ZJU-37 Promoted OPC Survival and Myelination in a Rat Neonatal White Matter Injury Model With hOPC Graft

Background: Recent evidence suggests that ZJU-37 plays an important role in inhibiting inammation and cell apoptosis. White matter injury (WMI), a leading cause of neurodevelopmental disabilities in preterm infants, which is characterized by extensive myelination disturbances and demyelination. Neuroinammation, leads to the loss and differentiation-inhibition of oligodendrocyte precursor cells (OPCs), represents a major barrier to myelin repair. Whether ZJU-37 can promote transplanted OPCs derived from human neural stem cells (hOPCs) survival, differentiation and myelination remains unclear. In this study, we investigated the effect of ZJU-37 on myelination and neurobehavioral function in a neonatal rat WMI model induced by hypoxia and ischemia. Methods: In vivo, P3 rat pups were subjected to right common carotid artery ligation and hypoxia, and then treated with ZJU-37 or/and hOPCs, and OPCs apoptosis, myelination, glial cell and NLRP3 inammasome activation together with cognitive outcome were evaluated at 12 weeks after transplantation. In vitro, the effect of ZJU-37 on NLRP3 inammasome activation in astrocyte induced by oxygen-glucose deprivation (OGD) were examined by western blot and immunouorescence. The effect of ZJU-37 on OPCs apoptosis induced by the conditioned medium from OGD-astrocyte was analyzed by ow cytometry and immunouorescence. Results: ZJU-37 combined with hOPCs more effectively decreased OPC apoptosis, promoted myelination in the corpus callosum and improved behavioral function compared to ZJU-37 or OPCs treatment. In addition, the activation of glial cells and NLRP3 inammasome was reduced by ZJU-37 or/and OPCs treatment in the neonatal rat WMI model. In vitro, it was also conrmed that ZJU-37 can suppress NLRP3 inammasome activation in astrocytes induced by OGD. Not only that, the conditioned medium from OGD-injured astrocytes (OGD-astrocyte-CM) treated with ZJU-37 obviously attenuated OPC apoptosis and dysdifferentiation caused by the OGD-astrocyte-CM. Conclusions: ZJU-37 may promote OPC survival, differentiation and myelination by inhibiting NLRP3 inammasome activation in a neonatal rat model of WMI with hOPC graft. in 2.5% glutaraldehyde at 4°C overnight for post-xation. After transferring to osmium tetroxide for 1 h at room temperature and dehydrating with increasing ethanol concentrations, the tissues were embedded in epoxy resin embedding medium. Ultrathin sections (50 nm) were made from the resin-embedded samples and observed under a transmission electron microscope. A total of 100 root axonal bers from three samples in each group were measured at a magnication of 15,000 × g. Following image acquisition, axon and myelin diameters were measured using Image J software. The g-ratio, which is a structural index of remyelination and dened as the ratio of the inner axonal diameter to the total outer ber diameter and lower ratios indicate more extensive myelination, was subsequently assessed. The average scores of ultrastructural myelin damage were


