Abstract
Germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most severe complications associated with prematurity. GM-IVH commonly results in neurodevelopmental impairments or neuropsychiatric disorders. However, GM-IVH has no successful treatment. Previous studies have shown that early intervention programs may have beneficial effects in preterm newborns (PT). Similarly, enriched environments (EE) increase neurogenesis and behavioral alterations in animal models. Therefore, we propose the study of the effects of long-term exposure to a complex EE in a murine model of induced GM-IVH. We analyzed brain pathological features and feasible markers of GM-IVH, as well as behavioral outcomes. In parallel, we analyzed the same plasma markers in PT with GM-IVH that followed an intervention program including parental bonding and stimulation. EE exposure limited brain atrophy, maintained the neuronal density, limited axonal damage and increased the neuronal complexity. Furthermore, neurogenesis was promoted and the inflammatory process was attenuated. Importantly, after the exposure to EE, cognition was improved, and the peripheral markers of brain damage, UCHL1 and plasma gelsolin, were also ameliorated. In line with this, plasma UCHL1 was early increased in patients with GM-IVH while plasma gelsolin was reduced and an overall reduction of peripheral markers was observed with time, both in Controls and in patients with GM-IVH. On the other hand, plasma tau levels were significantly reduced in those patients with GM-IVH that received parental support and nurturing environment. Altogether, our data show an improvement of pathological hallmarks, peripheral biomarkers and cognitive impairment associated with GM-IVH and support the beneficial effects of early intervention programs.
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Introduction
The advances of neonatal medicine along the last decades have significantly increased the survival rates of preterm newborns (PT), ultimately increasing the incidence of related problems and associated disabilities as well [1]. Among these, germinal matrix-intraventricular hemorrhage (GM-IVH) is one of the most common complications of prematurity, affecting about 20% of all PT (for review [2, 3]). Other studies have reported that GM-IVH may affect up to 40% of the PT [4] and these figures might also be larger in low resource settings [5].
The germinal matrix (GM) surrounds the fetal ventricular system and it is a highly vascularized region that has fragile capillaries with weak endothelial walls, prone to bleeding. The GM commences to involute by 26 gestational weeks until it disappears at full term [6], therefore GM-IVH occurs almost exclusively in PT. These patients are extremely vulnerable to ischemia and bleeding since they have an immature central nervous system, present hemodynamic instability, problems to regulate cerebral blood flow as well as great sensitivity to hypoxia and changes in osmolarity, among others [2]. Brain damage caused by GM-IVH includes the neurotoxic effects of hemoglobin and its derivates after bleeding [7,8,9], increased oxidative stress [10], white matter damage [11, 12], neuroinflammation [13] or compromised neurogenesis [14,15,16]. Importantly, up to one-third of PT with GM-IVH die [17]. Also, while it is hard to obtain consistent figures on the incidence of related neurodevelopmental disorders, some studies have reported that up 42% of the PT with periventricular hemorrhage will suffer these complications [18] and even kids with low-grade hemorrhages may show worse neurodevelopmental outcomes [19], including visual and hearing problems [20, 21], motor and cognitive impairments or epilepsy. These are life-lasting conditions and the patients will frequently need special care and continuous follow-ups [22]. Moreover, therapies for fetal neurodevelopmental disorders are extremely limited and mostly focus on screening and prevention to reduce the incidence and severity of the GM-IVH.
At this point, it is important to bear in mind that apart from the direct damage induced by GM-IVH, the PT leaves the protective maternal womb to be exposed to the stress of the neonatal intensive care units (NICU). Also, they are commonly separated from their mothers and may not have the much needed biological and emotional care that contributes to alterations in brain development, complicating the neurodevelopment of these kids and increasing the risk of sensory, cognitive and motor complications [2]. Thus, the participation of the parents in the NICU reduces the time the patients spend there and improves psychological, cognitive and behavioral outcomes of these infants [23]. In line with this, previous studies have reported the beneficial effects of early interventions to improve motor and cognitive skills in patients at risk of suffering cerebral palsy. Environmental enrichment (EE), skin-to-skin contact and family implication in behavioral tasks also have a positive effect, and in these patients the intervention should start as soon as possible [12, 24]. Whereas the beneficial effects of early developmental care to improve cognitive and motor outcomes in patients with GM-IVH seem out of question, to our knowledge, the effects of these approaches in the pathology of the PT with GM-IVH, that support the improved motor and cognition outcomes, have not been addressed. Therefore, we have analyzed the effects of exposure to EE in a murine model of GM-IVH and we have analyzed the effects on cognition as well as in brain pathology including brain atrophy and neuronal loss, white matter and vascular damage, and inflammation. We have also addressed possible peripheral markers of brain damage as neurofilament light (NfL), glial fibrillary acidic protein (GFAP), ubiquitin C-terminal hydrolase L1 (UCHL1) and plasma gelsolin (p-GSL) levels [15, 25, 26]. In parallel, we have analyzed the same markers in PT with GM-IVH that received parental support and nurturing environment for a period >6 h/day while they were in the NICU, measured by quantifying the amount of time parents perform skin-to-skin contact and holding their children. In our animal model of GM-IVH, we have observed a beneficial effect of exposure to EE when we analyzed brain atrophy and ventricle enlargement, accompanied by an increase of neuronal complexity as well as neurogenesis. Inflammation was also reduced in the brain from mice with GM-IVH exposed to EE, and an overall improvement of cognitive processes was detected. Interestingly, peripheral markers of brain damage, including UCHL1 and p-GSL are also normalized after EE. In patients with GM-IVH, we observed an overall early increase of peripheral markers of brain injury, including NfL, GFAP and tau, that reached statistical significance for UCHL1 levels. p-GSL was reduced in patients with GM-IVH. Also, while all peripheral markers were reduced in the long term, both in patients without GM-IVH (Control group from now on) and in patients with GM-IVH that received parental support and a nurturing environment for a longer period of time, we detected a significant reduction of tau levels.
Altogether, our data contribute to set the pathological basis that may underlie the beneficial effects of early developmental care in patients with GM-VIH, since brain atrophy, neuronal wellbeing and inflammation are improved. These beneficial effects also translate into changes in UCHL1, supporting them as feasible markers of GM-IVH pathology as well as the effects of the therapeutic treatments. Also, parental support results in the amelioration of peripheral markers of brain damage in the long term.
Materials and methods
Animals and EE setup
Animals were anesthetized with isoflurane and their reflexes were checked regularly. GM-IVH was induced in CD1 mice offspring at seven days of life (P7) by unilateral intracerebroventricular injection of 1 μl of collagenase (Col) (purified collagenase VII, batch C0773-1.5KU, Sigma Aldrich, St Louis, MO, USA) in TESCA (TES buffer 50 mM, calcium chloride anhydrous 0.36 mM) as described [15]. Sexing animals at the time lesions were performed (P7) is largely unreliable, which significantly limits the ability to form balanced sex subgroups. As a result, animals were randomly assigned to experimental groups that underwent surgery and exposure to an EE. A total of 16–17 animals per group were initially included in the study: Control (n = 16; 5 ♂, 11 ♀), Control + EE (n = 16; 9 ♂, 7 ♀), Col (n = 17; 8 ♂, 9 ♀), and Col + EE (n = 17; 7 ♂, 10 ♀). This sample size aligns with previous similar studies [27,28,29] and was determined based on a power analysis using a power value of 0.95, an eta-squared effect size of 0.25, and a significance level of 0.05, yielding a required minimum of 14 animals per group. The specific numbers and sexes of animals used are detailed for each experimental approach. All quantifications were performed blinded to the researcher conducting the analysis.
