CN118924898A - Neuron protection and function recovery method after focal ischemic cerebral apoplexy - Google Patents

Abstract

The invention relates to a neuron protection and function recovery method after focal ischemic stroke and a verification experiment method, wherein the neuron protection and function recovery method is to inhibit nerve injury by regulating miR-212-5p/PLXNA2 so as to promote nerve protection and function recovery after ischemic stroke injury.

Description

Neuron protection and function recovery method after focal ischemic cerebral apoplexy

Technical Field

The invention relates to the field of medical rehabilitation, in particular to a neuron protection and function recovery method after focal ischemic cerebral apoplexy aiming at a targeted gene technology.

Background

Cerebral stroke is a common cerebral vascular emergency that can cause serious brain tissue damage due to abrupt occlusion or rupture of blood vessels, preventing blood flow to the brain. Two main stroke types have been identified: hemorrhagic stroke and ischemic stroke. Ischemic stroke is characterized by high morbidity, mortality, and recurrence rates, accounting for about 80% of all strokes. Brain injury following permanent or transient focal cerebral ischemia can trigger a range of pathophysiological events including free radical release, microglial polarization, blood brain barrier dysfunction, neuronal apoptosis and neuroinflammation. Ischemic penumbra shows increased neuronal apoptosis during the acute phase of cerebral ischemia, a process which is considered an important component of cerebral ischemic injury. Rescue of ischemic penumbra is the heart of the neuroprotection concept of ischemic stroke.

Microglial cells serve as resident immune cells in the central nervous system, and have dual functions in brain tissue injury, regeneration and repair after ischemic stroke. Microglia are classified into pro-inflammatory M1 and anti-inflammatory M2. M2 microglial cells are thought to exert neuroprotective functions by promoting axonal regeneration, remyelination, neurogenesis and angiogenesis. The exosomes are inner vesicles of the multivesicular body with a diameter of 30-150nm, secreted by different types of cells in the brain, including microglia, neurons and astrocytes. Once the multivesicular body fuses with the plasma membrane, the exosomes are released into the extracellular space while protecting their contents from degradation. They are involved in the transport of functional biochemical substances such as messenger RNAs (mrnas), microRNA (miRNAs), cytokines and proteins and thus, in intercellular communication by direct transfer of genetic material to target cells.

Recent studies have shown that exosome miRNAs exhibit regulatory effects on target cells and thus may in fact represent a new way of intracellular communication. Notably, miRNAs are small non-coding RNA molecules that are processed into ribonucleoprotein complexes that bind to target mRNA, resulting in mRNA degradation or translational inhibition. One miRNA can be combined with a plurality of targets, and simultaneously, the miRNAs are inhibited from participating in biological processes functionally, so that the treatment effect on diseases is realized. According to increasing evidence, miRNAs have important functions in a wide range of neurological diseases, such as spinal cord injury, traumatic brain injury and cerebral stroke. They are the essential mediators of the endogenous neuroprotective response of ischemic preconditioning in the brain. Furthermore, miRNAs play a key role in the establishment of functional neurons by modulating neuronal morphology. Some miRNAs are very abundant in the brain and they can act as effector factors required for the development and maintenance of neuronal phenotypes. They are also expressed in dendrites, which are involved in synaptic plasticity by modulating synaptic and dendritic spine structures. Therefore, how to find out some miRNAs with protection and recovery effects on cerebral apoplexy patients through targeting experiments becomes a technical problem which needs to be solved currently.

Disclosure of Invention

The invention aims to solve the technical problems, and the neuron protection and function recovery method is to inhibit nerve injury by regulating miR-212-5p/PLXNA2 so as to promote nerve protection and function recovery after ischemic brain injury. The verification experiment method is used for quantitatively analyzing the phenotype change of microglial cells in a cortical ischemia semi-dark area after transient cerebral ischemia by detecting the levels of intracellular pro-inflammatory cytokines and anti-inflammatory cytokines through qRT-PCR on brain tissues so as to verify the neuroprotection and functional recovery conditions. The verification experiment method is characterized in that on the 3 rd day after operation, microglial exosomes are collected from ischemic penumbra of a cerebral apoplexy animal model, and Western blot analysis of NTA, TEM and exosome surface markers is used for identifying separated exosome samples. The verification experiment method is a behavior test to investigate whether miR-212-5p can improve the exercise function of the MCAO/R animal, and specifically a step walking test is used.

