B. Thisis the first study to identify a molecular modulation of
dopamine signaling pathways in the preterm brain due tointrauterine inflammation. Other studies have indicatedthe association of prenatal inflammation, developmental
perturbation of the dopaminergic system, and the eventual development of neuropsychiatric disorders suchas schizophrenia [36, 37]. Thus, the transcriptional dysregulation of the dopamine pathway provides a possible
mechanistic link between prenatal inflammatory eventsand neurodevelopmental disorders.
The effects of dopamine administration on the preterm
NVU

DA and vessels/BBB Dopamine infusion itself resulted
in morphological changes mainly to the cerebral microvasculature. Firstly, dopamine decreased vessel density
and size in the white matter suggesting a cerebrovascular vasoconstrictive response, as previously observed inhemodynamically stable unanesthetized preterm fetal
sheep treated with dopamine [38]. It is important to notethat our lambs were not hypotensive prior to dopamine

Tran et al. Journal of Neuroinflammation(2024) 21:142

treatment. Therefore, the dopamine-induced vasoconstriction could reflect an autoregulatory response to theincrease in systemic blood pressure [14], and/or the dominant activation of α-adrenergic receptors in the immature
cerebral vasculature [38].
Secondly, pericyte vascular coverage was increased following dopamine exposure in both control and LPS
lambs. Pericytes promote vascular structural stability
and protect the brain from blood-borne elements andbleeding. Notably, dopamine has a putative role in pericyte-vascular-neuronal function [39, 40]. Endogenous
dopamine mediates pericyte relaxation [40, 41]; andexogenous dopamine promotes mature pericyte vascular coverage via the D2 receptors [42]. However, we also
found that exogenous dopamine attenuated GLUT1
expression and downregulated OCLN tight junction
gene expression, suggesting disruption of the BBB and its
function. Of note, the reduced GLUT1 coverage within
vessel walls may be secondary to the decreased vessel
size and reflect vasoconstriction following the dopamine
infusion. In a similar respect, the decreased vessel size
may also explain the increase in Ki67+ cell density per
vessel perimeter of dopamine infused lambs, because
the smaller vessel perimeter results in a higher Ki67+ celldensity. Studies have also shown that exogenous dopamine crosses the preterm BBB, although it remains
unclear if the increased BBB permeability was due to prematurity or induced by dopamine [43, 44]. The actions ofexogenous dopamine on the preterm BBB and vasculature require further investigations.
DA and astrocytes Dopamine infusion was associatedwith astrogliosis in the cortex as indicated by the increased
overall coverage of GFAP immuno-positive labelling,whereas there was attenuated astrocytic vessel coveragein the white matter. The increased cortical GFAP coverage
may be due to changes in astrocyte morphology as dopamine exposure to cultured astrocytes can increase thegrowth of astrocytic processes growth (> 200% increase),although this does not necessarily mean an increased
interaction with the vasculature [45]. Indeed, while LPS
increased astrocytic vessel coverage in the white matter,
addition of dopamine reversed this change (Fig. 2D) which
may indicate a retraction of the astrocytic processes on
the vasculature. Interestingly, there was no exacerbationof vascular leakage in the LPSDA lambs which may be a
result of the overall cerebrovascular constriction, or theincreased pericyte coverage caused by dopamine treatment. In addition, the VEGFA mRNA expression in bothgrey and white matter was downregulated after dopamine exposure, which could affect the vascular–astrocyte
interactions as VEGF is closely associated with astrocyte
proliferation [46], and astrocyte-derived VEGF promotes
vascular growth and development [47].