At P14, the animals started to receive the EE exposure in that consisted of the use of different toys and setups that changed weekly up to P70 (Fig. 1A and B) (detailed information provided in supplementary material). Mice that were not exposed to an EE were placed in empty cages with water, food and sawdust. In all conditions (naïve, sham and Col mice) the mice remained in the cages in groups of 8 animals.
A Timeline for the studies of an EE in a murine model of GM-IVH induced by intracerebroventricular Col administration at P7, weekly EE changes, behavioral studies (P56-P70) and BrdU administration (P67-P70). B Cartoons representing the actual objects and conditions used in each setup for EE. C Individual daily assessment revealed that Col administration affected learning and memory, while EE improved this situation in Control and GM-IVH mice (day 1 [F(3,272) = 0.569, p = 0.636], day 2 [F(3,250) = 3.25, ┬p = 0.022 vs. Control+EE], day 3 [F(3,247) = 2.52, p = 0.058], day 4 [F(3,244) = 11.76, **p < 0.001 vs. rest of the groups, ††p < 0.001 vs. Control+EE]. D EE slightly improved memory in the retention phase of the MWM, although differences did not reach statistical significance 24 h after completing the acquisition phase (% time quadrant 2 [F(3,61) = 2.57, p = 0.062]; number of entries quadrant 2 [F(3,60) = 0.524, p = 0.668]) or 72 h after completing the acquisition phase (% time quadrant 2 [F(3,57) = 0.283, p = 0.042] no further differences detected; number of entries quadrant 2 [F(3,587) = 0.79, p = 0.504]). Data are representative of 16-17 animals/group (Control n = 16, Control+EE n = 16, Col n = 17, Col+EE n = 17). E GM-IVH significantly impairs the performance in the NOD test while long term EE improves episodic memory in “what” [F(3,182) = 3.63, ‡p = 0.014 vs. Control], “where” [F(3,178) = 2.92, ┬p = 0.035 vs. Control-EE] and “when” [F(3,179) = 2.93, ‡p = 0.035 vs. Control]. Data are representative of 16-17 animals/group (Control n = 16, Control+EE n = 16, Col n = 17, Col+EE n = 17).
Three days prior to sacrifice at P70 the animals received a daily dose of 5-bromo-2-deoxyuridin (BrdU) (B5002, Sigma-Aldrich, St. Louis, MO, USA) (70 mg/kg ip) (Fig. 1A). Long-term postmortem studies were also done in these young adults at P70. All experimental procedures were approved by the animal Care and Use Committee of the University of Cadiz in accordance with the guidelines for care and use of experimental animals (European Commission Directive 2010/63/UE and Spanish Royal Decree 53/2013) and Junta de Andalucia (protocol number 18/11/1019/188).
Patients and samples
Plasma samples from PT patients obtained in the NICU of the Puerta del Mar University Hospital between 2015 and 2016 were included in the study. Early plasma samples were collected at 0-3 days of life, before exposure to parental care, and late plasma samples were collected on 3-4 months after birth. This study was reviewed and approved by the local Research and Ethics Committee and with parental or legal guardian signed informed consent.
Inclusion criteria were PT with a birth weight equal or less than 1500 g and/or a gestational age at birth equal or less than 32 weeks. The exclusion criteria were defined as the presence of congenital or chromosomal anomalies, metabolic disorders, central nervous system infections and congenital infections. We also excluded those PT who presented posthemorrhagic ventricular dilatation or died. All neonates underwent transfontanellar ultrasound daily during the first 5 days of life, and weekly until the corrected age at term. Measurements were performed with a Voluson S8 BT18 ultrasound machine (General Electric Healthcare, Buckinghamshire, United Kingdom), with the Voluson transducer (S-VNA5-8B, 5 to 8 MHz).
The severity of GM-IVH was evaluated using the grading system of Papile et al [30]. Parents completed a Parent-Infant Closeness Diary during their child’s admission to quantify the time they performed skin-to-skin contact and holding their children [31]. PT patients were classified depending on whether or not their parents spent more than 6 h a day in skin-to-skin contact or holding their infants [32]. The number of patients initially included was as follows: PT without GM-IVH (Control group) whose parents spent less than 6 h performing skin-to-skin or holding their infants (n = 12), PT without GM-IVH whose parents spent more than 6 h doing skin-to-skin or holding their children in their arms (Control+EE group) (n = 6), PT with GM-IVH whose parents spent less than 6 h doing skin-to-skin or holding their children in their arms (GM-IVH group) (n = 17; hemorrhage grade II, n = 8, hemorrhage grade III/parenchymal hemorrhagic infarction (PHI), n = 9) and PT with GM-IVH whose parents spent more than 6 h doing skin-to-skin or holding their children in their arms (GM-IVH+EE group) (n = 13; hemorrhage grade II, n = 8; hemorrhage grade III/PHI, n = 5). Variables recorded included sex, gestational age, birth weight, Apgar 5 scores, cesarean or vaginal delivery, postnatal steroids, premature rupture of membranes (PROM), chorioamnionitis (CA), preeclampsia (PE), bronchopulmonary dysplasia (BPD), retinopathy of prematurity (ROP) and necrotizing enterocolitis (NEC) and sepsis. Due to the condition of some samples (e.g., hemolyzed or contaminated with blood) or the inability to obtain certain readouts, these samples could not be included in some analyses. All quantifications were performed blinded to the researcher conducting the analysis. The characteristics and details of the study sample are presented in Table 1. Supplementary table 1 includes information for all individual patients included in the study.
Morris water maze (MWM)
Behavioral studies started with the MWM at P56 and 16-17 animals were included in each group (Control n = 16 (5 ♂, 11 ♀), Control+EE n = 16 (9 ♂, 7 ♀), Col n = 17 (8 ♂, 9 ♀), Col+EE n = 17 (7 ♂, 10 ♀)). The acquisition phase took place for 4 consecutive days (4 trials/day). Twenty-four and 72 h after the end of the acquisition phase, the retention phase was performed. The platform was removed, and the mice were allowed to swim for 60 s. The time that mice spent in quadrant 2, the number of entries and the swimming speed were recorded using a SMART system (Panlab, Barcelona, Spain) as described [27].
New object discrimination test (NOD)
After completing the MWM, episodic memory was assessed by NOD test as previously described [33] and detailed in supplementary materials. Groups included 16-17 animals (Control n = 16 (5 ♂, 11 ♀), Control+EE n = 16 (9 ♂, 7 ♀), Col n = 17 (8 ♂, 9 ♀), Col+EE n = 17 (7 ♂, 10 ♀)).
Sample processing
Cerebrospinal fluid (CSF) was collected from cisterna magna as previously described [34], blood and brains were processed as described [27] and detailed in supplementary materials.
Cresyl violet staining
Six coronal sections (1.5, 0.5, 0.0, −1.5, −2.0 and −2.5 mm from Bregma) were selected to measure the sizes of the ipsilateral hemisection, cortex, hippocampus and ventricle to analyze the brain morphology using cresyl violet (sc-214775B, ChemCruz™ biochemicals, Heidelberg, Germany) solution (0.5% w/v) for 8 min as described [14]. Groups included 5-7 animals (Control n = 5-6 (1 ♂, 4-5 ♀), Control+EE n = 5 (2 ♂, 3 ♀), Col n = 6 (3 ♂, 3 ♀), Col+EE n = 6-7 (4 ♂, 2-3 ♀)).