Through the recovery method and the experimental method, whether PLXNA gene in miR-212-5p can inhibit nerve injury better or not can be further judged, so that ischemic brain injury is promoted.

Drawings

FIG. 1 determination of proinflammatory cytokine levels and anti-inflammatory cytokine levels 6h, 1,3, 5, 7 days post-operation;

FIG. 2 miRNA sequencing of microglial exosomes 3 days post MCAO/R surgery;

FIG. 3 treatment with agomir-212-5p may reduce neurological dysfunction 7 days after MCAO/R;

FIG. 4 treatment with agomir-212-5p reduces cerebral infarct volume 7 days after MCAO/R, reduces neuronal apoptosis;

FIG. 5 inhibition of PLXNA and RhoA/ROCK2 expression in ischemic penumbra 3 days after MCAO/R using agomir-212-5p treatment;

FIG. 6 treatment with agomir-212-5p promotes synaptic plasticity at day 7 after MCAO/R and reduces axonal degeneration;

FIG. 7 treatment with agomir-212-5p may promote the formation and growth of dendritic spines on day 7 after MCAO/R surgery;

FIG. 8 inhibition of PLXNA and RhoA/ROCK2 expression in PC12 cells using agomir-212-5p treatment;

FIG. 9 treatment with agomir-212-5p reduces neuronal apoptosis in PC12 cells after OGD/R and reduces axonal degeneration; FIG. 10 schematically illustrates the potential molecular mechanisms that can exert neuroprotective effects to reduce neuronal damage following ischemic stroke using agomir-212-5p therapy.

Detailed Description

FIGS. 1A-E are assays of proinflammatory cytokine levels and anti-inflammatory cytokine levels 6h, 1, 3, 5,7 days post-surgery. Pro-inflammatory cytokines (IL-1β, IL-6, INOS, CD32 and CD 86) released by microglia. Anti-inflammatory cytokines (anti-inflammatory cytokines TGF-beta, IL-10 and CD 206) released by F-H microglia. I the expression of CD86 (green) and Iba1 (red) in the cortical ischemia penumbra at different time points was examined by immunofluorescence staining. J immunofluorescent staining examined CD206 (green) and Iba1 (red) expression at different time points. Scale bar = 50 μm. Data are expressed as mean ± standard error (n=5 per group). ^*P<0.05,^** P <0.01 compared to sham.

FIG. 2 miRNA sequencing of microglial exosomes 3 days post MCAO/R surgery. A, B describe microglial exosomes using nanoparticle tracking analysis and transmission electron microscopy scanning. Scale bar = 200nm. The exosome markers CD9, CD63 and CD81 were detected by C Western blot. D microglial cell detection was performed by Western blot with microglial cell marker CD11 b. E shows a differential heat map of miRNAs expression in microglial exosomes 3 days post MCAO/R surgery. F adopts qRT-PCR to detect the expressions of miR-30c-5p, miR-126a-5p, miR-128-3p, miR-212-5p and miR-1949 in the semi-dark band of the cortical ischemia after MCAO/R operation. 3 days after G MCAO/R surgery, the expression levels of miRNA target genes PLXNA2, PTEN and FOXO3 in the cortical ischemia penumbra. H miR-212-5p is at a target site in the 3 'untranslated region (3' UTR) of PLXNA mRNA. I pmirGLO map of luciferase reporter vector. J double luciferase assay showed binding of miR-212-5p to the 3' UTR of PLXNA 2. Data are expressed as mean ± standard error (n=5 per group). ^*P<0.05,^** P <0.01 compared to sham surgery group; ^# P <0.05 compared to miR-NC group.

FIG. 3 treatment with agomir-212-5p reduces neurological dysfunction 7 days after MCAO/R and improves motor function. Flow chart of experiment a. Step B, step B walking test scoring standard schematic diagram. And C, testing results of a step walking test. The score for neurological deficit score was Zea Longa on the scale of Zea Longa. Waveforms of the respective sets of Motion Evoked Potentials (MEPs). Amplitude of each set of MEPs. Latency of MEPs of group G. Each group n=6. Schematic representation of H CatWalk gait analysis. I run duration(s). J average running speed (cm/s). K right forelimb stride (cm). L right hind limb stride (cm). Data are expressed as mean ± standard error (n=8 per group). And (5) analyzing the connectivity of the brain functions among the M four groups. ^*P<0.05,^** P <0.01 compared to sham surgery group; ^#P<0.05,^## P < value was 0.01 compared to the MCAO/R group.

