Background
Prematurity is associated with an elevated risk of suffering from brain injuries, and these brain injuries are associated with impaired quality of life due to disorders such as cerebral palsy and behavioral, social, attentional, and cognitive deficits. The most common form of brain injury in preterm infants is generally thought to consist primarily of periventricular leukomalacia (PVL), a specific form of cerebral white-matter injury that is often accompanied by elements of gray-matter injury [
1]-[
3]. PVL can be either cystic necrotic or non-cystic. The cystic necrotic form is associated with the loss of all cellular elements, but the non-cystic lesions are more diffuse and cell-specific. The diffuse non-cystic lesions are the most common form of injury seen in preterm infants at present, and these primarily affect premyelinating oligodendrocytes (preOLs) leading to either loss of these cells or their inability to differentiate into mature oligodendrocytes, which results in cerebral hypomyelination [
4]-[
6].
Hypotensive episodes and hypoxic events, inflammation and infection [
7]-[
9], and tissue hypoxia arising from inflammation/infection are the major initiating factors of preterm brain injury. The ischemia-reperfusion injury process has been confirmed by the presence of infarctions in the arterial end and border zones of the periventricular white matter [
10],[
11] and by the observation of pressure-passive circulation without autoregulatory function in newborn infants [
12]. The most commonly used method for studying hypoxia-ischemia (HI)-induced brain damage in the neonatal brain is a rodent HI model referred to as the Vannucci model [
13]. This model is very well characterized for mice and rats between postnatal day (PND) 7 and PND 10 [
14]-[
21], and adaptations in younger rodent pups including PND 3 to 6 rats [
22] and PND 6 to 7 mice [
23],[
24] have also been developed. The periventricular white matter in humans is highly susceptible to HI at gestational weeks 24 to 32 due to its high proportion of preOLs, and it is known that the number of preOLs is highest at PND 2 to 5 in rodents and that this number quickly declines after this developmental stage [
25]. By PND 6-7, extensive oligodendrocyte (OL) maturation occurs in rodents and this coincides with the onset of early myelination [
26]. Thus, white-matter vulnerability in the rodent at PND 2 to 5 would correspond to that in preterm human infants.
HI-induced injury to the immature brain has been found to induce the expression of various genes related to immune responses and inflammation, including macrophage and microglia-related genes, T-lymphocyte-related genes, and cytokines [
27]. In addition, inflammatory mediators have been suggested to contribute to injury after HI in the immature brain [
28] at a stage corresponding to human term and near-term infants. The immune system develops rapidly immediately after birth. For example, mice are not able to produce detectable levels of humoral antibodies in response to antigen until after one week of age, and the antigen-presenting dendritic cells do not reach normal levels as it is in adult rodent until one week after birth [
29]. This indicates that the inflammatory response is likely to be very different during the first days after birth compared to one week later, and the differences are even more pronounced compared to older rodents [
29]. However, the inflammatory and immune responses after HI brain injury in mice younger than PND 7 have not been characterized. The first aim of this study was to use the Vannucci model to find the duration of hypoxia that causes focal white/gray-matter injury in PND 5 mice, a developmental age in mice that corresponds to the preterm human infant. The second aim was to characterize the immune/inflammatory response after HI in this preterm injury model.
Methods
Animals
Pregnant C57BL/6 J mice were purchased from Charles River Laboratories (Sulzfeld, Germany) and these gave birth in the animal facility at the University of Gothenburg (Experimental Biomedicine, University of Gothenburg). The day of birth was defined as PND 1. Mice were housed with a 12-hour light/dark cycle, and free access to a standard laboratory chow diet (B&K, Solna, Sweden) and drinking water was provided. All animal experiments were approved by the Animal Ethical Committee of Gothenburg (Number 51/2012, 5/2013).
