Activin A Improves White Matter Injury in Neonatal Rats via Noggin/bmp4/id2 Signaling

Background Activin A (Act A) has been revealed to enhance the differentiation of oligodendrocyte progenitor cells (OPCs) in vitro. Here we aim to elucidate its roles and mechanisms in a rat model of white matter injury (WMI). Act A was injected into the lateral ventricle of a hypoxia-ischemia induced WMI rat model. Hematoxylin & eosin staining was used to detect pathological changes. Immunouorescence staining was used to assess OPC proliferation, migration, apoptosis, and differentiation. Myelin sheath and axon formation were detected via immunouorescence staining, Western blotting, and electron microscopy. Behavioral assessment of rats was performed with the Morris water maze test. immunouorescence staining Representative immunouorescence staining of Id2 (red) expression at P21. Quantitative analysis of the mean uorescence intensity of Id2 was performed. It showed that the expression of Id2 was signicantly decreased after Act A treatment. Scale bar, 20 µm. Detection of Noggin/BMP4/Id2 expression by Western blotting Western blotting was performed to quantify the expression of Noggin/BMP4/Id2. It showed that the expression of Noggin was signicantly upregulated in the Act A group compared with the PBS group, while BMP4/Id2 was signicantly downregulated.

mediators of signal transduction in the CNS, and their formation is fundamental for brain development and function [3]. The myelin sheath is the major component of the white matter, which is formed by mature oligodendrocytes (OLs) differentiated from oligodendrocyte progenitor cells (OPCs) [4]. Mounting evidence suggests that OPCs are the key insulted cells when WMI occurs, resulting in a wave of acute immature OLs death and the inhibition of OPCs differentiation, nally hindering myelination and blocking the formation of ensheathed axon [5]. Therefore, enhancing OPC differentiation via endogenous or exogenous molecules might be a powerful strategy to promote myelination in WMI.
Activin A (Act A) is a widely expressed homodimer composed of two β A chains. Sequence analysis showed that β subunit of Act A has the typical structural features of the transferring growth factor-β superfamily, and the mature human β A chain of Act A has 100 % amino acid sequence identity in cattle, cats, mice, pigs, etc., indicating its highly conserved structure [6]. In the nervous system, Act A can be secreted by both neurons and glial cells, which exert neuroprotective effect. Previous studies have found that adding recombinant Act A protein to OPCs cultured in vitro promoted OPC proliferation and differentiation [7]. However, the role of Act A in neonatal WMI and the mechanisms involved are unknown.
In the present study, we established a WMI model in 5-day postnatal (P5) Sprague-Dawley (SD) rats, and examined whether exogenous Act A treatment contributes to the recovery of WMI. Besides, the potential mechanisms underlying the role of Act A in WMI were also explored.

Methods
Animals and WMI modeling P5 SD rats (average weight 10-15 g) were purchased from Sichuan Dashuo Animal Science and Technology Co., Ltd (Chengdu, China). The rats were randomly divided into four groups: a Sham-operated group (Sham); a WMI model group (WMI); a group receiving Act A / phosphate-buffered saline (PBS) treatment after 24 h of WMI modeling (Act A/ PBS); and a group receiving Id2 overexpression lentiviral vector (Id2) and its corresponding vehicle(V), 6 h after Act A treatment (Id2/V).
The WMI model was established using the following procedure [8] (Back, 2017): First, P5 neonatal rats were xed on their backs after general anesthesia. The neck skin was then longitudinally incised for a length of about 1cm, and the right carotid artery was exposed and ligated after separation from glands and muscle tissue. After surgery, the rats were returned into an incubator for 30 min to recover. Then, they were placed in an 8%-oxygen and 92%-nitrogen cabin (8% O 2 and 92% N 2 ) with a gas ow rate of 3 L/min for 2 h to induce WMI. Rats were kept on a heating pad during surgical procedures to maintain the body temperature at 36-37 o C. The rats of the Sham group were only subjected to neck incision for dissociating the right carotid artery, without ligation or hypoxia. Following surgery, all neonatal rat pups were returned to their cages.