Immunohistochemistry studies: NeuN, ionized calcium binding adaptor molecule 1 (Iba1), BrdU and doublecortin (DCX)
Six sections contiguous to the used for cresyl violet staining were incubated with anti-NeuN antibody (ab279297, Abcam, Amsterdam, Netherlands) (1:500) overnight at 4oC, to label neurons as described [35]. Groups included 4-6 animals (Control n = 5-6 (1 ♂, 4-5 ♀), Control+EE n = 4 (1 ♂, 3 ♀), Col n = 5-6 (1 ♂, 4-5 ♀), Col+EE n = 4-5 (3-4 ♂, 1 ♀)).
Microglia was labeled with anti-Iba1 (Wako, Osaka, Japan, 019-19741) (1:1000) overnight at 4oC, as described [36]. Alexa Fluor goat anti-rabbit 488 (A11008, Invitrogen, Carlsbad, CA, USA) (1:1000) was used as secondary antibody for 1 h at room temperature. Microglia burden was quantified as % area covered by Iba1+ cells, respectively, in the cortex, hippocampus and SVZ using Image J software [36]. Groups included 5-6 animals (Control n = 6 (1 ♂, 5 ♀), Control+EE n = 5 (2 ♂, 3 ♀), Col n = 6 (2 ♂, 4 ♀), Col+EE n = 6 (4 ♂, 2 ♀)).
The six contiguous sections were used for the BrdU and DCX immunostaining and quantification as previously described [37, 38] and detailed in supplementary materials. Groups included 5-6 animals (Control n = 6 (1 ♂, 5 ♀), Control+EE n = 5 (2 ♂, 3 ♀), Col n = 5-6 (1-2 ♂, 4 ♀), Col+EE n = 6 (4 ♂, 2 ♀)).
Golgi-Cox staining
Neuronal complexity was studied by Golgi-Cox staining using Rapid Golgi Stain Kit (PK401, FD Neurotechnologies, Columbia, MD, USA) following the manufacturer’s indications and previously described [39]. Detailed procedures are included in the supplementary materials. Groups included 3 animals (Control n = 3 (2 ♂, 1 ♀), Control+EE n = 3 (2 ♂, 1 ♀), Col n = 3 (2 ♂, 1 ♀), Col+EE n = 3 (1 ♂, 2 ♀)).
Phospho-tau and total tau levels in animals
Total tau and phospho-tau [pS199] levels were measured in the cerebral cortex and striatum of mice using ELISA kits (KMB7011 and KMB7012 respectively, Thermo-Fisher Scientific, Waltham, MA, USA) as previously described [40] and detailed in supplementary materials. Groups included 4-6 animals (Control n = 5 (2 ♂, 3 ♀), Control+EE n = 4 (3 ♂, 1 ♀), Col n = 5-6 (3-4 ♂, 2 ♀), Col+EE n = 6 (1 ♂, 5 ♀)).
Prussian blue staining
Six sections next to those used in cresyl violet staining were chosen to detect small vessel hemorrhages by Prussian blue staining to label iron deposits as detailed in supplementary materials. Groups included 5-8 animals (Control n = 5 (1 ♂, 4 ♀), Control+EE n = 5 (2 ♂, 3 ♀), Col n = 8 (3 ♂, 5 ♀), Col+EE n = 6 (4 ♂, 2 ♀)).
NfL and GFAP levels in animals
Plasma levels of NfL (Control n = 4 (1 ♂, 3 ♀), Control+EE n = 4 (2 ♂, 2 ♀), Col n = 5 (2 ♂, 3 ♀), Col+EE n = 5 (2 ♂, 3 ♀)) and GFAP (Control n = 4 (1 ♂, 3 ♀), Control+EE n = 4 (2 ♂, 2 ♀), Col n = 5 (2 ♂, 3 ♀), Col+EE n = 5 (2 ♂, 3 ♀)) were measured in animals by single molecule array (SIMOA) using Neurology 2-Plex B (GFAP, NF-light) kit (103520, Quanterix, Billerica, MA, USA) in a SR-X equipment (SR-X™ Biomarker Detection and Analysis System, Quanterix, Billerica, MA, USA), following the manufacturer´s indications. For CSF samples (Control n = 8 (2 ♂, 6 ♀), Control+EE n = 5 (2 ♂, 3 ♀), Col n = 5 (2 ♂, 3 ♀), Col+EE n = 8 (4 ♂, 4 ♀)), NF-Light V2 kit (104073, Quanterix, Billerica, MA, USA) was used. Plasma samples were pre-diluted 1:4, and 1:100 dilution was used for CSF samples. NfL and GFAP levels were expressed in pg/ml.
Myelin basic protein (MBP) and UCHL1 in animals
To determine myelination status, MBP levels were measured by ELISA kit (ORB40937896, Biorbyt, Cambridge, UK) in the cortex, hippocampus and striatum from all groups of mice under study, according to the manufacturer´s instructions. MBP levels in the cortex (Control n = 7 (3 ♂, 4 ♀), Control+EE n = 8 (5 ♂, 3 ♀), Col n = 6 (3 ♂, 3 ♀), Col+EE n = 7 (2 ♂, 5 ♀)), hippocampus (Control n = 4 (2 ♂, 2 ♀), Control+EE n = 7 (4 ♂, 3 ♀), Col n = 6 (4 ♂, 2 ♀), Col+EE n = 5 (2 ♂, 3 ♀)) and striatum (Control n = 6 (3 ♂, 3 ♀), Control+EE n = 6 (3 ♂, 3 ♀), Col n = 5 (3 ♂, 2 ♀), Col+EE n = 6 (3 ♂, 3 ♀)) were calculated and expressed in ng/mg tissue.
UCHL1 levels were determined in plasma samples from all groups of mice under study using an ELISA kit (ABIN429877, Antibodies-online, Aachen, Germany) according to the manufacturer´s instructions. Groups included 6-8 animals (Control n = 8 (3 ♂, 5 ♀), Control+EE n = 6 (3 ♂, 3 ♀), Col n = 6 (3 ♂, 3 ♀), Col+EE n = 7 (4 ♂, 3 ♀)). Data were expressed in pg/ml.
Plasma NfL, tau, GFAP and UCHL1 levels in patients
Plasma levels of NfL, tau, GFAP and UCHL1 were measured by SIMOA using Neurology 4-Plex B kit (103345, Quanterix, Billerica, MA, USA) in a SR-X equipment (SR-X™ Biomarker Detection and Analysis System, Quanterix, Billerica, MA, USA), following the manufacturer´s instructions. Plasma samples were pre-diluted 1:4 and quality controls for each analyte were included. Data were expressed in pg/ml. Patient samples are detailed in Table 1.
p-GSL levels in patients and animals
To assess p-GSL levels, we used a colorimetric ELISA kit for mice (ABIN367662, Antibodies-online, Aachen, Germany) and human samples (CSB-E08160h, Cusabio Technology LLC, Houston, TX, USA) following the manufacturer’s recommendations. Plasma samples were diluted 1:500 (mouse samples) and 1:5 (human samples) and were measured at 450 nm in a spectrophotometer (MQX200R2, Biotek instruments, Burlington, VT, USA). Groups included 10-13 animals (Control n = 10 (4 ♂, 6 ♀), Control+EE n = 13 (7 ♂, 6 ♀), Col n = 12 (8 ♂, 2 ♀), Col+EE n = 13 (5 ♂, 8 ♀)). Patient samples are detailed in Table 1.