FIG. 4 treatment with agomir-212-5p reduced cerebral infarction volume 7 days after MCAO/R, and reduced neuronal apoptosis. At day 7 post AMCAO/R, brain sections were stained with TTC to observe ischemic lesions. The white areas show infarct cores. Quantitative analysis of infarct size percentage. C H & E staining. Scale bar = 1000 μm (low magnification) and 50 μm (high magnification). DNissl staining. Scale bar = 50 μm. Immunofluorescent staining of E, F NeuN showed survival of peri-infarct cortical and striatal neurons 7 days after MCAO/R. Data are expressed as mean ± standard error (n=3 per group). ^*P<0.05,^** P <0.01 compared to sham surgery group; ^#P<0.05,^## P <0.01 compared to the MCAO/R group.

FIG. 5 treatment with agomir-212-5p inhibits PLXNA and RhoA/ROCK2 expression in ischemic penumbra at day 3 after MCAO/R. The A asterisks indicate ischemic penumbra. B, analyzing the expression condition of PLXNA, rhoA and ROCK2 mRNA in the ipsilateral brain by adopting a qRT-PCR method. Representative images of Western blot of groups PLXNA, rhoA and ROCK2, C. Beta-actin was used as an internal reference. Quantitative analysis of DWestern blot results. Immunofluorescent staining of PLXNA, rhoA and ROCK2 proteins in each group E of neurons. Scale bar = 50 μm. Data are expressed as mean ± standard error (n=4-6 per group). ^*P<0.05,^** P <0.01 compared to sham surgery group; ^# P <0.05 and ^## P <0.01 compared to the MCAO/R group.

FIG. 6 treatment with agomir-212-5p promotes synaptic plasticity at day 7 after MCAO/R and reduces axonal degeneration. The ultrastructure of ischemic penumbra synapses was analyzed using transmission electron microscopy. Scale bar = 2 μm. B pre-synaptic (purple) and post-synaptic (green) structural schematic. The number of synapses in the cerebral cortical ischemic penumbra of each group C. Width of D synaptic space (nm). Each group n=3. E immunofluorescent staining showed expression of MAP-2 (green) in the cortical ischemic penumbra. Nuclei were stained with DAPI and blue was seen. Scale bar = 50 μm. Representative images of F Nogo-A, ngR and GAP-43 Western blots. And GWestern blot, quantitatively analyzing the detection result. Each group n=4-6. H-J immunofluorescent staining showed Nogo-A, ngR and βIII tubulin expression in the cortical ischemic penumbra. Nuclei were stained with DAPI and blue was seen. Scale bar = 50 μm. Data are expressed as mean ± standard error. ^*P<0.05,^** P <0.01 compared to sham surgery group; ^#P<0.05,^## P <0.01 compared to the MCAO/R group.

FIG. 7 treatment with agomir-212-5p promotes dendritic spine formation and growth of dendritic spines on day 7 after MCAO/R surgery. Representative golgi stained pictures of the brains of each group of rats. Scale bar = 50 μm and 20 μm respectively. B, calculating the top dendrite intersection point when the distance from the central point is continuously increased. Total number of intersections of the C-roof dendrites. D the basal dendrite intersection point is calculated as the distance from the center point increases. Total number of intersections of E-based dendrites. Total number of branches of the F roof dendrite. Total number of branches of G-base dendrites. Dendritic spine plot on H-roof dendrites. Scale bar = 10 μm. I is the density of dendritic spines along the top dendrites. Dendritic spine map on J-base dendrites. Scale bar = 10 μm. Density of dendritic spine along basal dendrites. Data are expressed as mean ± standard error (n=3 per group). ^*P<0.05,^** P <0.01 compared to sham surgery group; ^#P<0.05,^## P < value was 0.01 compared to the MCAO/R group.

FIG. 8 treatment with agomir-212-5p inhibited PLXNA and RhoA/ROCK2 expression in PC12 cells. Representative Western blot images of a show PLXNA, rhoA and ROCK2 levels for each group. Beta-actin is used as an internal reference. And BWesternblot, quantitatively analyzing the detection result. Immunofluorescent staining results of PLXNA, rhoA and ROCK2 (green) proteins in each group of neurons of C-E. Nuclei were stained with DAPI and blue was seen. Scale bar = 50 μm. F-H mRNA levels of PLXNA, rhoA, and ROCK2 were measured in each group of patients using qRT-PCR. Data are expressed as mean ± standard error (n=4-6 per group). ^* P <0.05 and ^** P <0.01 compared to sham surgery groups; ^# P <0.05 and ^## P < value was 0.01 compared to the MCAO/R group.