Drug treatment
To establish the Act A/PBS group, rats were injected with 5ul of Act A (12.5 mg/kg, 25 mg/kg, 50 mg/kg)/PBS after 24 h of WMI induction using a Hamilton syringe needle (Hamilton, USA) via the lateral ventricle (LV), located 2 mm posterior and 2 mm lateral (right) from the bregma with a 2 mm needle depth. 4 ul of Id2 overexpression lentiviral vector/vehicle was injected via the LV after 6 h of Act A treatment to establish the Id2/V group.
Hematoxylin & eosin staining At P7, rats were sequentially perfused with 0.9% normal saline and 4% paraformaldehyde (100 mL each), after which the tissues were extracted and post-xed in a 4% paraformaldehyde solution for 24-36 h at 4 o C. Then, the tissues were para n-embedded and serial sectioned in coronal position for 5mm, and three sections containing corpus callosum (CC) (0.26mm-1.80mm behind the anterior fontanelle according to the mapping of rat brain) were selected for analysis. Finally, the sectioned tissues were stained with hematoxylin & eosin (HE) and observed using a Leica inverted optical microscope (Leica, Germany). In each animal, four randomly selected elds were examined. Six animals per group were analyzed.

Immuno uorescence staining
Brains were taken at P7, P14, P21, P28, and P35 and post-xed in 4% paraformaldehyde at 4 o C for at least 48 h, then embedded in 2-3% agarose. Coronal brain sections (thickness 40 µm) were cut using an oscillating tissue slicer (Leica, Germany). Three sections containing CC (0.26 mm-1.80 mm behind the anterior fontanelle according to the mapping of rat brain) were selected for analysis. The sections were rst washed in PBS and incubated in 0.3% Triton X-100 at room temperature for half an hour, and then incubated for 1 h in fetal calf serum to inhibit non-speci c binding. Second, the brain sections were incubated with the primary antibodies (rabbit anti-Id2 polyclonal antibody,1:500, novusbio; rabbit anti-BMP4 polyclonal antibody, 1:1000, abcam; rabbit anti-Olig2 polyclonal antibody, 1:500, Millipore; rabbit uorescence imaging was performed using a confocal laser scanning microscope (Olympus, Japan) and FV-ASW-3.1 software (Olympus). The mean uorescence intensity was de ned as the ratio between the sum of the integral optical density of the target protein and the area. Positive cells and mean uorescence intensity counting were performed for each eld with a 40X objective lens ( eld size, 0.24 mm 2 ), using the Image J software. In each animal, four randomly selected elds from the CC were examined. Six animals per group were analyzed.

Western blotting
The isolated CC was treated with a brain tissue protein extraction kit (Chengdu beibokit, BB-31227-1). Lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. The protein concentration was determined through a BCA protein assay kit (Pierce) using bovine serum albumin (BSA) as the standard. Protein samples were separated on sodium dodecyl sulfate (SDS)-polyacrylamide gels. The protein was then transferred to polyvinylidene di uoride (PVDF) membranes, which were blocked in 5% non-fat dry milk in TBS containing 0.05% Tween 20 for 1 h at room temperature, with rotation. The membranes were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-Act A polyclonal antibody (1:500, novusbio), mouse anti-MBP monoclonal antibody (1:500, arigo), rabbit anti-MAG monoclonal antibody (1:100, CST), rabbit anti-PLP polyclonal antibody (1:500, abcam), mouse anti-Tau1 monoclonal antibody (1:500, Millipore), mouse anti-SMI31 monoclonal antibody (1:500, biolegend), mouse anti-SMI312 monoclonal antibody (1:500, biolegend), mouse anti-Noggin monoclonal antibody (Abcam,1:200), rabbit anti-BMP4 polyclonal antibody (1:500, abcam), rabbit anti-Id2 polyclonal antibody (1:500, novusbio), and a mouse anti-actin polyclonal antibody (Santa Cruz Biotechnology, 1: 5000) was detected as the loading control. Following washes, the membranes were incubated with peroxidase conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology, 1: 5000) in blocking solution for 1 h. The bound antibodies signals were developed by enhanced chemiluminescence (Pierce, Rockford, IL). The immunoreactivity of the signal bands was quanti ed using the Image J software. The relative expression level of the target protein was calculated as the target protein integrated density values (IDVs) relative to actin IDVs. All experiments were repeated at least three times to ensure the reproducibility of the results.
The rat brains were taken and sectioned to the size of approximately 1 mm 3 including the CC at 1.2 mm to 3.0 mm posterior to the bregma. The sectioned tissue was pre-xed with a mixed solution of 3% glutaraldehyde. Then post-xed in 1% osmium tetroxide, dehydrated in an acetone series, ltrated in Epox 812, and embedded. Next, the semi-thin sections were stained with methylene blue, the ultrathin sections were stained with uranyl acetate and lead citrate. Finally, the ultrathin sections were examined with a transmission electron microscope (EM) (H-600IV; Hitachi, Japan). Myelinated axons were counted for each eld using the Image Pro Plus 6.0 software. In each animal, four randomly selected elds from the CC were examined. Six animals per group were analyzed.