Statistical analysis
Two-way ANOVA (groupXday) was performed to analyze the acquisition phase in the MWM test. A one-way, two-sided ANOVA for independent samples was performed in all experimental studies, followed by either Tukey’s b or Tamhane’s test, as appropriate. Since no differences were observed when Sham and naïve mice were compared, they were all included in a single Control group. SPSS v.24 software was used for all statistical analyses. Data are expressed as the mean ± SEM.
Results
Exposure to EE improves learning and memory impairments following GM-IVH
We did not observe a significant timeXgroup effect when we analyzed the acquisition phase in the MWM [F(3,9) = 1.73, p = 0.078]. Nevertheless, when we analyzed individual days we observed an improvement in mice with GM-IVH that were exposed to an EE. Importantly, the EE also had a beneficial effect in Control animals (Fig. 1C). Separate analysis of male and female subjects revealed similar outcomes for males in the acquisition phase of the MWM [F(3,9) = 1.70, p = 0.086] and females [F(3,9) = 1.53, p = 0.132], as well as similar results when individual days during acquisition phase were analyzed (suppl. figure 1A). In the retention phase, animals with GM-IVH spent shorter times and a lower number of entries in quadrant 2 (where the platform was located), both at 24 and 72 h after completing the acquisition phase, and a slight improvement was observed after exposure to EE, although the differences did not reach statistical significance (Fig. 1D). Although similar profiles were observed when males and females were analyzed separately, statistically significant differences were found only in the number of entries among males. (suppl. figure 1B). After Col administration, episodic memory at long-term in the NOD test was also affected in the injured animals, and EE significantly improved this situation in all the paradigms under study (“what”, “where” and “when”) (Fig. 1E). When we analyzed males and females independently, we observed an impairment in different paradigms, that were more severe for “what” and “when” paradigms in females and for “where” paradigm in males (suppl. figure 1C).
Exposure to EE attenuates brain atrophy in animals with GM-IVH
We analyzed the brain weight/body weight ratio and we observed a significant reduction in animals with induced GM-IVH, while long-term exposure to EE restored this situation (Fig. 2A). A similar profile was observed when males and females were analyzed independently (suppl. figure 2A). Assessment of brain morphology by cresyl violet staining revealed a slight reduction of the ipsilateral hemisection size after the GM-IVH, although differences did not reach statistical significance (Fig. 2B). No significant differences between groups were observed when hippocampus size was measured (Fig. 2C). While cortex size was compromised in animals with GM-IVH, EE did not have a significant effect at this level (Fig. 2D). Similar profiles were observed when males and females were analyzed independently (suppl. figure 2B–D). Since brain atrophy observed in animal models and patients with GM-IVH is accompanied by an important ventricular enlargement, we also analyzed the ventricle size and we observed an overall enlargement in animals with GM-IVH, whereas exposure to EE significantly reduced the ventricle sizes, reaching control values (Fig. 2E and F). Similar profiles were observed when males were analyzed independently, although differences differences did not reach statistical significance (suppl figure 2B–E).
A Brain/body weight ratio was slightly reduced in animals with GM-IVH and EE limited this situation [F(3,50) = 3.72, \({\bar{\rm {T}}}\) p = 0.017 vs. Col+EE]. Data are representative of 10-16 animals/group (Control n = 10, Control+EE n = 14, Col n = 14, Col+EE n = 16). B Hemisection size was slightly reduced in animals with GM-IVH although differences did not reach statistical significance [F(3,122) = 1.25, p = 0.292]. Data are representative of 5-6 mice/group (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6). C No differences were observed among groups when hippocampus size was analyzed [F(3,60) = 1.67, p = 0.183]. Data are representative of 5-7 mice/group (Control n = 7, Control+EE n = 5, Col n = 6, Col+EE n = 7). D Cortex size was reduced in animals with GM-IVH and EE did not significantly affect this situation [F(3,129) = 8.91, ††p < 0.01 vs. Control and Control+EE]. Data are representative of 5-7 mice/group (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 7). E A significant enlargement of the ventricle was observed in GM-IVH animals while EE limited this situation reaching control values [F(37,60) = 4.77, ††p = 0.005 vs. Control and Control+EE]. Data are representative of 5-6 mice/group (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6). F Illustrative example of ventricle enlargement after GM-IVH and the reduction of ventricle size after EE treatment. Scale bar=50 µm. G NeuN/DAPI ratio was significantly reduced in the cortex from animals with GM-IVH and no significant effect was observed after exposure to EE [F(3,2370) = 14.34, ††p < 0.01 vs. Control and Control+EE]. Data are representative of 4-6 mice/group (Control n = 6, Control+EE n = 4, Col n = 6, Col+EE n = 5). H Illustrative example of the NeuN-DAPI immunostaining in the cortex, where NeuN are in red and DAPI are in blue. Scale bar=50 µm. I NeuN/DAPI ratio was significantly reduced in the SVZ from animals with GM-IVH and while a slight improvement was observed after treatment with EE, differences were not statistically significant [F(3,444) = 2.27, ┬p = 0.041 vs. Control+EE]. Data are representative of 4-6 mice/group (Control n = 6, Control+EE n = 4, Col n = 6, Col+EE n = 5). J Illustrative example of the NeuN-DAPI immunostaining in the SVZ. NeuN are in red and DAPI are in blue. Scale bar=50 µm. K P-tau/total tau levels were slightly increased in the cortex and striatum from animals with GM-IVH and lower levels were observed after EE, however differences did not reach statistical significance in the cortex [F(3,17) = 1.44, p = 0.266] (Control n = 5, Control+EE n = 4, Col n = 6, Col+EE n = 6) or striatum [F(3,16) = 1.37, p = 0.285] (Control n = 5, Control+EE n = 4, Col n = 5, Col+EE n = 6). L We analyzed overall neurite complexity by Sholl analysis up to 60 µm from the neuron soma [F(3,2182) = 33.57, **p < 0.001 vs. rest of the groups, ‡‡p < 0.001 vs. Control and Control+EE, ††p < 0.01 vs. Control+EE]. Data are representative of 3 mice/group. M We analyzed neurite complexity in the cortex by Sholl analysis and we did not detect a significant groupXdistance effect [F(15,2162) = 11.33, p = 0.519]. However, we observed an overall significant increase in neurite complexity in those animals that grew up on an EE ( < 10 µm [F(3,365)=12.57, ‡‡p < 0.001 vs. Control and Control+EE, ††p < 0.01 vs. Control+EE], 10-20 µm [F(3,367) = 15.94, ‡‡p < 0.001 vs. Control and Control+EE, ††p < 0.01 vs. Control+EE], 20-30 µm [F(3,360) = 8.20, ‡‡p < 0.001 vs. Control and Control+EE, ††p < 0.01 vs. Control+EE], 30-40 µm [F(3,357) = 5.69, ‡‡p = 0.001 vs. Control and Control+EE], 40-50 µm [F(3,362) = 5.93, ‡‡p = 0.001 vs. Control and Control+EE], 50-60 µm [F(3,351) = 7.13, ‡‡p < 0.001 vs. Control and Control+EE, ††p < 0.01 vs. Control+EE]). Data are representative of 3 mice/group. N Illustrative example of Golgi-Cox staining where a reduction in the number of crossings can be observed in Col-animals while exposure to an EE reverts this situation. Scale bar=25 µm.