FIG. 9 treatment with agomir-212-5p reduced neuronal apoptosis in PC12 cells following OGD/R and reduced axonal degeneration. ACLEAVED CASPASE-3 immunofluorescent staining (green). Nuclei were stained with DAPI and blue was seen. Scale bar = 50 μm. Expression levels of Nogo-A and NgR genes in B-C PC12 cells. The gene expression level, β -actin, was detected by qRT-PCR as an internal control. D-F immunofluorescent staining showed PC12 cell Nogo-A, ngR and βIII tubulin expression levels. Nuclei were stained with DAPI and blue was seen. Scale bar = 50 μm. Data are expressed as mean ± standard error (n=5 per group). ^*P<0.05,^** P <0.01 compared to sham surgery group; ^#P<0.05,^## P <0.01 compared to the MCAO/R group.

FIG. 10 schematically illustrates the potential molecular mechanisms that can exert neuroprotective effects to reduce neuronal damage following ischemic stroke using agomir-212-5p therapy.

The levels of intracellular pro-inflammatory cytokines and anti-inflammatory cytokines were detected by qRT-PCR and the time course of the cortical ischemic penumbra microglial phenotypic changes after transient cerebral ischemia was quantified. mRNA expression of M1-type microglial-related pro-inflammatory mediators IL-6, IL-1. Beta., iNOS increased significantly and gradually after cerebral ischemia, peaking at 24h (FIGS. 1A-C). The expression of CD32 and CD86 increased gradually after ischemia and remained elevated for at least 7 days (fig. 1D and E). For M2 microglial-related markers, including TGF- β, IL-10 and CD206, we found in this study that TGF- β expression peaked 24 hours post-surgery (FIG. 1F). IL-10 and CD206 were induced after MCAO/R and peaked 3-5 days after injury (FIG. 1G, H). Immunofluorescent staining of cerebral cortical ischemic penumbra CD86/Iba1 and CD206/Iba1 showed that expression of CD86 increased gradually over time for at least 7 days (FIG. 1I). Furthermore, CD206 was significantly lower in expression on day 7 than on days 3 and 5 (fig. 1J). Thus, M2 microglial markers were expressed abundantly on day 3 post-surgery. On day 3 after MCAO/R, the expression of miR-212-5p in microglial exosomes from the ischemic penumbra is reduced; Microglial exosomes were collected from ischemic penumbra cortex 3 days after MCAO/R, further investigating the role of microglial exosomes miRNAs in pathological changes after ischemic stroke. Isolated exosome samples were identified using Western blot analysis of NTA, TEM and exosome markers (CD 9, CD63, CD 81). As shown in FIG. 2A, B, the exosomes are in the form of typical cup-shaped membrane vesicles, approximately 30-120nm in diameter. Exosome specific markers CD9, CD63 and CD81 were all at high levels in the pellet (fig. 2C). Western blot analysis also showed high expression of CD11b in the pellet (FIG. 2D). The expression level of miRNA in pseudomodule and MCAO/R mouse microglial exosomes was detected by sequencing. The heat map of miRNA expression is shown in figure 2E. The change in miRNA expression was further verified by qRT-PCR. We screened miR-30c-5p, miR-126a-5p, miR-128-3p, miR-212-5p and miR-1949, which could be the cause of neurodegeneration. Experimental results showed significant downregulation of ischemic penumbra miRNAs (fig. 2F). Based on our miRNA sequencing and qRT-PCR results, we found that miR-212-5p expression was significantly reduced in the ischemic penumbra, which we selected for further study. Searching the predictive database found several predicted mRNA targets of miR-212-5p, which might be involved in the neuroprotective effect of miR-212-5 p. Expression levels of PLXNA, PTEN, and FOXO3 in ischemic penumbra were examined (fig. 2G). Then, a dual luciferase reporter system was performed to verify PLXNA2 as a direct target for miR-212-5 p. transfection of miR-212-5p significantly reduced luciferase activity in PC12 cells transfected with PLXNA2 wild-type 3' -UTR, but did not inhibit luciferase activity in cells containing the mutant structure, indicating that PLXNA is a direct target for miR-212-5p (fig. 2H-J).