Introduction
Preterm birth is a global public health issue [1]. According to estimates by the World Health Organization, there are nearly 15 million preterm births annually worldwide, with an overall incidence of 11.1% [2]. As many as 25-50% of preterm birth survivors develop chronic neurodevelopmental disorders, which manifest as cognitive, motor, and sensory disorders [3,4], among which the white matter injury (WMI) is the most common [5]. It characterized by extensive myelination disturbances, demyelination and in ammatory reaction which can damage axons [6]. The period of highest risk for WMI is 23-32 weeks post-conception, during which time the pre-oligodendrocytes (pre-OLs) are more abundant than other cells [7]and the development of central nervous system (CNS) exhibits vulnerability to various insults such as hypoxia, ischemia, infection and in ammation [8]. Unfortunately, the pathogenesis of neonatal WMI is unclear and no speci c therapies are currently available other than supportive treatment [9]. Studies thus far have suggested that remyelination is one of the pivotal mechanisms in promoting functional recovery following neonatal WMI [6, 10,11] and that the inhibition of demyelination is one of the key challenges of therapeutic peptides.
As precursors of oligodendrocytes (OLs), oligodendrocyte precursor cells (OPCs) can proliferate and generate immature OLs, which then differentiate into mature OLs and wrap around axons to form the myelin sheath [12]. After in ammation-induced white matter damage, myelin regeneration occurs in demyelinating lesions, with endogenous OPCs maturing into myelin-producing OLs; however, endogenous OPCs are in a state of relative insu ciency and gradually begin to failing to differentiate properly, and thus remyelination is incomplete [13]. Therefore, transplantation of exogenous OPCs may promote remyelination in the area of white matter lesions area [14]. Moreover, transplantation of embryonic stem cells(ESCs) [15], mesenchymal stem cells [16,17], and neural stem cells (NSCs) [18] has been shown to enhance the repair of neurological de cits resulting from perinatal brain injury. Recently, OPC transplantation therapy has been investigated, including for lysophosphatidylcholine-induced demyelination [19], spinal cord injury [20][21][22], and radiation-induced injury [23], with therapeutic effects achieved in all animal models. Chen et al. [11] showed that transplanted mouse ESC-derived OPCs can migrate in vivo, differentiate into myelin, and ultimately provide neuroprotection to WMI mice. Given the species-speci c differences in myelin development and regeneration between human and mouse OPCs [7], transplantation of OPCs derived from human neural stem cells (hOPCs) is clearly more conducive to clinical translational research. Moreover, our previous studies demonstrated that hOPCs alleviates demyelination, but are not abundant enough to completely repair this damage [24], so stimulating an endogenous regenerative response may be an effective treatment and many small-molecule compounds can promote OPC differentiation and/or remyelination.
Numerous studies have reported that neuroin ammation results in myelin-producing OLs undergoing apoptosis and the loss of the myelin sheath. When the myelin sheath fails to regenerate, this ultimately leads to neurological disability [9]. Previous studies have reported astrocyte and microglial activation in demyelinating diseases. The diminishing of the in ammatory response during demyelination can contribute to nervous system recovery and prevent demyelination deterioration [25,26]. In ammasomes are a family of cytosolic pattern recognition receptors (PRRs) [27]. The Nod-like receptor pyrin domain containing 3 (NLRP3) recruits and activates pro-caspase-1 into caspase-1 through ASC (apoptosis associated speck-like protein containing caspase recruitment domain) and then forms the in ammasome, which leads to the release of IL-1β and IL-18 into the extracellular environment and induces neuroin ammation and damage to axons [25,28,29]. It was recently veri ed that the NLRP3 in ammasome is overactivated during demyelinating disorders [26]. Our previous studies demonstrated that inhibition of NLRP3 in ammasome activation can alleviate demyelination in the corpus callosum induced by cuprizone [9,25,30]. Therefore, molecules that regulate NLRP3 may prevent demyelination and restore neurological dysfunction of neonatal WMI by reducing in ammatory reactions. ZJU-37 is a new type of threonine/serine protein kinase-1/-3 (RIP1/RIP3) dual inhibitor and a potential candidate as a therapeutic drug for neurological diseases [31]. RIP3 can promote RIP1 phosphorylation and then form a stable phosphorylated RIP1 and RIP3 complex with a signi cant increase in intracellular reactive oxygen species. An inhibitor of RIP1 effectively regulated in ammation in microglial cells and apoptosis in oligodendrocytes in a recent study [32], suggesting its therapeutic bene t for demyelination in vitro and in vivo [33][34][35]. ZJU-37 is an essential mediator of cell death processes and participates in in ammatory responses, but does not induce apoptosis by preventing RIP1/RIP3 complex formation. More speci cally, ZJU-37 alleviates lipopolysaccharide-induced acute liver injury in mice, effectively protects the myelin sheath, and promotes remyelination in lysophosphatidyl choline-induced demyelination and a multiple sclerosis animal model (unpublished data). Whether ZJU-37 can participate in demyelination in the neonatal rat WMI model remains unclear, and relatively few studies have focused on the effects of ZJU-37 on the injury and functional recovery of OPCs.
The evidence indicates that ZJU37 plays a critical role in inhibiting in ammation and cell apoptosis. To determine whether ZJU37 promotes remyelination more e ciently with hOPC graft, we transplanted hOPCs into WMI rat brains with ZJU37 intraperitoneal injection, and investigated the myelination and neurobehavioral functions. Furthermore, we intended to provide novel insights into the role of ZJU37 in the survival, differentiation and myelination of hOPCs in vitro and explored the related mechanism. The data presented here demonstrate for the rst time that ZJU-37 not only promotes OPC survival, differentiation, and myelination by inhibiting NLRP3 in ammasome activation in a neonatal rat WMI model with hOPC graft, but also promoted myelination and improved behavioral function with hOPC graft compared to ZJU-37 or OPCs alone.