Hypoxia-ischemia procedure
At PND 5, mice of both sexes were subjected to HI insult according to a method described previously with some modifications [
13]. Briefly, mice were anesthetized with isoflurane (5.0% for induction and 1.5 to 3.0% for maintenance) in a 1:1 mixture of nitrous oxide and oxygen. The left common carotid artery was ligated, and the mice were returned to their cage and allowed to recover for one hour. The mice were then placed in an incubator perfused with a humidified gas mixture (10 ± 0.01% oxygen in nitrogen) at 36°C for 50 minutes, 70 minutes, or 80 minutes. After HI, the pups were returned to their dam until being killed. The combination of artery ligation and hypoxia resulted in injury only in the hemisphere ipsilateral to the artery ligation (the left hemisphere), while no injury was produced in the contralateral hemisphere (the right hemisphere). PND 9 mice were subjected to hypoxia for 50 minutes to produce a comparable degree of injury in the hippocampus as seen after 70 minutes HI in the PND 5 mice [
30].
Assessment of brain damage
Mice were deeply anesthetized and perfused intracardially with saline and 5% buffered formaldehyde (Histofix; Histolab Products AB, Gothenburg, Sweden). The brains were dissected, paraffin-embedded, and cut into 10- μm coronal sections throughout the whole brain. Immunohistochemical staining for microtubule-associated protein 2 (MAP-2) and myelin basic protein (MBP) was performed on every 50th section.
The extent of white- and gray-matter injury was analyzed by quantitative measurements of the injury area (at 4 × magnification) using Micro Image version 4.0 (Micro-macro AB, Gothenburg, Sweden). The MAP-2-positive or MBP-positive area (remaining tissue) was measured in each hemisphere as previously described [
31],[
32], and total tissue loss was calculated as:
(1)
Coronal sections at the hippocampal level were used for all other staining. Thionin/acid fuchsin staining was performed as described previously [
33]. Briefly, the sections were dipped in 1% thionin/toluidin solution, dipped into acid fuchsin solution, and dehydrated in a gradient of ethanol before mounting.
Immunohistochemical staining
After antigen recovery and blocking, the following primary antibodies were used: mouse anti-MAP-2 (clone HM-2, Sigma-Aldrich, Stockholm, Sweden), mouse anti-MBP (SMI94, Covance, NJ, USA), goat anti-triggering receptor expressed on myeloid cells-2 (TREM-2, clone G-18, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-ionized calcium binding-adapter molecule 1 (Iba-1, Wako Chemicals, Richmond, VA, USA), and mouse anti-cluster of differentiation 4 (CD4, Vector Laboratories, Burlingame, CA, USA). After incubation with the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA, USA), visualization was performed using Vectastain ABC Elite with 3,3’-diaminobenzidine (DAB) enhanced with ammonium nickel sulfate, beta-D glucose, ammonium chloride, and beta-glucose oxidase (all from Sigma-Aldrich, Stockholm, Sweden).
For fluorescent triple staining, the sections were incubated with goat anti-TREM-2, mouse anti-DNAX activating protein 12 antibody (DAP12, clone B-2, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-Iba-1. The sections were then incubated with the appropriate Alexa Fluor 594 donkey-anti-rabbit or Alexa Fluor 488 donkey-anti-mouse (Molecular Probes, Leiden, Netherlands) secondary antibody in PBS, and a coverslip was mounted using ProLong Gold antifade reagent with 4,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Carlsbad, CA, USA).
RT-PCR
Changes in cytokine gene expression in the brain at 6 hours, 24 hours, or 7 days after HI in PND 5 and control mice were analyzed by RT-PCR. After a brief intracardial perfusion with saline, the brains were rapidly dissected, divided into ipsilateral and contralateral hemispheres, snap frozen, and stored at -80°C until use.