Morris water maze
Behavioral testing using the Morris water maze (MWM) was performed from P29 to P35. The testing facilities includes a circular tank (1.5 m in diameter) and a location-constant platform (14 cm in diameter) placed 1.5 cm under the surface of the water. The water temperature was maintained at 25±1 o C during testing. The test consists of two parts, namely place navigation training and space exploration, both of which are aimed to test spatial learning and memory ability.
The place navigation training was conducted during the rst 6 days (P29-P34), For which the rats were trained to swim in the four alternating quadrants. From each quadrant, the rats swim in the water for 120 s. If the platform is successfully found during this period, the escape latency is recorded as the time at which the rats nd the platform. If the rats fail to nd the platform within 120 s, it is guided to it by a researcher and stays on the platform for 30 s, and the escape latency time being recorded as 120 s. The time at which the rat found the platform in each training session was recorded, and the average of the four quadrant latency periods was computed as a daily nal score representing the ability to acquire the spatial information.
The platform was removed and the space navigation test was conducted at P35 to test the memory retention ability of the rats, 24 h after the last place navigation training. The rats were free to swim in the tank for 120 s from the third quadrant starting point. The trials were recorded using a video camera on the ceiling, and the platform crossing time was calculated and analyzed using the tracking system (Mengtai, China).

Quanti cation analysis and statistics
All images were acquired from the same CC area. All data were presented as mean ± standard deviation (SD). All graphs were produced using GraphPadPrism 8.0. A Student's t-test was used when comparing between two groups. Analysis of variance (ANOVA) was used when comparing more than two groups, followed by the Student's t-test if homogeneity of variance was assumed or by Dunnett's test if homogeneity of variance was not assumed. All statistical analyses were performed using SPSS 23.0. Pvalues *P <0.05, or **P < 0.01 were considered statistically signi cant.

Results
HI attenuated the expression of endogenous Act A in the rat brain According to the developmental characteristics of rat brain, we set a time course for each assay, displayed as a schematic diagram in Figure 1A. We conducted Western blotting to detect the endogenous expression of Act A. It showed that Act A expression was reduced in the WMI group compared with the Sham group ( Figure 1B), indicating that HI leads to evident reduction of Act A expression in neonatal rats.