OPC preparation and cyclosporine injection
NSCs were generated from the CNS tissue of spontaneously aborted human female fetuses at gestational week 10-12 from the Pediatrics Laboratory of the Sixth Medical Center of PLA General Hospital (Beijing, China). Informed written consent was obtained from the mothers, and differentiated in vitro according to a modi ed version of a previously published protocol [36]. Brie y, the brain was homogenized into a suspension of single cells by mechanical dissociation. Cells were trypsinized and transferred to a neural differentiation medium containing DMEM/F12, leukemia inhibitory factor, basic broblast growth factor, epidermal growth factor, and nonessential amino acids (all from Gibco, Waltham). Ten-day old neurospheres were used for this experiment. NSC isolation, culturing, and differentiation were performed as previously described [36]. Purity and viability of transplanted NSCs was con rmed by cell morphology and immuno uorescence staining of speci c markers nestin (mouse IgG1, 1:1000, Abcam), A2B5 (rabbit IgG, 1:200, Abcam), platelet-derived growth factor receptor (PDGFRα, rabbit IgG, 1:800, Cell Signaling Technology), neuron-glial antigen 2 (NG2, rabbit IgG, 1:200, Millipore), O4 (mouse IgG, 1:200, Sigma), and glial brillary acidic protein (GFAP, mouse IgG, 1:500, Santa Cruz). All transplant recipients received cyclosporine (i.p, 10 mg/kg) daily three days before transplantation and continuing for a total of four weeks. Subsequently, the rats were administered cyclosporine (100 μg/mL) via their drinking water until perfusion. During the immunosuppression period, the cyclosporine dose was appropriately reduced or discontinued if the rats were found to have sustained weight loss, red eyelids, or nasal damage.

Neonatal rat WMI model and treatment
Clean-grade SD rat pups (P3) obtained from the Center of Experimental Animals of Xuzhou Medical University were randomly divided into control (Ctrl), WMI and vehicle-treated (Vehicle) group, WMI with ZJU-37-treated (ZJU-37) group, WMI with OPC transplantation (OPCs) group and WMI with ZJU-37treated and OPC transplantation (ZJU-37+OPCs) group (n=12 per group). Animals were housed in an airconditioned room with a 12 h light/dark cycle, and provided with adequate food and water. All efforts to reduce the number of animals used and minimize animal suffering were made. All WMI groups were generated as described previously [36]. Brie y, P3 pups were placed in a refrigerator at -20°C for 7-10 min. After freezing anesthesia, the right common carotid artery was carefully isolated from the surrounding tissue and ligated. Then, the wound was sutured with an 8-0 suture and the time of operation was controlled to within 5 min. Upon completion of the surgery, pups were moved to the recovery area under a heat lamp for 10 min, returned to their mother, and allowed to recover for an additional 1 h before exposure to 6% oxygen (94% nitrogen saturation) at 37°C for 90 min in a humidi ed chamber. After monitoring recovery, the pups were returned to cages to continue feeding. Sham-operated rats without hypoxia were used as the Ctrl group. At P12, the ZJU-37 group was injected intraperitoneally with ZJU-37 (i.p, 10 mg/kg) once daily for the rst month, every other day for the second month and every third day for the third month until euthanasia. The Vehicle group was administered DMSO (i.p, 10 mg/kg) whereas rats in the Ctrl group received no treatment. At P4, P6, P8 and P10, the degree of injury (mild/moderate to severe) was determined by observing the general condition of animals, neurobehavioral evaluation, and histopathology.
Moderately to severely injury rats were xed on a stereotaxic apparatus after narcotization with 4% chloral hydrate (4 mL/kg) at P12. A small incision was made through the midline to expose the skull. The anterior fontanelle was used as a guide to determine the puncture point whose coordinates from bregma were as follows: anteroposterior-1.0 mm; mediolateral-1.5 mm; dorsoventral-2.0 mm by brain stereotaxic apparatus. A 5 μL microsyringe was used to withdraw 3 μL cell suspension (approximately 3×10 5 OPCs), which was then slowly injected into the transplant site. After injection, the needle was left at the injected site for an additional 5 min then slowly withdrawn. Subsequently, the scalp wound was closed and the rats were placed back into home cages and nursed after fully awaking from anesthesia under a heat lamp. The transplantation time for each rat was approximately 30 min. In the ZJU-37+OPCs group, ZJU-37(i.p, 10 mg/mL) was administered daily for the rst month after transplantation, every other day for the second month, and every third day for the third month until euthanasia. In the OPCs group, DMSO was administered instead of ZJU-37.

Behavioral tests
All behavioral experiments were performed during the 11 weeks after transplantation of eight rats each group.

Morris water maze (MWM) test
This test was performed as previously described [37]. Brie y, each rat entered the water training from four different quadrants and underwent two training sessions per day for ve consecutive days. The latency to escape the water maze (the time from the rats entering the water to standing on the platform) was counted for each trial. On day 6, a probe test was performed by removing the platform and allowing each rat to swim freely for 60 s after entering the water from the rst quadrant. The number of platform crossings and number and time that rats crossed through the platform quadrant were recorded. All data were recorded with a computerized video system.

Limb-use asymmetry test (cylinder)
The forelimb-use asymmetry test was used to assess sensorimotor function and behavioral symmetry. Rats were placed in a transparent Plexiglas cylinder (40 cm high, 20 cm diameter) [38] and initial forepaw (left/right/both) preference for weight-bearing contacts was scored. The forelimb asymmetryscore was calculated as: (right-left)/total of number of contacts.