The brain tissue was homogenized with Qiazol lysis reagent homogenizer (Qiagen, Sollentuna, Sweden), and total RNA was extracted using the RNeasy Lipid Tissue Mini Kit (Qiagen, Sollentuna, Sweden). RNA was measured with a spectrophotometer at 260 nm absorbance. cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen, Sollentuna, Sweden) following the manufacturer's instructions, and each sample was run in duplicate. The following primers were used: RAR-related orphan receptor gamma (RORγt, QT00197722), T-bet (QT00129822), GATA binding protein 3 (GATA3, QT00170828), forkhead box P3 (Foxp3, QT00138369), IL-4 (QT00160678), IL-5 (QT00099715), IL-6 (QT00098875), IL-10 (QT00106169), IL-12a (p35) (QT01048334), IL-13 (QT02423449), IL-17α (QT00103278), IL-21 (QT01758036), IL-22 (QT00128324), IL-23a (QT01663613), transforming growth factor beta (TGF-β, QT01038821), IFN-γ (QT00145250), B- and T-lymphocyte attenuator (BTLA, QT00117635), CXCR5 (QT00253449), CXCL13 (QT00107919), inducible T-cell co-stimulator (ICOS, QT02253552), and 18S ribosomal RNA (QT01036875) (all from Qiagen, Sollentuna, Sweden). Melting curve analysis was performed to ensure that only one PCR product had been produced. A standard curve was generated for quantification and for estimating amplification efficiency using increasing concentrations of cDNA, and the amplification transcripts were quantified with the relative standard curve and normalized to the 18S ribosomal RNA reference gene.
Statistics
The Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL, USA) v19.0 software package was used for all analyses. Comparisons between groups were performed by Student's t-test, and data with unequal variance were compared with the Mann-Whitney U-test. Analysis of variance followed by the LSD post-hoc test was used for comparison of data from more than two groups. P < 0.05 was considered statistically significant.
Discussion
We have established a novel PND 5 mouse preterm brain injury model to show that both the innate and adaptive immune responses are activated after HI at this age. This model has allowed us for the first time to examine the responses of different subsets of Th cells in a neonatal HI model, and we found that HI induced a primarily Th1/Th17-type immune response in the neonatal mice.
Fifty minutes of HI produced mild injury localized in the white and gray matter, and this degree of injury is similar to that previously reported in PND 6 HI mice [
23]. However, only 25% of the mice were damaged and most of the pups did not display any signs of injury. Furthermore, no significant differences were seen in the white-matter volume between the ipsilateral and contralateral hemispheres, and this limits the usefulness of the 50-minute HI exposure in neuroprotection studies. When the HI duration was extended to 80 minutes, the injury in the ipsilateral hemisphere was very extensive and included widely distributed infarction of the hippocampus, cortex, and thalamus that is rarely seen in preterm infants.
The model using 70 minutes of HI produced diffuse white-matter injury combined with focal neuronal loss in the cerebral cortex and hippocampus injury (Table
1). Indeed, at 7 days after HI (PND 12), the time when myelination becomes detectable in mice, we found impaired myelination in the subcortical white matter of the ipsilateral hemisphere (Figure
4B and
4D). Seventy minutes of HI significantly reduced both white- and gray-matter volume in 93% of the pups (Figure
3A and B), and this makes the model reliable and suitable for neuroprotection studies. Interestingly, we found that the degree of white-matter vulnerability after 70 minutes of HI at PND 5 (Figure
3D and
3E) was more obvious compared to that seen at PND 9 in mice (Figure
3F and
3G). This observation further confirms that PND 5 is within the time window when the white matter in the brain is more vulnerable to injury [
35].
TREM-2 is expressed on the cell membrane of monocyte-derived dendritic cells, osteoclasts, and microglia and is associated with the signaling molecule DAP12 [
36],[
37]. DAP12 in turn plays a signal transduction role in dendritic cells and macrophages and is considered to be both pro- and anti-inflammatory [
38]. Apart from their function in the inflammatory response, DAP12 and TREM-2 clearly play an important role in white-matter disease and oligodendrocyte pathology. Mutations in TREM-2 and DAP12 cause sclerotic lesions in the white matter of the central nervous system (CNS) in human polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL)/Nasu-Hakola disease [
39],[
40] and cause hypomyelinosis in mice [
41]. DAP12
+ and TREM-2
+ microglia/macrophages surround myelinating oligodendrocytes at PND 10 [
42]. TREM-2 is expressed by microglia in the CNS both during normal development in mice [
43] and in experimental autoimmune encephalomyelitis (EAE) - a mouse model of multiple sclerosis (a white-matter disease) [
44],[
45]. Blockage of TREM-2 signaling using an anti-TREM-2 antibody increases the severity and progression of EAE [
44].