MiR-212-5p can improve motor function recovery and relieve brain injury;

FIG. 3A is a flow chart of an experiment. We performed behavioral tests to investigate whether miR-212-5p could improve motor function in MCAO/R rats, including the step walking test (FIG. 3B). The right-hand step rate was significantly higher after MCAO/R than in the sham-operated group, while the MCAO/R+ agomir-212-5p group was significantly lower on day 7 post-operation (FIG. 3C). The MCAO/R rats had higher neurological deficit scores, while the MCAO/r+ agomir-212-5p group had a lower neurological deficit score than the MCAO/R group (fig. 3D). After MCAO/R, MEP latency of animals was prolonged and amplitude peaks were reduced. Animals in the MCAO/R+ agomir-212-5p group were significantly improved 7 days after MCAO/R (FIG. 3E-G). Furthermore, we performed CatWalk gait analysis to assess motor function (fig. 3H). The longer the run time after MCAO/R surgery, the slower the average run speed. Agomir-212-5p shortens the run time to some extent (fig. 3I) and also increases the average run speed (fig. 3J). At all measurement time points after MCAO/R, the stride of the affected limb was significantly reduced. The stride of the rats treated with miR-212-5p was significantly improved compared to the MCAO/R rats (FIG. 3K, L). The FC analysis results showed that MCAO/R rats had reduced functional connectivity of the motor cortex on the affected side with the cortex after healthy side compression, sensory cortex on the affected side, bilateral caudate nucleus, and ventral portion of the healthy side hippocampus compared to sham surgery group. In addition, MCAO/r+ agomir-NC group showed reduced connection of the affected motor cortex to the affected motor cortex, the affected prefrontal cortex, and the healthy caudate nucleus function in rats after molding, compared to sham group. However, compared with the MCAO/R group, the MCAO/R+ agomir-212-5p group of rats had elevated functional connection of the motor cortex on the affected side to the sensory cortex on the healthy side, the motor cortex on the healthy side and the piriform cortex on the affected side. At the same time, the motor cortex of the MCAO/R+ agomir-212-5p group had an elevated gray matter function connection around the motor cortex of the affected side and the healthy side aqueduct compared to the MCAO/R+ agomir- + group (FIG. 3M and Table 3). In summary, treatment with agomir-212-5p promotes MEP recovery, enhances the connectivity of the brain neural network and significantly improves motor behavior by activating functional connections between the affected motor cortex and sensory cortex, the full brain motor cortex.

We collected rat brains on day 7 post MCAO/R and stained with TTC to examine the effect of agomir-212-5p treatment on cerebral infarct volume (FIG. 4A). TTC staining showed a significant increase in the white area of the MCAO/R group, while administration of agomir-212-5p significantly reduced the cerebral infarct volume in the MCAO/R rats (FIG. 4B). This change may indicate that agomir-212-5p has neuroprotective effects. Tissue sections were stained with H & E and Nissl, showing histological changes in neurons, and further confirming neuroprotection by miR-212-5 p. H & E staining showed a disorder of cell alignment, interstitial edema, and massive cell necrosis in MCAO/R rats (FIG. 4C). Nissl staining showed a decrease in the number of Nissl individuals in the ischemic penumbra of the MCAO/R group. Agomir-212-5p treatment was effective in improving brain tissue and neuronal damage (FIG. 4D). NeuN staining further showed reduced neuronal apoptosis after agomir-212-5p treatment of the cortex (FIG. 4E, F) and striatum (FIG. 4G, H) compared to the MCAO/R group. Taken together, these results indicate that miR-212-5p can effectively alleviate brain damage induced by cerebral ischemia. MiR-212-5p promotes neuroprotection in MCAO/R rats by targeting PLXNA;

The mechanism of neuroprotection of miR-212-5p is further studied by detecting the expression of miR-212-5p target gene PLXNA. On day 3 post MCAO/R, qRT-PCR analysis showed significant increases in PLXNA, rhoA and ROCK2 levels, whereas agomir-212-5p treatment significantly down-regulated their expression in ischemic penumbra (fig. 5B). Western blot results were consistent with qRT-PCR results (FIGS. 5C, D). Finally, we detected the expression of PLXNA, rhoA and ROCK2 in neurons by immunofluorescent staining and labeled ischemic penumbral neurons with NeuN antibodies. Immunofluorescent staining showed that agomir-212-5p attenuated PLXNA, rhoA and ROCK2 expression in neurons 3 days after MCAO/R (fig. 5E). The results suggest that PLXNA and RhoA/ROCK2 may be involved in the neuroprotection of miR-212-5 p.