Adhesive removal test
Sensory and motor functions were measured as described previously [39]. All rats were familiarized with the testing environment for three days in a Perspex box. Two adhesive tapes were placed with equal pressu recovering the hairless parts on both forelimbs. The time to remove the adhesive tapes from each forelimb was recorded within a maximum of 120 s.

Histological examination
Rats from each group were deeply anesthetized with chloral hydrate at 12 weeks after transplantation and intracardially perfused with Phosphate-buffered saline(PBS) followed by xation with 4% cold paraformaldehyde. Brains were dissected and post-xed in the same solution for 12 h at 4°C, then sequentially dehydrated in sucrose (15% and 30%) until permeated. Coronal sections (14 µm thickenss) were cut on a freezing microtome (Leica Microsystems, Nussloch, Journal Pre-proof Germany) from the bregma anterior-posterior coordinates +1.0 to -1.0, collected on 3-aminopropyltriethoxysilane-coated slides (Sigma, St. Louis, MO, USA), and stored at -80°C in cryoprotectant solution. For protein analyses, fresh corpus callosum from sacri ced rats (4 from each sub-group) were isolated and stored at -80°C.
TUNEL staining OPC apoptosis was assessed by TUNEL staining via a Dead End Fluorometric TUNEL System (Roche, Switzerland). Brain tissues were incubated overnight at 37℃ with the anti-PDGFRα (1:800) antibody. A standard TUNEL procedure was performed as described previously [9] after probing with a relevant secondary antibody. Nuclei were stained using 4', 6-diamidino-2-phenylindole (DAPI, Beyotime Biotechnology, Shanghai, China). TUNEL-positive cells were counted at ve randomly chosen microscopic elds. OPC apoptotic rate was calculated as TUNEL and PDGFRα double-positive cells/total PDGFRαpositive cells per eld × 100%.

Immunohistochemistry
Immuno uorescence staining was performed on the above-described sections of brain tissue (20 µm).

Transmission electron microscopy (TEM)
Samples were prepared for electron microscopy according to previous protocols [40]. In brief, brains were removed quickly after perfusion with 2% PFA/2.5% glutaraldehyde. The corpus callosum corresponding to the transplantation site (n=4 per group) were dissected and placed in 2.5% glutaraldehyde at 4°C overnight for post-xation. After transferring to osmium tetroxide for 1 h at room temperature and dehydrating with increasing ethanol concentrations, the tissues were embedded in epoxy resin embedding medium. Ultrathin sections (50 nm) were made from the resin-embedded samples and observed under a transmission electron microscope. A total of 100 root axonal bers from three samples in each group were measured at a magni cation of 15,000 × g. Following image acquisition, axon and myelin diameters were measured using Image J software. The g-ratio, which is a structural index of remyelination and de ned as the ratio of the inner axonal diameter to the total outer ber diameter and lower ratios indicate more extensive myelination, was subsequently assessed. The average scores of ultrastructural myelin damage were determined as described previously [9].
Primary cell culture and drug treatment Extraction and culturing of primary rat astrocytes and OPCs from newborn 0-2 day-old rat cerebral cortices were performed as previously described [41]. Digestions were stopped with DMEM/F12 (1:1, HyClone) containing 10% fetal bovine serum (FBS, Clark Bioscience, Richmond). Cells were centrifuged at 1200 × g for 5 min, and supernatant was discarded. The medium was changed once every 2-3 days. After 9-11 days, microglia were dislodged using an orbital shaker (200× g for 1 h, 37°C), and OPCs were harvested by collecting the cell suspension after shaking the on ahorizontal orbital shaker for 18 h at 200× g and 37℃. The cells were then digested with 0.25% trypsin and seeded into 24-well plates at an appropriate density; the remaining adherent cells were astrocytes. Cells after the third generation were used for experiments and were divided into normal (Ctrl) and oxygen and glucose deprivation (OGD) groups. OGD-treated astrocytes were further divided into OGD, OGD-ZJU-37(5 µM) and OGD-ZJU-37(10 µM) groups and incubated in OGD-DMEM for 6 h. Finally, cells were collected for cellular immuno uorescence staining and western blot analysis. The astrocyte supernatant was mixed with DMEM/F12 at a ratio of 1:1. After oligodendrocytes grew for 2 days in DMEM/F12 containing 10% FBS, the mixed medium without OGD-astrocyte was used for the control (Ctrl) group and the medium with OGD-astrocyte with DMSO or ZJU-37 were used for the conditional medium-vehicle (CM-vehicle) group, the CM-ZJU37 (5μM) group and the CM-ZJU37 (10μM) group respectively for 24 h. Finally, the OPCs were collected for cellular immuno uorescence staining of PDGFRα and MBP and the Annexin V-FITC/PI to detect apoptosis by ow cytometry, separately.