Act A alleviated pathological damage of WMI rats
To detect the distribution of Act A after injected via the LV, we made Act A-EGFP protein and conducted immuno uorescence tracing. Fluorescence scanning showed that Act A-EGFP distributed in the cortex and white matter (including CC) from day 1 to day 28 after LV injection ( Figure 1C). To select the optimal usage of Act A for WMI therapy, we used three dose concentrations (low, 12.5 mg/kg; medium, 25 mg/kg; and high, 50 mg/kg) and three time points (12 h, 24 h and 48 h) for Act A administration after HI. We examined the pathological changes in the brain white matter and liver via hematoxylin & eosin (HE) staining, and detected rat weight at P14. HE staining for white matter showed that Act A improved necrosis and liquefaction after 24 h of HI ( Figure 1D). The medium and the high doses of Act A gave better results than the low dose, while the difference between the medium and the high dose was not obvious ( Figure 1D). However, hepatic HE staining at P21 and body weight analysis showed that the high dose rats underwent more liver damage, with lower body weight and poor state ( Figure 1D). Therefore, the subsequent experiment was performed with the medium dose of Act A (25 mg/kg). To detect the overall expression level of Act A in the brain after exogenous Act A injection, we conducted western blotting at P6. It showed that Act A was abundantly expressed in the Act A group, as compared with the PBS group ( Figure 1E). Collectively, these results suggest that Act A was successfully upregulated in WMI via exogenous Act A supplement.
Act A treatment promoted OPC proliferation and differentiation in the neonatal rat brain after HI OPCs are the primarily insulted cells when WMI occurs. We next examined whether Act A affects OPC function in vivo. At P7, we investigated the proliferation, migration and apoptosis of OPCs via double immunostaining of Olig2 with Ki67, Vimentin, and CC3, respectively. It showed that the number of Ki67/Olig2 positive cells was signi cantly increased in the Act A treatment group compared with the PBS group ( Figure 2A). However, the number of Vimentin/Olig2 ( Figure 2B) and CC3/Olig2 positive cells ( Figure 2C) was not different in the Act A group compared with the PBS group. Furthermore, we examined whether Act A contributes to the differentiation of OPCs to OLs. At P7 and P14, we quanti ed the number of OPCs in the white matter via double immunostaining of Olig2 with the OPCs speci c marker NG2 among the experimental groups. It showed that the number of NG2/Olig2 positive cells and the mean NG2/Olig2 uorescence intensity at both time points were signi cantly increased in the Act A group compared with the PBS group ( Figure 3A). Then, at P14 and P21, we quanti ed the number of preoligodendrocytes (pre-OLs) in the white matter by double immunostaining Olig2 with the pre-OLs speci c marker O4 among the experimental groups. As expected, the number of O4/Olig2 positive cells and the mean O4/Olig2 uorescence intensity were also signi cantly increased at both time points in the Act A group compared with the PBS group ( Figure 3B). Next, at P21 and P28, we performed double immuno uorescence staining of the mature OLs marker CC1 with Olig2 in the white matter. It showed that the number of CC1/Olig2 positive cells and the mean CC1/Olig2 uorescence intensity were signi cantly increased in the Act A group compared with the PBS group ( Figure 3C). Together, these results indicate that Act A promotes the proliferation and differentiation of OPCs, whereas it shows no signi cant effect on OPC migration and apoptosis.
Act A treatment promoted myelination and axon formation in the neonatal rat brain after HI We then examined the myelination of the white matter by assessing the expression of MBP, PLP, and MAG through immunostaining and western blotting. At P28, the expression of MBP, PLP, and MAG was signi cantly enhanced in the Act A group compared with the PBS group ( Figure 4A). Consistently, at P35, the expression of the axon markers Tau1, SMI31, and SMI312, was signi cantly increased in the Act A group compared with the PBS group ( Figure 4B). At P35, EM also showed more myelinated axons in the CC of brains in the Act A group compared with the PBS group ( Figure 4C). Collectively, these results indicate that exogenous Act A supplement in neonatal WMI contributed to the long-term recovery of myelination and axon formation.
Act A treatment enhanced the behavioral performance of WMI rats.
The MWM test was conducted to compare the behavioral abilities among the experimental groups from P29 to P35, by calculating the average escape latency and platform crossing times during swimming. It showed that the average escape latency was signi cantly decreased from P31 in the Act A group compared with the PBS group ( Figure 5A), and the frequency of platform crossing was signi cantly increased at P35 in the Act A group compared with the PBS group ( Figure 5B). These results suggest that the exogenous Act A supplement contributes to the long-term behavioral improvements in terms of learning and memory.
Act A treatment enhanced the expression of Noggin, while inhibited BMP4/Id2 expression.
We then examined the possible down-stream effectors of Act A. At P21, we performed immuno uorescence staining to assess the expression of bone morphogenetic protein 4(BMP4) and inhibitor of DNA binding 2(Id2) among Sham, PBS and Act A groups. We found that they were signi cantly increased in the PBS group compared with the Sham group ( Figure 6A-B), while signi cantly decreased after Act A treatment ( Figure 6A-B). Consistently, Western blotting was conducted to detect Noggin/BMP4/Id2 expression. It showed that Noggin expression was signi cantly enhanced in the Act A group compared with the PBS group, whereas both BMP4/Id2 proteins showed a signi cant downregulation in the Act A group compared with the PBS group ( Figure 6C). Collectively, these ndings suggest that Id2 might be the down-stream effector of Act A, which might be activated through Noggin/BMP4 signaling.