MiR-212-5p promotes synaptic plasticity in MCAO/R rats and reduces axonal degeneration;

The synaptic structure of the sham is complete, including complete pre-synaptic and post-synaptic structures. Fewer synaptic vesicles, blurring or absence of synaptic clefts, and abnormal synaptic structure following MCAO/R surgery (fig. 6A). The presynaptic and postsynaptic structures are represented in light purple and green, respectively (FIG. 6B). After agomir-212-5p treatment, both the number of synapses and the inter-synaptic cleft were improved (FIGS. 6C, D). We observed a significant decrease in MAP-2 staining density, however, agomir-212-5p treatment could reverse this phenomenon (fig. 6E). Next, we studied the effect of agomir-212-5p on the attenuation of axonal degeneration. Western blot results showed that the expression levels of Nogo-A and NgR were significantly lower in the MCAO/R groups after the dry prognosis of the MCAO/R+212-5p group (FIGS. 6F, G). The results of Nogo-A and NgR immunofluorescence staining were consistent with the results of Western blot experiments (FIG. 6H, I). Treatment with agomir-212-5p was effective in reducing MCAO/R-induced axonal injury (figure 6J). Taken together, these findings suggest that increasing miR-212-5p expression may be a potential strategy to promote synaptic plasticity and inhibit axonal degeneration following MCAO/R-induced focal cerebral ischemia.

MiR-212-5p promotes formation and growth of dendritic spines in rats after MCAO/R surgery;

We measured the complexity of the dendritic structures in vivo by performing golgi staining on brain sections to assess the effect of miR-212-5 p. On day 7 post MCAO/R, the complexity of the apical and basal dendrites was significantly reduced, the number of dendrite intersections was reduced in MCAO/R rats (fig. 7A-E), and dendrite branches were reduced (fig. 7f, g). We also compared the density of dendritic spines on the apical and basal dendrites. In calculating the number of dendritic spines, a significant decrease in dendritic spine density was found after MCAO/R. However, the application agomir-212-5p can reduce the decrease in dendritic spine density (FIGS. 7H-K). Taken together, these data suggest that miR-212-5p may play an important role in promoting dendritic spine formation and promoting dendritic growth.

In vitro experiments further prove that miR-212-5p promotes neuroprotection through targeting PLXNA < 2 >;

In an in vitro study, we used PC12 cells of the OGD/R model to assess the neuroprotective effects of miR-212-5 p. We first studied PLXNA, rhoA and ROCK2 protein expression levels using Western blot analysis. PLXNA2, rhoA and ROCK2 were expressed in cells with similar results to rat brain tissue (fig. 8a, b). In addition, after OGD/R injury, the intensity of immunofluorescent staining was increased for PLXNA, rhoA, and ROCK2, but reversed for agomir-212-5p (FIGS. 8C-E). These results were further confirmed by qRT-PCR (FIG. 8F-H). Taken together, PLXNA2 may be involved in the action of miR-212-5 p.

MiR-212-5p can reduce apoptosis and axonal degeneration in vitro;

As shown in FIG. 9A, immunofluorescent staining showed a significant increase in C-caspase3 levels after OGD/R, whereas agomir-212-5p treatment significantly reduced cell death. nogoA and NgR mRNA levels were higher in the OGD/R group than in the agomir-212-5p treated group (FIGS. 9B, C). Also, the results were further confirmed by cellular immunofluorescence experiments. OGD/R increased Nogo-A and NgR mRNA expression and decreased βIII tubulin expression, while agomir-212-5p treatment significantly reversed these changes (FIG. 9D-F). These results indicate that miR-212-5p has neuroprotective effects on cell death in vitro.

Materials and methods;

Animals

We obtained adult male SD rats weighing 260-280 grams from Shanghai laboratory animal research center. Only male rats were used in this study, as previous studies demonstrated the neuroprotective effect of estrogens on brain ischemic animal models. We raised all rats under standard laboratory conditions of 23.+ -. 2 ℃, 40% -50% humidity and 12-12 hours light-dark cycle. These animals can obtain food and water. All protocols were reviewed and approved by the institutional animal ethics committee of the Shanghai university of traditional Chinese medicine, and all animal procedures followed guidelines of national institutional laboratory guidelines for animal care and use.