We found increased expression of TREM-2 and DAP12 in the brain after HI with maximum expression at 24 hours after HI in the injured areas of the hippocampus and the white matter. DAP12 was co-expressed with microglia in the brain parenchyma, and this agrees with previous findings in the normal neonatal mouse brain [
43] and in the rodent model of HI that is more representative of the type of brain injury seen in full-term infants [
27]. The expression of DAP12 is found not only in injured areas but also in the meninges. Together with the finding that TREM-2 is also expressed in the choroid plexus and along the blood vessels, this indicates that the TREM and DAP12 pathway might serve as a sensor/communicator at the interface between the periphery and the CNS and might participate in the development of white-matter injury in preterm infants.
The accumulation of TREM-2, DAP12, and CD4
+ T-cells in the meninges and choroid plexus and adherence to blood vessels might also have implications for the status of cerebrovascular reactivity. Similarly, another recent report showed that TREM-2 was expressed by some microglia/macrophages inside and around the blood vessels of the hippocampal fissure in PND 1 mouse brains, and such expression diminished with age [
43]. The vasculature of the blood-brain barrier (BBB) protects the CNS from the systemic circulation by restricting entry of unwanted molecules and immune cells into the brain. Reports have shown that TREM-2 knockout mice have reduced invasion of CD3
+ T-cells [
46] through yet unknown mechanisms. Together with our finding that TREM-2 is located close to the blood vessels in the choroid plexus, this indicates that TREM-2 might actively participate in the invasion of peripheral T-cells into the brain through the choroid plexus and BBB by either modulating the BBB permeability in the brain or directly regulating the blood flow.
T-cells have been observed infiltrating the brain in the infarction area in animal models of stroke, and it is now widely accepted that T-cells play a role in both ischemic brain injury (stroke) and white-matter injury (multiple sclerosis) in both adult animal models and humans [
47],[
48]. For example, T-cell-deficient mice exhibit significant reductions in infarct volume [
49],[
50] in rodent models of HI brain injury. T-cells and other peripheral immune cells were found to infiltrate into the neonatal rat brain and to remain there for hours to months following HI [
51]-[
53].
In this study, we found that there were low, yet significantly increased, numbers of CD4+ cells in the brain after HI at PND 5 and that this was accompanied by changes in spleen weight, one of the most important organs in the immune system. At the time points examined in the present study, which are all relatively early for the adaptive immune response, the CD4+ T-cells are mostly located adhered to the blood vessels in the brain instead of in the brain parenchyma. This might well indicate that CD4+ cells are beginning to infiltrate into the brain at a relatively early stage in the adaptive immune response in addition to other cell types that are also involved in the early immune response in the brain.
Mammalian immune responses are often grouped into type 1 and type 2 responses [
54]. These responses differ broadly in their mechanisms of induction as well as their innate cell types, cytokines, effector molecules, and Th types. The classical type 1 and type 2 responses are mediated by TCR αβ CD4
+ Th cells and are thus generally referred to as Th1 and Th2 responses, respectively. It is now clear that many innate cell types such as γδ T-cells and innate lymphoid cells (ILCs) broadly parallel the known CD4
+ Th cell subsets in terms of their signature cytokine secretion profiles. An overlapping series of transcription factors is also used to drive the differentiation of the various ILC and Th cell subsets. The results of our experiments show that HI leads to an imbalance between Th1/Th17-type responses and Th2/Treg-type responses, and these differences might be due to the recruitment and proliferation of different innate-type immune cells.