Page 13 of 16

DA and neuronal dendrites Dopamine decreased neuronal dendritic coverage within grey matter regions,consistent with in vitro observations of primary cortical neuronal cultures where dopamine receptor activation decreased dendritic extension via increased MAP2
phosphorylation [48]. Disturbance to neuronal dendritic
development and increased astrogliosis with dopamine
infusion may have compounded effects on the alreadyvulnerable neuronal network maturation that is known tobe present in preterm infants [49].
DA and dopamine genes In the caudate, dopamine
administration upregulated the DRD1 gene which
encodes for the dopamine receptor D1, and also upregulated NOX1 and SOD3 genes which are involved in mediating oxidative stress. Major dopaminergic pathways withhigh densities of dopaminergic receptors are found in thebasal ganglia [50]. Our findings suggest that increased
extracellular dopamine and its degradation may lead toelevated levels of oxidative metabolites [51], given thatNOX catalyses the production of reactive oxygen species
and that SOD3 in an antioxidant enzyme. Future studies
are needed to fully elucidate the long-term effects on the
dopaminergic systems at cellular and functional levels in
the preterm brain after exposure to exogenous dopamine.
Clinical implications

This study has revealed an important interaction between
intrauterine inflammation and postnatal exposure to
dopamine, with significant and regional effects on neurovascular structure in the preterm lamb. The NVU
maintains brain homeostasis by facilitating neurovascular coupling and the trafficking of blood-borne molecules and cells into and out of the brain. Alterations
to the NVU due to intrauterine inflammation has long
been thought to be the basis of long-term neurological disabilities [52, 53], including neuropsychologicaldisorders such as autism [54] and schizophrenia [55] inwhich NVU impairments have been identified [56, 57].
BBB breakdown may also be an early biomarker for later
cognitive dysfunction [58]. Importantly, we showed thata treatment commonly used to support the hypotensive
preterm infant - intravenous dopamine - is likely to haveadditional effects on the NVU already compromised byexposure to inflammation. Future research should aim tounderstand the impact and interactions of other medical interventions on the preterm NVU after intrauterine
inflammation, such as, for example, antenatal and postnatal steroid treatments, given that the glucocorticoid
receptor is important for the stabilization of vascularendothelial cells and proliferation of neural stem/progenitor cells [59]. Protection of the NVU could therefore bea potential therapeutic target to reduce long-term neurodevelopmental deficits in preterm infants.

Tran et al. Journal of Neuroinflammation(2024) 21:142

Strengths and limitations

Sheep have been used extensively to study the preterm
cerebral circulation and neuropathies [60]. The pretermlamb at 126–127 dGA (0.8 gestation) displays oligodendrocyte maturation similar to a late preterm human babyof ~ 35 weeks, and also cerebral vascular pathophysiology and immature neurovascular coupling comparable toa preterm human baby [61, 62], and is thus an excellent
model for the study of clinical conditions and therapies
to assess their effects in the preterm brain. Our model
simulated clinical management for preterm delivery following intrauterine inflammation. However, the pretermlambs in this study were normotensive rather than hypotensive, and received dopamine infusion for a relatively
brief duration of 90 min [8, 14], which may have limited
our findings of the impact of dopamine. Also, we wereable to characterized the NVU at only a single timepoint
at 3–4 h after birth. Assessment at later time-points ina maturing brain would delineate the further developmental impact on the NVU. We assessed transcriptionalchanges within whole brain regions which contain a mixture of cellular populations; further insights using single
cell transcriptomics would confirm the exact cell populations with the major transcriptional changes found in thisstudy. Importantly, we demonstrate that both intrauterine inflammation and postnatal dopamine exposure independently disrupt the NVU which is a critical component
in the pathophysiology of preterm brain injury.