Western blot analysis
Western blotting was performed as previously described [9]. Total protein was extracted from the corpus callosum of the WMI rats and primary rat astrocytes were lysed with a lysis buffer and homogenized and centrifuged at 12,000 × g for 15 min at 4℃. The supernatants were collected and used for protein detection. Samples were run on a 10% SDS-PAGE gel and transferred tonitrocellulose membranes. Primary antibodies were: anti-NLRP3 (IgG, 1:500, Novus Biologicals), anti-ASC (IgG, 1:500, Santa Cruz), anti-caspase-1 (IgG, 1:500, Santa Cruz), anti-cleaved caspase-1 p20 (IgG, 1:1000, Cell Signaling Technology) and anti β-actin (IgG, 1:1000, Santa Cruz). The gray value of every band was analyzed with Image J software and reported as relative optical density of the speci c proteins.

Statistical analysis
Three independent replicates were conducted for each experiment. Experimental data were analyzed with GraphPad Prism® software. After variation similarity was compared, one-way analysis of variation (ANOVA) followed by either the Newman-Keuls or Tukey honestly signi cant difference post hoc test was used for comparisons among multiple groups. A two-way ANOVA was used for escape latency in the Morris water maze training task. Quantitative data are expressed as mean±standard error of mean. Statistical signi cance was set at p < 0.05 for all tests.