Id2 is the crucial downstream effector of Act A in WMI
To further verify that Id2 is the key downstream effector of Act A in WMI, we overexpressed it in the Act A group with an Id2 overexpression lentiviral vector (1x10 9 TU/ml), a corresponding vehicle-only (V) group was set up as the control. Fluorescence scanning showed that Id2-EGFP (green uorescence) distributed in the cortex and white matter (including CC) for up to 4 weeks. On the rst day after injection, Id2-EGFP has already appeared in the CC (white arrow) and cortex (red arrow). From the 7th day to the 21st day after injection, the uorescence intensity of Id2-EGFP in the CC and cortex is signi cantly increased, whereas on the 28th day after injection, the uorescence intensity of Id2-EGFP in the CC and cortex is reduced signi cantly ( Figure 7A). At P21, we performed immuno uorescence staining and Western blotting to detect Id2 expression in the V and Id2 groups. It showed that Id2 was signi cantly increased in the Id2 group compared with the V group ( Figure 7B).
The effects of Id2 overexpression were further examined. HE staining at P7 showed more white matter necrosis in the Id2 group compared with the V group ( Figure 7C). Id2 overexpression did not affect the proliferation of OPCs, as indicated by the immuno uorescence staining and statistical analysis of Ki67/Olig2 positive cells, which were not signi cantly different between the V and Id2 groups ( Figure 8A).
Regarding OPC differentiation, using different time points (from P7 to P28) corresponding to speci c differentiation stages, we found that the number of positive cells and the mean uorescence intensity for NG2/Olig2, O4/Olig2 and CC1/Olig2 ( Figure 8B) were signi cantly decreased in the Id2 group compared with the V group. Consistently, we found that overexpression of Id2 attenuated the expression of MBP, PLP, and MAG at P28 (Figure 9A), as well as that of Tau1, SMI31, and SMI312 at P35 ( Figure 9B) after Act A treatment, as indicated by immuno uorescence staining and western blotting. Similarly, at P35, EM showed less myelinated axons in the Id2 group compared with the V group ( Figure 9C). The MWM test showed that the average escape latency was signi cantly increased from P31 in the Id2 group compared with the V group ( Figure 10A), and the frequency of platform crossing signi cantly decreased at P35 in the Id2 group compared with the V group ( Figure 10B). The performance of the Id2 group was similar to that of the PBS group, suggesting that Id2 overexpression triggers the restoration of behavioral dysfunction. Collectively, these results indicated that Id2 overexpression reversed the effect of Act A in WMI, suggesting that Id2 is the crucial downstream effector of Act A in WMI.

Discussion
As OPCs are the major insulted cells in WMI, protecting OPCs has become the key strategy for WMI recovery [9]. Recent reports of the involvement of Act A in the regulation of OPC maturation in vitro led us to examine its role in WMI in vivo [3]. In an adult rat model of focal cerebral ischemia that simulates a stroke, it was found that the expression of Act A protein around the infarction was higher than that of normal rats [10]. However, in this study, we found that the expression of endogenous Act A was signi cantly reduced after WMI in newborn rats. We suspect that this discrepancy may be due to the differences in the age of rats and the models constructed. We try to replenish a certain dose of Act A externally to compensate for the decrease in Act A caused by WMI. Considering the obstacles of the blood brain barrier, we tried to inject Act A via the lateral ventricle in the rat model of WMI. The Act A-EGFP tracing experiment showed that Act A-EGFP protein was distributed in the cerebral cortex and white matter (including the CC) from day 1 to day 28 after injection, indicating that after lateral ventricle injection, Act A can enter and exist in the brain for up to 4 weeks, ensuring its e cacy after just one injection. To explore the therapeutic time window of Act A molecules, we set up injections before and after molding, single injection and continuous multiple injections in our preliminary study, and found that the effect of injection before molding is better than injection after molding, but the effect of multiple injections and single injection is not obvious. Considering that the injection before the model is di cult to simulate the actual clinical treatment, we decided to adopt the injection program after the model. Next, three different concentrations of Act A were analyzed to determine the optimal dosage. We found that the medium dose of Act A led to WMI recovery without obvious side effects. Although lateral ventricle injection is a powerful medication method, it is a traumatic operation, which will limit its clinical usage.
Recently, some non-traumatic methods bringing drugs into the brain overcoming the blockage of the blood brain barrier have emerged, mostly based on material-based deliveries. For example, in 2012, Wang et al. effectively delivered a glial cell-derived neurotrophic factor to the brain of rats via conjugatedbiotinylated lipid-coated microbubbles [11]. Our future work will explore more feasible pathways to deliver Act A into the brain.
According to the brain developmental characteristics of rats, the formation of myelin sheath wrapped axons undergoes several successive stages, starting from OPC differentiation [8]. OPC differentiation occurs mainly in the rst week after birth and continues into the next week. The main process during the late second week after birth is the formation of pre-OLs, while the third week after birth is used for the formation of OLs. The myelin sheath, which wraps the axons to provide the basis for the transmission of neural signals, is mostly formed later [12]. In the present study, we set different time points for the detection of the progressive differentiation and maturation of OLs: P7 and P14 for the detection of OPCs, P14 and P21 for the detection of pre-OLs, P21 and P28 for the detection of OLs, P28 for the detection of myelin formation, and P35 for the detection of myelin-wrapped axons. This experimental design allowed us obtaining an overall perspective of how Act A affects the progression of WMI.
Previous studies revealed that Act A exerts its neuroprotection roles mainly through Smad-dependent pathways. Recently, it was indicated that Act A can exert its function through Smad-independent pathways such as nuclear factor-κB, extracellular signal-regulated kinase (ERK1/2), ubiquitin-proteolytic, mitogen-activated protein kinase (MAPK), and AKT pathways [13]. Our study revealed that the repairing effect of exogenous Act A on WMI of newborn rats is mainly achieved by promoting the differentiation and maturation of OLs. Previous studies have reported that the Id2 participates in different stages of OLs differentiation and is a key molecule that regulates the differentiation and maturation of OLs [14]. Id2 can inhibit the expression of myelin formation genes and keep OPCs in an undifferentiated state, thereby inhibiting the differentiation of OPCs and the production of mature OLs [15]. Based on these existing reports, we overexpressed Id2 in WMI rat model and con rmed that it was the crucial downstream effector of Act A. Then we want to clarify how Act A regulates Id2. Numerous studies have found that Act A can regulate cell differentiation by interacting with BMP4 [16][17][18][19], while BMP4 can regulate the differentiation and maturation of OLs by regulating its downstream target molecule Id2. The BMP4/Id2 signal will hinder the differentiation of OPCs into OLs [20]. At the same time, Noggin is a key upstream molecule regulated by BMP4. Increased Noggin expression can inhibit BMP4 expression, while Act A can enhance Noggin expression [21,22]. Therefore, we speculate that the regulatory effect of Act A on Id2 may be achieved through the Noggin/BMP4 pathway. Therefore, we detected the expression of Noggin/BMP4/Id2 after WMI or Act A treatment. It showed that the expression of Noggin was inhibited after WMI, while the expression of BMP4 and Id2 was increased. After Act A treatment, the expression of Noggin was signi cantly upregulated, while the expression of BMP4 and Id2 was signi cantly downregulated. Besides, Overexpression of Id2 blocked the repairing effect of Act A on WMI. Collectively, these results suggest that Act A might regulate Id2 through Noggin/BMP4 signaling. We speculate that Act A enters the intercellular space through diffusion after it is injected into the lateral ventricle, and binds to Act A receptors on the surface of OPCs, then activates the expression of Noggin, inhibits the expression of BMP4 and Id2, thereby relieves the negative regulatory factors modulating OPC differentiation, then promoted the formation of myelin sheath, reduced the pathological damage of brain white matter, and nally repaired the neurobehavioral ability of rats. However, since the effects of blocking or overexpression of Noggin and BMP4 have not been tested, it is only a possible speculation that Act A regulating Id2 through the Noggin/BMP4 signaling. More evidence is needed to clarify its causality.