Establishing an animal model of ischemic cerebral apoplexy and injecting lateral ventricles;

Rats were anesthetized with sodium pentobarbital (30 mg/kg, intraperitoneal injection). The rats were then placed in a supine position with a midline incision made in the neck, exposing the left External Carotid Artery (ECA), internal Carotid Artery (ICA), and Common Carotid Artery (CCA). ECA was ligated with surgical wire to block blood flow, and left ICA and CCA were temporarily clamped with a microcatheter. In ECA, a small incision is made. The thrombus was then gently advanced from the ECA stump to the ICA to occlude blood flow in the MCA. After the fixation of the wire bolt for 2 hours, ICA perfusion was resumed. The sham rats were subjected to the same procedure except for the insertion of the plug.

Animals were randomly divided into 4 groups: sham, MCAO/R, MCAO/R+ agomir-NC, MCAO/R+ agomir-212-5p. According to previous reports, agomir-NC and agomir-212-5p were injected through the lateral ventricle 30min before MCAO/R. The skull is exposed by making an incision along the midline of the scalp. The injection coordinates were 1mm posteriorly and 1.5mm laterally (to the left) from the anterior fontanel and 3.5mm below the skull. Agomir-212-5p or agomir-NC (10. Mu.M in 7. Mu.l) was slowly injected. At the same time, 7. Mu.l of 0.9% sodium chloride was injected into the ventricles of both the sham-operated and MCAO/R groups. The needle was left in place for 5min after injection.

Isolation of microglial exosomes in the injured brain; . The ischemic penumbra of the cortex rapidly separated from the left hemisphere. The tissue was digested with collagenase at 37℃for 15 min. Then, the mixture was centrifuged at 2000 Xg at 4℃for 10min to remove cellular fibers and debris, and the supernatant was collected and centrifuged again at 10000 Xg at 4℃for 30min to remove cellular debris. After collecting the supernatant, it was filtered through a 0.22 μm filter. The sample was centrifuged at 100000 Xg at 4℃for 70min and the supernatant was removed. The pellet was resuspended in 0.35. Mu.l Dulbecco's PBS to obtain an exosome suspension. Mu.g of CD11b antibody was added to 50. Mu.l of 3% BSA and the overlaid samples were incubated at room temperature for 60min to isolate microglial exosomes. Subsequently, 10. Mu.l Pierce ^TMStreptavidin Plus UltraLink^TM Resin was added to 40. Mu.l 3% BSA and the covered samples incubated for 30min at room temperature. Then, the mixture was centrifuged at 800 Xg for 10min. After centrifugation, the supernatant was discarded, and microglial exosomes were recovered and stored at 4 ℃ for subsequent analysis.

Exosome identification;

Exosomes were identified using nanoparticle tracking analysis, transmission electron microscopy and Western blot analysis. First, exosomes were analyzed by NTA. To measure the size and concentration of exosome particles we used NTAZetaView PMX and zetaview8.04.02. Isolated exosome samples were diluted appropriately with 1 XPBS. NTA detection was performed at 11 positions and the measurement results were recorded. The ZetaView system was calibrated using 110nm polystyrene particles. The temperature is maintained at about 26.26 deg.c to 27.21 deg.c. And analyzing the exosome morphology by a transmission electron microscope. In 50 μl of 2% PFA, the exosomes were resuspended, and 5 μl of exosomes were dropped onto Formvar-carbon-loaded copper mesh and washed 3 times with PBS. The copper mesh was placed on 50 μl 1% glutaraldehyde droplets for 5min, and then washed. After washing with distilled water, the copper mesh was placed on 50. Mu.l uranium oxalate droplets (pH 7) for 5min, and then transferred to 50. Mu.l methylcellulose droplets for 10min. Excess liquid was removed with filter paper and the sample was allowed to air dry for 10min. Electron micrographs were obtained by JEOL 1230 transmission electron microscopy.

Expression of exosome marker proteins CD9, CD63 and CD81 was analyzed by Western blot. We used CD11b to identify microglial cells. Exosome samples were first lysed with RIPA buffer, total proteins separated on a 12% sds-PAGE gel, transferred to PVDF membrane, blocked, and primary incubated overnight. Antibodies include CD9, CD63 and ITGAM/CD11b. PVDF membrane was then incubated with secondary antibody (1:5000) for 1 hour at room temperature. Protein signals were detected using a UVP imaging system.