Conclusion
We report the cellular and molecular impact of intrauterine inflammation and dopamine, together and separately, on the preterm NVU. LPS exposure predominately
resulted in vascular leakage in the presence of increased
vessel density and vascular remodelling; while dopamine
infusion resulted in decreased vascular density and sizewith increased pericyte coverage, but decreased astrocytic vascular coverage and integrity of the blood-brain
barrier. Notably, we did not find any synergistic effects of
LPS and dopamine, and we speculate that they may contribute independently to neurovascular disruption which
could predispose the brain to further developmental
impairments.
Abbreviations%	percent
α-SMA	alpha-smooth muscle actinµm	micrometerµg	microgram
ANOVA	analysis of variance
BBB	blood-brain barrier
CAT#	catalogue number
CBF	cerebral blood flow
CLDN1	claudin 1
CTL	controlDA	dopamine
DAT	dopamine transporter

Page 14 of 16

dGA	days of gestation
dH2O	deionized waterdL	decilitre
DRD1	Dopamine receptor D1
FOV	fields of viewg	grams
GFAP	Glial fibrillary acidic protein
GLUT1	glucose transporter 1GM	grey matterh	hour
HCO3−	BicarbonateHR	heart rate
IBA-1	ionized calcium binding adaptor molecule-1
IgG	Immunoglobulin Gkg	kilogramL	litre
LPS	lipopolysaccharideM	moles
MABP	mean arterial blood pressure
MAP2	microtubule-associated protein 2mg	milligrammin	minuteml	millilitremm	millimetremM	millimole
mmHg	millimetres of mercury
MMP9	matrix metallopeptidase 9mRNA	messenger ribonucleic acidn	number of
NeuN	neuronal nuclei
NVU	neurovascular unit
OCLN	occludin
PaCO2	partial pressure of arterial carbon dioxide
PaO2	partial pressure of arterial oxygen
PVWM	periventricular white matterRNA	Ribonucleic acid
RT-qPCR	Reverse transcription-quantitative polymerase chain reaction
SCWM	subcortical white matterSD	standard deviation
SpO2	peripheral oxygen saturation
VEGFA	Vascular Endothelial Growth Factor A
WM	white matter

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s12974-024-03137-0.
Supplementary Fig. 1. Schematic indicating fields sampled for histological assessmentField of view (FOV) indicated in red boxes were sampled
for assessment of cortical grey matter (cortex; yellow), subcortical (SCWM;
green) white matter regions within the first, second, third and fourthparasagittal gyri and FOVs also assessed in the periventricular white matter(PVWM; pink) and caudate (blue).
Acknowledgements
The authors gratefully acknowledge the technical assistance of Dr. Ilias Nitsos,
Mr. Dalibor Stanojkovic and the Monash Health Translation Precinct Histology
Platform.
Author contributions
NTT, NH, DWW, GP and FYW conceptualised and designed the study. NTT,
NH, MW, GP and FYW undertook the experiments and were involved in dataacquisition. NTT, NH and AAWM undertook the immunohistochemistry, cell
quantification, analysis and preparation of figures. MCM and FYM provided
overall oversight of the research. NTT, NH, FYW contributed to drafting andrevising the manuscript. All authors critically reviewed the manuscript andapproved the final manuscript as submitted and agree to be accountable for
all aspects of the work.

Tran et al. Journal of Neuroinflammation(2024) 21:142

Funding
This study was supported by the Victorian Government’s Operational
Infrastructure Support Program. F.Y.Wong has been supported by NHMRC
Career Development Fellowships (1159120), the Victor Yu Clinical Research
Fellowship and the RACP Career Development Fellowship.
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).

Page 15 of 16

9.
10.
11.
12.

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14.

15.

Consent for publication
Not applicable.

16.

Competing interests
The authors declare no competing interests.

17.

Author details
1
The Ritchie Centre, The Hudson Institute of Medical Research,
Melbourne, Australia
2
Department of Obstetrics and Gynaecology, Monash University,
Melbourne, Australia
3
Department of Paediatrics, Monash University, Melbourne, Australia
4
Monash Newborn, Monash Medical Centre, Melbourne, Australia
5
Monash Children’s Hospital, Level 5, 246 Clayton Rd, Clayton, VIC
3168, Australia
Received: 4 April 2024 / Accepted: 22 May 2024

18.
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