Conclusions
In summary, our study indicated that exogenous Act A treatment could protect WMI via Noggin/BMP4/Id2 signaling. Besides, our study also found that OPC proliferation was not signi cantly altered after Id2 overexpression, suggesting the presence of another mechanism by which Act A regulates OPC proliferation. Although Act A has been used as a diagnostic and prognostic biomarker for some brain diseases [23], it has not been used to treat brain damage in clinical practice. Our ndings demonstrated for the rst time that exogenous Act A treatment could alleviate WMI in the developing brain, providing a potential agent to treat neonatal WMI in the future. Besides, its roles in other types of white matter damage, such as multiple sclerosis and hereditary multi-infarct dementia, are worthy to be explored.    Act A treatment promoted myelination and axon formation in the neonatal rat brain after WMI (A) Act A   Overexpression of Id2 attenuated axon formation Representative immuno uorescence staining images of the expression of axon markers Tau1, SMI31, and SMI312(red) at P35. The mean uorescence intensity for Tau1, SMI31, and SMI312 was quanti ed. Western blotting and corresponding quanti cation were performed to measure the expression of Tau1, SMI31, and SMI312. It showed that the expression of Tau1, SMI31, and SMI312 was signi cantly attenuated in the Id2 group compared with the V group. Scale bar, 1000 μm, 20 µm. (C) Overexpression of Id2 attenuated myelinated axon formation Representative EM images at P35. It showed that the Id2 group with less myelinated axons compared with the V group.

Figure 10
Overexpression of Id2 compromised the behavioral performance of rats after Act A treatment (A) Daily average escape latency of SD rats during the training period It showed that the average escape latency was signi cantly increased in the Id2 group compared with the V group. (B) The average platform crossing times of SD rats during the test period It showed that the frequency of platform crossing was signi cantly decreased in the Id2 group compared with the V group.