EE improves neuronal alterations observed after GM-IVH
To deepen in the effects of exposure to EE, we analyzed the neuronal population and we observed that GM-IVH causes a reduction in the NeuN/DAPI ratio, in both the cortex and the SVZ, as previously observed in this model [14, 40]. EE did not have a significant effect in the cortex after the injury (Fig. 2G and H) while a slight improvement was observed after EE treatment in the SVZ, although it did not reach statistical significance (Fig. 2I and J). When males and females were analyzed separately, females appeared to exhibit a more consistent profile with that observed when all animals were analyzed together (suppl. figures 2F and 2G).
Tau hyperphosphorylation is a marker of neuronal damage. However, when we analyzed the levels of phospho-tau/total-tau in a distant region of the injured area such as the cortex we did no observe differences between groups. Tau phosphorylation levels were slightly elevated in the striatum from animals with GM-IVH and whereas exposure to an EE reduced tau levels, differences did not reach statistical significance (Fig. 2K). When males and females were analyzed separately, males appeared to exhibit a more consistent profile with that observed when all animals were analyzed together (suppl. figure 2H).
On the other hand, EE significantly improved altered neuronal architecture after the induction of GM-IVH and exposure to EE significantly improved neuronal complexity, suggesting that EE might have more subtle changes on neuronal wellbeing. Analysis of neurites complexity by Sholl analysis after Golgi-Cox staining revealed an overall reduction of crossings up to 60 μm from the soma (Fig. 2L and N). While we did not observe a significant groupXdistance effect [F(15,2162) = 11.33, p = 0.519], further analysis in 10 μm steps revealed a reduction of dendrite branching in animals with GM-IVH that was increased after exposure to EE both in animals with GM-IVH and in Control mice (Fig. 2M and N). When males and females were analyzed separately, males appeared to exhibit a more consistent profile with that observed when all animals were analyzed together (suppl. figure 2I).
Proliferation and neurogenesis compromise is reversed by exposure to EE
Proliferation and neurogenesis were analyzed in neurogenic niches, the SVZ and the dentate gyrus (DG) of the hippocampus. As previously described [15], GM-IVH induced by Col administration directly affects and compromises the SVZ. When we analyzed the SVZ we observed that the number of BrdU+ cells was not affected in any of the groups, indicating that this cell proliferation was not affected in this region (Fig. 3A and D). DCX burden was slightly compromised after Col injury and EE improved this situation although differences did not reach statistical significance (Fig. 3B and D). On the other hand, when the BrdU/DCX ratio was evaluated, a significant compromise was detected in GM-IVH mice, and EE reversed the situation (Fig. 3C and D). We also analyzed the proliferation and neurogenesis state in the DG of the hippocampus. As we observed in the SVZ, the number of BrdU+ cells was not affected in the DG (Fig. 3E and H). Whereas a significant decrease in DCX burden occurred in the DG from GM-IVH animals, EE treatment resulted in a successful reversal of this compromise (Fig. 3F and H). Differences were not statistically significant when we analyzed the BrdU/DCX ratio (Fig. 3G and H). When we analyzed the cortex, we found no differences in the number of BrdU+ cells between the different groups (Fig. 3I). However, the number of DCX+ cells was significantly reduced in GM-IVH mice, and exposure to EE treatment improved this situation (Fig. 3J). Similarly, we found a reduction in the number of BrdU+/DCX+ cells in the cortex of injured mice (Fig. 3K), while EE treatment reverted this situation in the cortex. When analyzed separately, males displayed a profile in the SVZ that more closely resembled the overall pattern observed when all animals were considered, whereas in the cortex, females showed a more similar profile (suppl. figure 3A–I).
A The number of BrdU+ cells was not significantly affected in the SZV in any of the groups under study [F(3,60) = 0.335, p = 0.800]. B Whereas a slight compromise was observed in animals with GM-IVH when we analyzed DCX burden, differences did not reach statistical significance [F(3,52) = 0.687, p = 0.564]. C However, a significant compromise was observed when we analyzed BrdU/DCX ratio in mice with GM-IVH, and EE successfully reversed this situation [F(3,57) = 2.67, \({\bar{\rm {T}}}\) p = 0.039 vs. Col+EE]. Data are representative of 5-6 mice (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6). D Illustrative image of BrdU (green) and DCX (red) immunostaining in the SVZ from all groups under study. Scale bar=100 µm. E The number of BrdU+ cells was not significantly affected in the DG in any of the groups under study [F(3,57) = 0.54, p = 0.651]. F A compromise was observed in animals with GM-IVH when we analyzed DCX burden [F(3,55) = 2.88, †p = 0.049 vs. Control]. G However, differences were not significant when we analyzed BrdU/DCX ratio in mice with GM-IVH or after exposure to EE [F(3,57) = 0.732, p = 0.537]. Data are representative of 5-6 mice (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6). H Illustrative image of BrdU (green) and DCX (red) immunostaining in the DG from all groups under study. Scale bar=100 µm. I We detected differences among groups when we analyzed the number of BrdU+ cells in the cortex, although posthoc analysis did not reveal further differences [F(3,119) = 3.49, p = 0.018]. J We observed a reduction in the number of DCX+ cells in mice with GM-IVH that was reversed by exposure to EE treatment [F(3,110) = 6.29, \({\bar{\rm {T}}}\) \({\bar{\rm {T}}}\) p = 0.001 vs. Control+EE and Col+EE]. K The same profile was observed when we analyzed the number of BrdU+/DCX+ cells [F(3,119) = 6.88, \({\bar{\rm {T}}}\) \({\bar{\rm {T}}}\) p = 0.001 vs. Control+EE and Col+EE]. Data are representative of 5-6 mice (Control n = 6, Control+EE n = 5, Col n = 5, Col+EE n = 6).
EE reduces the presence of hemorrhages in the brain with GM-IVH
Hemorrhage burden (% area covered by hemorrhages) was significantly increased in the cortex from animals with GM-IVH. The slight reduction in hemorrhage burden observed after exposure to EE did not reach statistical significance (Fig. 4A and B). When the hippocampus was analysed, no differences were observed among groups (Fig. 4C), as previously described in this model [27]. However, in the SVZ we observed a significant increase of hemorrhages while EE slightly reduced the hemorrhage burden in GM-IVH animals, although did not reach statistical significance (Fig. 4D and E). A similar profile was observed in the striatum, where EE slightly decreased the presence of hemorrhages after injury but did not reach statistical differentiation (Fig. 4F). When analyzed separately, males displayed a profile that more closely resembled the overall pattern observed when all animals were considered (suppl. figure 4A–D).