Results
Transplanted hOPCs produce myelin sheath The hOPCs showed a bipolar or multipolar morphology under phase-contrast microscopy and expressed the NPC or OPC marker A2B5, the pre-oligodendrocyte marker NG2, the OPC marker PDGFRα and the immature oligodendrocyte marker O4 uorescence. No astrocyte marker GFAP -positive cells were found in vitro. To determine whether ZJU-37 could promote a more effective engraftment in vivo, hOPCs were injected into the corpus callosum of WMI rats. The retention of hOPCs was indicated by the presence of TEM121-positive cells at 1 and 12 weeks after transplantation at the injection site (Fig. 1A). At 1 week after transplantation, grafted cells revealed by STEM121 were found near the transplant site in all transplanted animals (Fig. 1A). The animals without cell transplantation did not exhibit such labelling. At 12 weeks after transplantation, STEM121-positive cells were detected in the contra lateral and anteroposterior directions, and were also observed migrating along the white matter tract of the corpus callosum (Fig. 1B, C), con rming that transplanted hOPCs can penetrate the blood-brain barrier (BBB) and migrate into the host brain. More STEM121-positive cells were observed in the ZJU-37 group, and both groups exhibited long-term survival. At higher magni cation, the STEM121-positive cells adopted a typical bipolar branched OPC morphology (Fig. 1D). Teratomas, tumours, and non-neuronal tissue formation were not observed in the transplant recipients over the course of the experiment.
MBP, a structural protein with an indispensable role in myelin thickening and compaction, was observed to detect the differentiation of OPCs and the status of myelin by immuno uorescence staining at 12 weeks after transplantation. The remyelination uorescence signal was observed in all WMI groups, and the bundles of MBP-positive nerve bers were denser in the ZJU-37+OPCs group than the other groups (Fig. 2B, E). The percentage of the MBP-positive area in the Vehicle group was signi cantly lower than in the Ctrl group. Additionally, compared with the Vehicle group, the percentages of the MBP-positive area in the three treatment groups were signi cantly increased to different degrees, and which were more pronounced in the two transplantation groups (Fig. 2E). The results suggest that transplanted hOPCs can perform myelination and ZJU-37 effectively promote oligodendrocyte differentiation in vivo.
ZJU-37 combined with hOPCs more effectively decreased OPCs apoptosis, and promoted remyelination of WMI rat compared to ZJU-37 or OPC treatment alone OPC apoptosis of the neonatal rat WMI model was detected by TUNEL and PDGFRα immuno uorescence staining, which con rmed that there was a clear increase in the percent of PDGFRα and TUNEL doublepositive cells in the Vehicle group. The three treatment groups showed marked reductions in OPC apoptosis to varying degrees, with the ZJU-37+OPCs group showing the lowest percent compared to the Vehicle group ( Fig. 2A, D). Based on the results obtained for MBP immuno uorescence staining, ZJU-37 appeared to not only suppress the apoptosis of OPCs, but also promote the differentiation of OPCs and the maturation of OLs in WMI rats. Under transmission electron microscopy, the structure of the myelin sheath in the Ctrl group maintained its integrity. The Vehicle group exhibited signi cant ultrastructural alterations in the myelin and axons, such as reduced thickness of the myelin sheath, a clearly disordered and loosened of myelin lamellar arrangement, and an irregular and sporadic arrangement of myelin sheath regeneration around the axons. In contrast, evidence of structural repair was observed in demyelinated areas in the three treatment groups, with some of the myelin sheath appearing normal in shape and at an increased abundance compared to the Vehicle group (Fig. 2C). However, some areas of myelin sheath still exhibited an abnormal structure, including insu cient myelin formation, division, and vacuolation. The g-ratio increased markedly in the Vehicle group compared to the Ctrl group. More thicker and compact myelin sheaths and less disturbed myelin sheaths were found in the ZJU-37+OPCs group (Fig. 2C). The three treatments led to a signi cant decrease in the g-ratio. Additionally, the ZJU-37+OPCs group exhibited a more compact and thicker myelin structure compared to the ZJU-37 and OPCs groups (Fig. 2E). The average score of ultrastructural myelin damage was signi cantly higher in the Vehicle group and was signi cantly attenuated in the ZJU-37+OPCs group, which nearly returned to the Ctrl group (Fig. 2F). These data indicate that ZJU-37 combined with hOPCs transplantation promoted remyelination more e ciently than ZJU-37 or OPC transplantation alone.
ZJU-37 combined with hOPCs more effectively improved cognitive and motor function of WMI rat compared to ZJU-37 or OPC treatment alone To determine whether ZJU-37 combined with hOPCs could recover long-term neurological damage due to WMI, we performed behaviour analysis. Morris water maze tests were performed on rats for ve consecutive days to evaluate place navigation. The results showed that all of the rats had same performance at Day 1. The Vehicle group rats had longer escape latencies than the Ctrl group from Days 2. The three kinds of treatments decreased the escape latency of WMI rats from Day 3 to Day 5 in the acquisition/learning phase (Fig. 3B), whereas the ZJU-37+OPCs group and the Ctrl group had no signi cant differences at Day 5. On the sixth testing day, the platform was removed for the probe test, and the number of times that the rats crossed the target region was signi cantly increased in the ZJU-37+OPCs group compared to the Vehicle group (Fig. 3C) and the time spent in the target quadrant was markedly longer in the three treatment groups than in the Vehicle group. Furthermore, this time period was longer in the ZJU-37+OPCs group than ZJU-37 and OPCs groups (Fig. 3D). These results indicate that ZJU-37 combined with hOPCs treatment signi cantly reversed learning ability and reference memory function.
The cylinder test showed that forelimb movement in the control group was symmetrical, that WMI exacerbated forelimb-use asymmetry in the animals, and that ZJU-37 combined with hOPCs prevented advancements in forelimb-use asymmetry but not to normal levels (Fig. 3E). The adhesive removal test, which is a sensitive approach used to evaluate sensorimotor de cits, revealed functional de cits in the Vehicle group; ZJU-37 combined with hOPCs reduced the mean time to remove the patch (Fig. 3F). Based on these results, ZJU-37 combined with hOPCs signi cantly ameliorated neurological de cits and improved somatosensory functions in the WMI rats.
The activation of glial cells and NLRP3 in ammasome was reduced by ZJU-37 or/and OPCs treatment in a neonatal rat WMI model It has been reported that astrocytes, microglia and the NLRP3 in ammasome are activated in demyelination animal models [25,26]. Immuno uorescence staining was used to determine whether ZJU-37 alleviated demyelination by inhibiting glial activation. As shown in Fig. 4A, increased numbers of positive cells for GFAP (a marker of astrocytes) and Iba-1 (a marker of microglia cells) were observed in the corpus callosum of the Vehicle group, demonstrating that astrocytes and microglia were signi cantly over-activated, but ZJU-37 or/and OPCs treatment signi cantly decreased the number, with the ZJU-37+OPCs group close to the Ctrl group (Fig. 4D, F). Western blotting showed that NLRP3, ASC, and cleaved caspase-1 (p20) protein expression was signi cantly increased in WMI rats, whereas treatment with ZJU-37 combined with hOPCs clearly reduced this level (Fig. 4B, E). This suggests that the NLRP3 in ammasome was activated in the neonatal rat WMI model and ZJU-37 may alleviate demyelination by inhibiting activation of the NLRP3 signalling pathway.