Type 1 immune responses are usually pro-inflammatory and are mediated by the type 1 cytokines such as IFN-γ, TNF-α, IL-6, and IL-17 [
55]. In contrast, type 2 responses have immune-modulating functions and mediate allergic inflammatory diseases such as asthma, allergic rhinitis, and atopic dermatitis. Th2 cells regulate type 2 responses through the secretion of various type 2 cytokines, including IL-4, IL-5, IL-9, and IL-13. For many years, neonates have been considered to be immune deficient. It is now clear, however, that the newborn is capable of raising an immune response. Neonatal mice that have primary immune responses to certain foreign antigens normally mount Th2-type-dominant secondary responses when re-exposed to the same antigens. However, under certain pathogenic infections or after immunization with certain adjuvants that promote strong Th1-cell responses, such as DNA vaccines or oligonucleotides containing CPG motifs
, neonates develop Th1-type responses that often induce the onset of diseases [
54],[
56],[
57]. In HI-induced brain injury in neonatal mice, we observed an increased expression of Th1/Th17-related transcription factors that may reflect a dominant Th1-type response that could contribute to the brain injury. Indeed, high expression of Th1 and Th17-type cytokines has been found in PVL patients [
58]-[
60], and inhibition of Th17-type cytokines reduces brain injury in a neonatal mouse model [
61]. Moreover, Th17 lymphocytes traffic to the CNS [
62] and participate in the pathogenesis of CNS inflammatory demyelination disease [
63]. These indicate that different types of immune responses might play different roles in preterm brain injury.
In this study, we examined different types of immune responses by examining the stimulatory cytokines that are present in the microenvironment during activation, transcription factors that prime naive precursor cells for differentiation, and the signature cytokine profiles for each type of response. We observed significantly increased levels of Th1-related (T-bet, IL-12a) and Th17-related (RORγt, IL-6, IL-23, and IL-22) transcription factors and cytokines after HI, and most Th2/Treg-related transcription factors and cytokines were either decreased (IL-4) or unchanged (GATA3, IL-5, Foxp3, TGFβ, and IL-10). Notably, the signature cytokine for Th17-related response (IL-17A) was not detectable in the neonatal mouse brain in the current study. We carefully analyzed the IL-17A expression in normal neonatal mice, but could not detect any mRNA under our experimental conditions. However, analysis of IL-17A expression in the present study focused on the early time points within 7 days post-HI when the adaptive immune responses are not initiated, and the early Th1/Th17-type response in the brain after HI might be due to innate-type cell types, for example, γδ T-cells and ILCs. Possibly, IL-17A could be expressed at later time points after HI when massive infiltration of T-cells into the CNS starts, since unlike its other analogs (that is, IL-17F), IL-17A is produced mainly in T-cells [
64]. For the Th2-type response, we did find significantly increased levels of the Th2-type cytokine IL-13 in the brain at 6 hours after HI. IL-13 is a mediator of allergic inflammation and disease, and in the brain IL-13 has been found to induce oxidative stress and contribute to the death of activated microglia [
65],[
66]. In general, it seems that the physiological Th2/Treg-type bias is lost in preterm brain injury after HI in mice and that the balance is instead skewed towards a type 1 (Th1/Th17) immune response (Figure
8).
Altogether, these results indicate that HI triggers a robust immune response in the brain that is characterized by an imbalance between Th1/Th17- and Th2/Treg-type responses. This imbalance occurs early in the immune response and is probably caused by an innate type of response as indicated by the increased yet low number of CD4+ cells mostly located in the blood vessels.
Acknowledgements
This work was supported by the Swedish Medical Research Council (VR 2008-2286 and VR 2013-2475 to XW, VR 2012-3500 to HH, and VR 2012-2992 to CM), the Bill & Melinda Gates Foundation Grand Challenge Explorations and Global Health (OPP1036135 to XW), Swedish governmental grants to researchers in the public health service (ALFGBG-367051 to XW, ALFGBG-2863 to HH, and ALFGBG-142881 to CM), VINNMER-Marie Curie international qualification (VINNOVA, 2011-03458 to XW), Wilhelm and Martina Lundgren (37/2013 to XW and 35/2012 to AM), Wilhelm and Martina Lundgren Foundation (HH), the Åhlén Foundation (HH, CM), Wellcome Trust (WT094823 HH), the Frimurare Barnhus Foundation (HH), the Byggmästare Olle Engqvist Foundation (HH, CM), the Leducq Foundation (DSRR-P34404 to CM and HH), and the Swedish Brain Foundation (FO2013-0095 to CM, 2013-0035 HH).
Competing interests
The authors declare that they have no competing interests.