A Hemorrhage burden was significantly increased in animals with GM-IVH and EE did not significantly reduce the presence of hemorrhages in the cortex [F(3,122) = 2.89, ‡p = 0.032 vs. Control]. Data are representative of 5-8 animals (Control n = 5, Control+EE n = 5, Col n = 8, Col+EE n = 6). B Illustrative images of cortical regions stained with Prussian blue. Green arrows point at individual hemorrhages. Scale bar=100 µm. C Hemorrhage burden in the hippocampus was not significantly affected in any of the groups under study [F(3,59) = 0.993, p = 0.403] (Control n = 5, Control+EE n = 5, Col n = 8, Col+EE n = 6). D Hemorrhage burden was significantly increased in the SVZ from animals with GM-IVH whereas EE slightly reduced the presence of hemorrhages [F(3,60) = 0.294, ‡p = 0.017 vs. Control and Control+EE]. Data are representative of 5-8 animals (Control n = 5, Control+EE n = 5, Col n = 8, Col+EE n = 6). E Illustrative images of the subventricular regions stained with Prussian blue. Green arrows point at individual hemorrhages. Scale bar=100 µm. F Hemorrhage burden was slightly increased in the striatum from animals with GM-IVH although differences did not reach statistical significance [F(3,61) = 1.21, p = 0.312]. Data are representative of 5-8 animals (Control n = 5, Control+EE n = 5, Col n = 8, Col+EE n = 6). G Microglia burden was increased in the cortex from mice with GM-IVH and the EE ameliorated this situation [F(3,3126) = 8.61, **p < 0.01 vs. rest of the groups]. Data are representative of 5-6 mice/group (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6). H Illustrative images of microglia in the cortex. Scale bar=50 µm. I Microglia burden was increased in the SVZ from mice with GM-IVH while animals in the EE reached control values [F(3,407) = 4.73, ††p = 0.003 vs. Control and Control+EE]. Data are representative of 5-6 mice/group (Control n = 6 Control+EE n = 5, Col n = 6, Col+EE n = 6). J Illustrative images of microglia in the SVZ. Scale bar=50 µm. K Microglia burden was increased in the hippocampus from mice with GM-IVH and the EE ameliorated this situation [F(3,470) = 5.92, **p < 0.01 vs. rest of the groups]. Data are representative of 5-6 mice/group (Control n = 6, Control+EE n = 5, Col n = 6, Col+EE n = 6).
Exposure to EE reduces microglia burden in animals with GM-IVH
We analyzed the inflammatory process by quantifying the burden of Iba1+ cells. We analyzed the microglia burden in the cortex of animals with GM-IVH and we observed a significantly increase after injury, while EE improved this situation (Fig. 4G and H). Similarly, in the SVZ of injured mice the microglia burden was also increased, values that were reduced after EE treatment reaching control values (Fig. 4I and J). In the same way, microglia burden values were significantly higher in the hippocampus after GM-IVH and EE ameliorated this situation (Fig. 4K). When analyzed separately, females displayed a profile that more closely resembled the overall pattern observed when all animals were considered (suppl. figure 4E–G).
Exposure to EE restores MBP levels in the cortex of animals with GM-IVH
When we evaluated MBP levels in mice with GM-IVH, we observed that MBP levels were reduced in the cortex and EE contributed to increased MBP levels (Fig. 5A). No differences in MBP levels were observed in the hippocampus (Fig. 5B) or the striatum (Fig. 5C). When analyzed separately, males and females displayed a profile that resembled the overall pattern observed when all animals were considered (suppl. figure 5A–C).
A MBP levels were reduced in the cortex from mice with GM-IVH and EE improved this situation [F(3,24) = 3.08, ‡p = 0.047 vs. Control]. Data are representative of 7-8 mice (Control n = 7, Control+EE n = 8, Col n = 6, Col+EE n = 7). B No differences were observed when MBP levels were determined in the hippocampus [F(3,24) = 0.316, p = 0.814]. Data are representative of 4-7 mice (Control n = 4, Control+EE n = 7, Col n = 6, Col+EE n = 5). C MBP levels were not affected in the striatum [F(3,19) = 0.191, p = 0.901]. Data are representative of 5-6 mice/group (Control n = 6, Control+EE n = 6, Col n = 5, Col+EE n = 5). D NfL levels in CSF were slightly increased in mice with GM-IVH and EE limited this increase, although differences did not reach statistical significance [F(3,21) = 1.47, p = 0.250]. Data are representative of 5-8 mice (Control n = 8, Control+EE n = 5, Col n = 5, Col+EE n = 6). E NfL plasma levels were slightly increased in mice with GM-IVH and EE limited this increase, although differences did not reach statistical significance [F(3,20) = 1.39, p = 0.273]. Data are representative of 4-5 mice (Control n = 4, Control+EE n = 4, Col n = 5, Col+EE n = 5). F GFAP plasma levels were slightly increased in mice with GM-IVH and EE limited this increase, although differences did not reach statistical significance [F(3,14) = 1.76, p = 0.200]. Data are representative of 4-5 mice (Control n = 4, Control+EE n = 4, Col n = 5, Col+EE n = 5). G p-GSL was significantly reduced in mice with GM-IVH and long-term EE returned p-GSL levels to control values [F(3,42) = 3.19, †p = 0.033 vs. Control and Control+EE]. Data are representative of 10-13 mice (Control n = 10, Control+EE n = 13, Col n = 12, Col+EE n = 13). H UCHL1 plasma levels were significantly increased in mice with GM-IVH and EE ameliorated this situation [F(3,23) = 4.17, *p = 0.017 vs. rest of the groups]. Data are representative of 6-8 mice (Control n = 8, Control+EE n = 6, Col n = 6, Col+EE n = 7).
EE restores NfL and GFAP levels after GM-IVH in animals
NfL levels were higher in the CSF from mice with GM-IVH and a reduction in NfL levels was detected after exposure to EE, although differences did not reach statistical significance (Fig. 5D). A similar profile was observed when we analyzed plasma NfL levels (Fig. 5E). Also, GFAP plasma levels were slightly increased after injury, a situation that improved after exposure to EE without reaching statistical differences (Fig. 5F). When analyzed separately, males and females displayed a profile that resembled the overall pattern observed when all animals were considered (suppl. figure 5D–F).
p-GSL and UCHL1 plasma levels are restored after exposure to EE
p-GSL levels in plasma were significantly reduced in GM-IVH animals and we observed a recovery after exposure to EE (Fig. 5G). Also, plasma UCHL1 levels were significantly higher after GM-IVH, whereas exposure to EE treatment restored control levels (Fig. 5H). When analyzed separately, males and females displayed a profile that resembled the overall pattern observed when all animals were considered (suppl. figure 5G, H).
Parental support reduces plasma tau levels in patients
We detected an overall increase of acute plasma GFAP, NfL and tau levels in patients with GM-IVH, that reached statistical significance when UCHL1 levels were compared with Control patients (Fig. 6A–D), supporting the early diagnostic value of these markers. We also observed that GFAP, NfL and UCHL1 were reduced when analyzed in the long term, independently of the exposure to parental support and nurturing environment. On the other hand, tau levels were only significantly reduced in patients with GM-IVH that received parental support and nurturing environment (Fig. 6C). p-GSL levels were reduced in patients with GM-IVH, as previously observed [15] and while patients that received an EE showed slightly higher levels in the long term, no differences were detected (Fig. 6E).