ZJU-37 attenuated OPC apoptosis and dysdifferentiation caused by the CM from OGD-astrocyte
We used the OGD-astrocyte-CM treated with ZJU-37 to incubate oligodendrocytes and then detected the apoptosis and differentiation of oligodendrocytes. The percentage of apoptotic oligodendrocytes was markedly increased in the CM-Vehicle group compared to the Ctrl group. ZJU-37 reversed the increase in the apoptotic rate of oligodendrocytes induced by OGD-astrocyte, with the 10 µM group showing more effects (Figures 6A, C). The immuno uorescence double staining showed that more PDGFRα-positive cells and less MBP-positive cells were observed in the CM-Vehicle group while less PDGFRα-positive cells and more MBP-positive cells were observed in the ZJU-37 groups ( Figures 6B). In addition, the percentage of MBP-positive cells was signi cantly increased in CM-ZJU37(10μM) group ( Figure 6D), which means that ZJU-37 promoted OPCs differentiation into mature oligodendrocytes in vitro. In accordance with observations from in vivo experiments, the results indicate that ZJU-37 suppressed apoptosis and promoted the survival and differentiation of OPCs.

Discussion
White matter damage is a clinically important aspect of several CNS diseases in preterm infants, for which no speci c treatments are available [6]. With improvements in obstetrics and intensive medical care, the condition has improved but approximately 35% of preterm infants suffer chronic neurological disorders [5]. Studies show that nervous system development in SD rats at postnatal 2-5 days is equivalent to that in human preterm infants at 23-32 weeks of gestation [42]. Current studies using neonatal animal WMI models have employed electrocoagulation of the right common carotid artery in P3 SD rats after hypoxia to discover new therapeutic strategies [43]. Recent evidence suggests that ZJU-37 plays an important role in inhibiting in ammation and cell apoptosis. Here, we show for the rst time that ZJU-37 not only suppresses NLRP3 in ammasome activation, attenuates OPC apoptosis and dysdifferentiation in vitro and in vivo but also promotes myelination and improves behavioral function in a neonatal rat WMI model with hOPC graft. Thus, this could be a feasible therapeutic approach to neonatal WMI.
Adverse factors in brain injury, such as infection and hypoxia, lead to OPC damage and subsequent termination of the maturation process of OLs, resulting in pathological changes including myelin dysplasia, decreased white matter volume, and ventricular enlargement in preterm infants [8]. Meanwhile, oligodendrocytes degeneration and necrosis and abnormal OPC differentiation and maturation are major histological features. Stimulating the differentiation of OPCs into myelinating oligodendrocytes is a viable therapeutic option in WMI. Previous studies have reported [11,14,22] the differentiation of endogenous OPCs into myelin through drug therapies, which was previously tested in many animal models and clinical trials. For instance, Najm et al. reported that miconazole and clobetasol promoted OPC differentiation and remyelination in an experimental autoimmune encephalomyelitis model [44]. For genetic or congenital defects whose endogenous remyelination is unsuccessful, transplantation of exogenous cells is a promising treatment. Increasing evidence suggests that OPC transplantation exerts its actions through reducing the loss of endogenous oligodendrocytes and stimulating endogenous progenitors proliferation [21]. Extensive animal model studies have demonstrated the feasibility and relative effectiveness of OPC transplantation as re ected by increased remyelination [11,38]. Currently, human cell sources for OPC transplantation include human embryonic stem cells [20], human induced pluripotent stem cells [45], and NSCs [46]. However, the clinical application of these cells shows many limitations, such as the risk of tumorigenesis, the low survival rate of transplanted cells because of the lack of a more pro-survival milieu for OPCs, and high costs. Our previous studies indicated that although hOPCs transplantation helps to improve demyelination in mice with leukodystrophy, it cannot fully reverse the pathological status of damaged myelin [24]. Additionally, individual small-molecule functions have been revealed in OPCs [33] and ZJU-37 has been proved inhibiting in ammation and cell apoptosis, which effectively protects myelin sheath in animal models of several demyelinating diseases. However, it is unknown whether ZJU-37 exerts positive effects on hOPCs by preserving both structural and functional white matter integrity following neonatal WMI. To this end, upon the transplantation of hOPCs into the neonatal rat WMI model, ZJU-37 was injected intraperitoneally, and by 12 weeks, an increased MBP positive nerve ber bundles was detected in the corpus callosum. In the present study, hOPC grafts effectively survived, migrated to the injured region and differentiated into mature oligodendrocytes expressing MBP in vivo with ZJU-37 treatment is exciting. Ultrastructural studies further con rmed the presence of new myelin sheaths. The g-ratio and score of myelin damage decreased after ZJU-37 combined with hOPCs graft in the neonatal rat WMI model. ZJU-37 exerted a positive effect that exert remyelination function. Hypoxia and ischemia-induced oligodendrocytes progressively undergo apoptosis [5]. Our data show that the apoptosis OPCs increased in WMI rats, which is consistent with the results of previous reports. ZJU-37 combined with hOPCs more effectively decreased OPCs apoptosis compared to ZJU-37 or OPCs treatment alone, as con rmed by PDGFRα immuno uorescent and TUNEL staining. One limitation of the present study was that we did not evaluate the differentiation of transplanted cells. Therefore, whether myelin repair occurred because of the transplanted cells or the differentiation of endogenous cells requires further analysis.
Previous research has shown that the neonatal rat WMI model display decreased motor function and impaired spatial working memory [11,40]. In the present study, we provided in vivo evidence that ZJU-37 combined with hOPCs improved cognitive and motor function compared to ZJU-37 or OPCs alone in WMI rats, as shown by behavioural tests. The ZJU-37+OPCs group showed greater improvement in the MWM test, cylinder test and adhesive removal test than the scramble group, suggesting that ZJU-37 facilitated cognitive functional improvement by decreasing OPCs apoptosis, and promoting oligodendrocyte differentiation and remyelination. However, the question remains: What is the mechanism underlying this recovery phenomenon?
The ischemia-hypoxia mediated oligodendrocytes apoptosis appears to be related to in ammation [41].
Increasing evidence has demonstrated that glial activation reactions are important self-protection mechanisms, which is a key factor in the promotion of functional recovery; however, the hyperactivation of glial cells is detrimental and induces demyelination by inhibiting OPC migration and differentiation, as well as inducing oligodendrocytes death [13,41,47]. Our previous studies demonstrated that astrocyte and microglia activation are consistently observed and the inhibition of NLRP3 in ammasome activation alleviated cuprizone-induced demyelination [9,25,30]. This later study was particularly important, as it provided a foundation for the present study; Here, we found that ZJU-37 or/and OPCs treatment signi cantly decreased the numbers of GFAP-positive and Iba-1-positive cells in the neonatal rat WMI model. Therefore, we explored how ZJU-37 protects the myelin sheath against in ammation by detecting the protein levels of NLRP3, ASC and cleaved-caspase-1 in the corpus callosum. Our data indicated that ZJU-37 inhibited glial and NLRP3 in ammasome activation and promoted oligodendrocytes survival and myelin regeneration in WMI rats with hOPC graft. This contributed to the functional integrity of white matter and recovery of neurological function and suggests that NLRP3 is involved in glial activation and neuroin ammation in WMI rats.
Astrocytes are highly secretory cells thought to be important in oligodendrogenesis following white matter damage [48]. Several studies have observed excessive astrocyte gliosis and in ammation in animal models of demyelination [9,28]. Furthermore, apoptosis of OPCs is fundamental to the progression of demyelinating diseases, and astrocytes can induce oligodendrocytes death [48]. To investigate whether ZJU-37 decreased oligodendrocytes apoptosis induced by over-activation and in ammation of astrocytes, we used medium derived from OGD-astrocyte to treat oligodendrocytes. The results suggested that ZJU-37 suppressed NLRP3 in ammasome activation in OGD-induced astrocyte, and obviously attenuated OPC apoptosis and dysdifferentiation caused by the OGD-astrocyte-CM. This suggests that NLRP3 in ammasome activation in astrocytes triggers apoptosis in OPCs, which is critical for the pathogenesis of neonatal WMI and ZJU-37 suppressed this apoptosis of OPCs. Besides this, the immuno uorescence double staining showed ZJU-37 promoted OPCs differentiation into mature oligodendrocytes in vitro.