A Plasma GFAP levels were initially increased in patients with GM-IVH, although differences with the Control group did not reach statistical significance (p = 0.138). GFAP levels were reduced in Control patients with and without EE when compared with initial Control values [F(2,33) = 7.63, **p = 0.002 vs. Control initial]. A similar profile was observed in patients with GM-IVH, although differences did not reach statistical significance [F(2,35) = 2.40, p = 0.105]. No differences were observed when Control patients or patients with GM-IVH were compared in the long term [F(3,30) = 0.555, p = 0.649]. Data are representative of 5-20 patients (Control initial n = 19, Control long term n = 11, Control+EE long term n = 6, GM-IVH initial n = 20, GM-IVH long term n = 13, GM-IVH+EE long term n = 5). B Plasma NfL levels were initially increased in patients with GM-IVH, although differences with the Control group did not reach statistical significance (p = 0.054). NfL levels were reduced in Control patients with and without EE when compared with initial Control values [F(2,34) = 6.47, **p = 0.004 vs. Control initial] or initial GM-IVH values [F(2,37) = 6.61, **p = 0.004 vs. GM-IVH initial]. No differences were observed when Control patients and patients with GM-IVH were compared in the long term [F(3,30) = 0.759, p = 0.526]. Data are representative of 6-21 patients (Control initial n = 20, Control long term n = 11, Control+EE long term n = 6, GM-IVH initial n = 21, GM-IVH long term n = 13, GM-IVH+EE long term n = 6). C Plasma tau levels were initially increased in patients with GM-IVH, although differences with the Control group did not reach statistical significance (p = 0.122). Tau levels were lower in the long term when Control patients were compared [F(2,33) = 6.89, **p = 0.003 vs. Control initial]. A similar profile was observed in patients with GM-IVH although differences did not reach statistical significance [F(2,36) = 23.92, *p = 0.029 vs. GM-IVH initial]. No differences were observed when patients were compared in the long term [F(3,32) = 0.855, p = 0.475]. Data are representative of 6-20 patients (Control initial n = 19, Control long term n = 11, Control+EE long term n = 6, GM-IVH initial n = 20, GM-IVH long term n = 13, GM-IVH+EE long term n = 6). D Plasma UCHL1 levels were initially increased in patients with GM-IVH (†p = 0.019 vs. Control initial). UCHL1 levels were lower in the long term when Control patients were compared, although differences did not reach statistical significance [F(2,33) = 4.38, p = 0.020, no further differences detected]. A similar profile was observed in patients with GM-IVH [F(2,37) = 5.36, **p = 0.009 vs. GM-IVH initial]. No differences were observed when patients were compared in the long term [F(3,32) = 1.043, p = 0.387]. Data are representative of 7-20 patients (Control initial n = 19, Control long term n = 11, Control+EE long term n = 6, GM-IVH initial n = 20, GM-IVH long term n = 13, GM-IVH+EE long term n = 7). E P-GSL levels were initially reduced in patients with GM-IVH (†p = 0.032 vs. Control initial). P-GSL levels were lower in Control patients with and without EE although differences did not reach statistical significance [F(2,28) = 4.56, p = 0.019, no further differences detected]. In patients with GM-IVH we observed a slight increase of p-GSL after EE although differences did not reach statistical significance [F(2,28) = 0.954, p = 0.397]. No differences were observed when Control patients or patients with GM-IVH were compared in the long term [F(3,30) = 1.22, p = 0.319]. Data are representative of 3-14 patients (Control initial n = 14, Control long term n = 14, Control+EE long term n = 3, GM-IVH initial n = 13, GM-IVH long term n = 12, GM-IVH+EE long term n = 6).
Discussion
GM-IVH is one of the most common complications of prematurity that may affect over 40% of all PT [4]. Besides, the improved survival rates of extremely PT have secondarily resulted in a larger number of patients with this condition. Observed brain damage includes a wide range of alterations such as white matter damage, neuroinflammation, neuronal loss, brain atrophy, proliferation and neurogenesis impairment, oxidative stress or glutamate excitotoxicity, among others, as well as related behavioral and developmental complications that may include cerebral palsy, cognitive impairment, hearing and visual impairment, or neuropsychiatric disorders. However, there is no optimal therapy to prevent GM-IVH or to treat its consequences (for review [2, 12]). Currently, early intervention programs, including parental bonding and physical, occupational and speech therapy are common approaches to improve motor, cognitive and communication skills in these patients [12]. The beneficial effects of different enriched environments have been reported in PT patients [41, 42] and positive impact has also been reported in animal models resembling other newborn challenges [43, 44]. Nevertheless, to our knowledge, the effects of such therapies in brain pathology and derived outcomes after GM-IVH have not been analyzed. Therefore, we have assessed the consequences of long-term exposure to EE in brain pathology and cognitive impairment in a murine model of GM-IVH. We have also analyzed feasible peripheral markers of brain damage after exposure to EE. Despite the obvious differences with the approaches used in the clinic, we have also analyzed peripheral markers (GFAP, NfL, tau, UCHL1 and p-GSL) in Control patients and in patients with GM-IVH exposed to early parental stimulation and in patients that did not have this stimulation. We conducted a parallel study analyzing all pathological, cognitive, and peripheral markers separately in males and females (see supplementary data). While in many cases the results were similar to those observed when all animals were analyzed together, we cannot obviate that the limited number of animals in these subgroup analyses constrained the interpretability and scope of the findings.
In our hands, GM-IVH resulted in a reduction of the brain/body weight ratio, and long-term exposure to EE restored these to control values. Further assessment of brain morphology revealed no effect of the lesion in hippocampus size, in line with previous observations [15, 27, 40], suggesting that the hippocampus is relatively spared in this model. EE had a limited repercussion on hemisection and cortical sizes; however we observed that after 8 weeks of EE, ventricle enlargement, a common feature of patients with GM-IVH [45] and of this model [15, 27, 40], was significantly ameliorated. Our data are in agreement with previous studies showing that EE tends to reduce ventricle enlargement after a hemorrhagic insult. Similarly, EE preserves striatum integrity in rats submitted to neonatal hypoxia-ischemia [46]. Also, while we detected an increase in plasma and CSF NfL levels after GM-IVH and a slight improvement was observed after exposure to EE, this effect did not result in significant differences. The overall beneficial outcomes of EE seem limited when brain atrophy is assessed. Nevertheless, our observations are in line with studies showing that EE might not overtly restore brain volumes but it may induce a wide range of more subtle structural and functional benefits in brain after the lesions [47]. In this sense, we observed that EE had a beneficial effect in maintaining the neuronal population in the SVZ. Similarly, tau phosphorylation, as an indication of axonal wellbeing, is increased in the striatum after GM-IVH, and EE partially limits this effect. Previous studies have shown mixed results in transgenic models of tauopathy [48, 49], however, no previous study has assessed the effect of exposure to EE in tau phosphorylation after GM-IVH. On the other hand, EE significantly increased neuronal complexity in the cortex. Interestingly, this effect was not limited to animals with GM-IVH, but it was also observed in Control mice, supporting the capacity of long-term exposure to EE to facilitate neuronal plasticity. Similarly, other studies have reported that EE ameliorates Purkinje cell dendritic atrophy in a model of prenatal stress and increases dendritic spine density in the CA1 region of the hippocampus in a model of neonatal hypoxia-ischemia [50].
Whereas it has been shown that moderate GM-IVH may increase cell division in the neonate SVZ [51], the majority of the studies show the deleterious effects of GM-IVH in proliferation and neurogenesis, both in patients and animal models [10, 15, 16, 40, 52]. We observed an overall compromise of neurogenesis in mice with GM-IVH that affected the SVZ, the DG and the cortex, and long-term exposure to EE has a beneficial effect at this level. To our knowledge, the effects of EE have not been tested in models of GM-IVH. However, in line with our observations, previous studies have shown that EE improves neurogenesis in the adult brain [53, 54] and studies in neonate models have also reported that early exposure to EE results in higher number of neurons in the hippocampus, with a large impact on hippocampal development [55].
We also analyzed the effects of long-term exposure to EE in other pathological features commonly associated with GM-IVH. In this sense, bleeding and inflammation are increased in patients and animal models [2, 52]. Hemoglobin is a potent activator of inflammation and released iron can induce oxidative damage. Iron and hemoglobin are also involved in ventricular dilation [9]. We observed an increase of small vessel bleeding after the lesions, in line with previous observations [27] and the effects of EE were limited at this level when different brain regions were analyzed. On the other hand, the increased presence of microglia after the lesions was significantly ameliorated by long-term exposure to EE. Whereas, with different paradigms and approaches, other studies addressing the stressor factors associated with NICU have revealed that early stimulation also results in a reduction of neonatal inflammation [56]. Similarly, EE seems to reduce blood levels of IL-1B in neonate males [57]. Other studies have also reported that EE may reduce hippocampal inflammatory response after stroke [58] in line with our observations.
GM-IVH results in grey matter damage by directly inducing neuronal loss [15, 27, 59] as well as by disrupting corticogenesis while reducing the number of neurons in the upper cortical layer, ultimately diminishing the volume of the cerebral gray matter [10]. However, widespread white matter damage is also observed in patients and animal models [12, 27, 60]. Therefore, we analyzed MBP levels and we observed lower levels of MBP in the cortex from mice with GM-IVH, in line with previous observations [27, 61]. Also, previous studies have reported that oligodendrocyte lineage proliferation and maturation are interrupted after GM-IVH, preventing proper myelination [62]. Long-term exposure to EE ameliorated this situation, suggesting a beneficial effect on white matter damage, in line with studies showing the beneficial effects of EE in the volume of the corpus callosum and the myelinated fibers, as well as the total volume of the myelin sheaths and total length of the myelinated fibers after middle cerebral artery occlusion [63]. Similarly, EE promotes oligodendrocyte maturation and myelination after perinatal hypoxia [64]. Whereas differences did not reach statistical significance, we also detected that plasma GFAP levels were increased after the lesions and reduced by exposure to EE. These observations are in line with previous studies showing increased immunoreactivity for GFAP and S100β in rats after GM-IVH induced by Col administration [65, 66] and those reporting that EE may reduce astrocyte reactivity after a neonatal hypoxic-ischemic insult [67].
GM-IVH results in cognitive impairment in patients, largely depending on the severity of the lesion [68]. We detected learning alterations in our animals when analyzed spatial memory in the MWM as well as episodic memory impairment in the NOD test, in line with previous observations [14, 15], and EE significantly improved these limitations. The beneficial effects of EE in cognition have been largely described in newborn complications [43, 69] and it has been recognized as a non-pharmacological neuroprotective strategy in different animal models of neurological and psychiatric diseases [70]. However, to our knowledge no previous work has assessed the effects of EE on GM-IVH.
We also analyzed peripheral p-GSL and UCHL1 as feasible markers of GM-IVH severity and prognosis. GSL is a circulating actin-binding protein that plays a protective role in response to tissue injury and may ameliorate harmful inflammatory responses by scavenging pro-inflammatory mediators [71]. Reduced levels of p-GSL are detected in patients with subarachnoid [72] or intracerebral [73] hemorrhage. Also, reduced p-GSL levels during the first month after birth may be linked to adverse outcomes in PT infants [74]. In our hands, GM-IVH results in a decrease of p-GSL levels, as previously observed in this model [14, 15] and in patients with GM-IVH [15], and similar results have been reported in neonate patients with hypoxic-ischemic encephalopathy [75], respiratory stress syndrome [74] or bronchopulmonary dysplasia [76]. Importantly, exposure to EE results in an increase of p-GSL levels and previous studies have hypothesized that p-GSL might be a prognostic marker for different complications while it may also qualify as a general health marker [77]. UCHL1 is widely expressed in neurons with limited presence in other tissues, therefore it has been proposed as a neuron-specific biomarker for detecting brain injury (for review [78]). Plasma UCHL1 levels were increased as previously observed in patients with GM-IVH [15] or hypoxic-ischemic encephalopathy [79]. Previous studies have suggested that UCHL1 might also be a biomarker of brain hemorrhage-related complications in the adult [72] and the fact that long-term exposition to EE reduces UCHL1 levels may support the predictive value of UCHL1 after GM-IVH.
We performed a parallel study on feasible plasma markers of brain damage in Control patients and in patients with GM-IVH. We detected an overall acute increase of NfL, GFAP and tau levels in patients with GM-IVH, although differences did not reach statistical significance due to large variabilities among patients. On the other hand, UCHL1 levels were significantly increased after GM-IVH, in line with previous observations [15]. We observed an overall reduction of plasma NfL, GFAP and UCHL1 in the long term, independently of whether patients received parental support and nurturing environment. While we cannot obviate that the number of patients and the lack of division in GM-IVH grades, importantly hampers these observations, we did detect a significant reduction of plasma tau levels in patients with GM-IVH that had parental support and nurturing environment, suggesting that different markers might be differentially affected by same conditions. Our data show that early exposure to EE reduces cognitive impairment and the neuroinflammatory process. It also limits brain atrophy by ameliorating neuronal loss and neuronal complexity while promoting neurogenesis. Altogether, these results provide an insight on the neuropathological features that are improved by long-term exposure to EE. Importantly, EE also results in the improvement of feasible peripheral markers of the disease and its prognosis, including p-GSL and UCHL1 and tau.
Data availability
all information would be provided upon reasonable request.
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Acknowledgements
We thank the animal facility: Servicio de Produccion y Experimentacion Animal (University of Cadiz) for their technical support.
Funding
Instituto de Salud Carlos III. Ministerio de Economia y Competitividad. Proyectos de Investigación en Salud (Codigo PI21/01499), co-funded by the European Union (IBF). Ministerio de Ciencia e Innovacion. Programa Estatal de Generacion de Conocimiento y Fortalecimiento Cientifico y Tecnologico del Sistema de I+D+i y del Programa Estatal de I+D+i Orientada a los Retos de la Sociedad, del Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion (PID2020-115499RB-I0). Ministerio de Ciencia, Innovación y Universidades. Programa Estatal para la Investigacion y el Desarrollo Experimental, del Plan Estatal de Investigación Científica, Técnica y de Innovación. Proyectos de Generación de Conocimiento (PID2024-160890OB-I00). Universidad de Cadiz. Plan Propio de Apoyo y Estimulo a la Investigacion y la Transferencia, financiados por el Programa Operativo FEDER Andalucia 2021-2027, con la colaboracion economica del Fondo Europeo de Desarrollo Regional (FEDER) y de la Consejeria de Universidad, Investigacion e Innovacion de la Junta de Andalucia (FEDER-UCA-2024-A2-10) (MGA). Plan Propio INIBICA 2023 (CO18/CO20) (MGA/IBF).
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IA-N: experiment design, data acquisition, analysis and interpretation, and manuscript drafting; LM and AdM: data acquisition, analysis and interpretation; AR-M: data acquisition, analysis. IB-F: design and analysis, data acquisition and critical revision of the manuscript for intellectual content; SL-L: study concept and design, data acquisition, analysis and interpretation, and critical revision of the manuscript for intellectual content; MG-A: study concept and design, analysis and interpretation, drafting and critical revision of manuscript. All authors provided critical feedback and helped shape the research, analysis, and manuscript.
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All experimental procedures were approved by the animal Care and Use Committee of the University of Cadiz in accordance with the guidelines for care and use of experimental animals (European Commission Directive 2010/63/UE and Spanish Royal Decree 53/2013). Studies with patients were approved by the local Research and Ethics Committee and had parental or legal guardian signed informed consent.
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Atienza-Navarro, I., Maeso, L., Del Marco, A. et al. Exposure to enriched environment and parental care ameliorates brain pathology and behavioral impairment after germinal-matrix intraventricular hemorrhage. Mol Psychiatry (2025). https://doi.org/10.1038/s41380-025-03353-x
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DOI: https://doi.org/10.1038/s41380-025-03353-x