Conclusions
In conclusion, the results demonstrated here show that the RIP1/RIP3 kinase dual inhibitor ZJU-37 can effectively promote OPC survival and differentiation with hOPC graft, which alleviate hypoxia and ischemia-induced myelination and improves behavioural functions by targeting the NLRP3 in ammasome complex. Furthermore, these results support the potential therapeutic value by suggesting the positive neuroprotective effects of ZJU-37 combined with hOPCs transplantation for ischemia and hypoxia caused neonatal WMI. As both astrocytes and microglia have a crucial role in demyelination, and because ZJU-37 inhibited NLRP3 in ammasome activation of astrocytes and microglia in vivo, we only evaluated its effect on astrocytes and not its direct effect on microglia. This should be addressed in further studies.

Consent for publication
Not applicable.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding authors upon reasonable request.

Con ict of interest
The authors declare that they have no con ict of interest.  PDGFRα double-positive cells. E Quanti cation of the percentage area positive for MBP immunoreactivity. F g-ratio of the myelin sheath. G Ultrastructural myelin-damage scores for all groups. Data are presented as the mean ± SEM (n= 4). *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Ctrl group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. Vehicle group; &P < 0.05, &&P < 0.01 vs. ZJU-37 group; △P < 0.05 vs. OPCs group.  ZJU-37suppresses NLRP3 in ammasome activation in astrocyte induced by OGD. A Representative immuno uorescence images for GFAP and NLRP3 staining in the corpus callosum for all groups. Scale bar=100 µm. B-C Western blotting and quanti cation for the expression of NLRP3 and cleaved caspase-1 (p20) in the corpus callosum for all groups (n=4). Data are presented as the mean ± SEM. *P<0.05,