MiRNA sequencing analysis and target gene prediction;

MiRNA sequencing was performed by Kangshen Biotech company in Shanghai, china. Total RNA samples were extracted with TRIzol reagent, quality and quantification of the extracted total RNA samples were performed by agarose electrophoresis and Nanodrop, library was constructed, library quality was assessed using an Agilent 2100Bioanalyzer, the library was denatured with 0.1M NaOH to generate single stranded DNA, and finally 51 cycle sequencing was performed on an Illumina NextSeq 500 sequencer. . TargetScan (http:// TargetScan. Org /) and miRDB (http:// mirdb. Org/miRDB /) were used to predict the miRNA target genes, and the consensus predicted genes were selected as potential target genes for miRNA.

Double luciferase reporter gene experiments;

wild-type PLXNA 'UTR (WT) or PLXNA' UTR Mutant (MUT) of miR-212-5p binding site was inserted into the dual-luciferase pmirGLO vector. Luminescence was detected using a GloMax instrument (Promega) with a dual luciferase reporter assay.

Cell culture and oxygen glucose deprivation/reoxygenation (Oxygen glucose deprivation/reoxygenation, OGD/R) modeling;

We obtained PC12 cells from Shanghai cell biology institute of China academy of sciences and cultured them in DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells were cultured in 5% CO ₂ at 37 ℃. For the cell transfection experiments, cells were transfected with either agomir-212-5p or agomir-NC at 100 nM. Cells were used for subsequent experiments 24h after transfection. We then changed the cell culture broth to sugar-free medium. Cells were incubated for 4 hours (1%O ₂,94％N₂ and 5% CO ₂) in a hypoxic incubator at 37 ℃. Subsequently, the sugarless medium was changed to a normal medium and reoxygenation was performed with maintenance under standard conditions (5% CO ₂ and 37 ℃).

Behavioral testing;

Neurological deficit was scored using Zea Longa scores: 0 = no signs of neurological deficit; 1 = right front paw cannot be fully straightened; 2 = right turn; 3 = toppling to the right; 4 = inability to walk spontaneously, loss of consciousness.

A junction walking test;

Each group had 8 rats. We tested the ability of rats to walk on irregularly spaced (1-3 cm) ladders and recorded video to assess impairment of right forelimb function after stroke. Scoring was based on the following criteria: 0 = complete miss; 1 = severe slipping; 2 = light slipping; 3 = reset; 4 = correction; 5 = partial placement; 6 = correctly placed. The step rate refers to the number of steps with a step score of 0, 1 or 2. To calculate the step rate, we use the following formula: (number of wrong steps/total steps for right forelimb) ×100%.

Catwalk gait analysis;

Each group included 8 rats. During the experiment, the room was black and quiet. Prior to the formal experiments, rats were subjected to an adaptive training for one week. The rats are placed at one end of a glass runway, food is taken as bait at the other end of the runway, and running conditions of the rats are recorded under the runway through a high-speed camera. The test was performed on day 1, 3, 5, and 7, respectively, before and after the operation. Data were collected and analyzed using catwalk version 10.6 software.

Exercise evoked potential (MEP);

Exercise evoked potential testing was performed using a Keypoint 9033A07 electromyography/evoked potential apparatus manufactured by Denmark at day 7 post-molding. Rats were anesthetized by intraperitoneal injection with sodium pentobarbital (30 mg/kg) for detection, and then fixed in the supine position. The recording electrode was placed on the right bicep muscle and the stimulating electrode was penetrated by the rat palate (here near the left motor cortex of the brain). The stimulation is performed by applying a single square wave electric pulse, the wave width is 100 mu s, the stimulation intensity gradually increases to the latency period and the wave amplitude are not changed obviously any more. The motor evoked potential latency and amplitude changes of the rats were recorded.

Resting state functional magnetic resonance imaging (fMRI);

Rat brain scans were performed using an 11.7T magnetic resonance scanner (Bruker, karlsruhe, germany). Rats were anesthetized using 5% isoflurane inhalation induction, fixed on a magnetic resonance scanning table, and kept anesthetized using 1.5% isoflurane in combination with 0.05mg/kg dexmedetomidine in prone position. Resting fMRI employs a single shot planar Echo imaging sequence (Echo PLANAR IMAGING, EPI) of the barrier scan, the parameters of which are shown below: flip angle=90°, scan layer thickness=0.3 mm, average number of times 1,repetition Time (TR) =3000 ms, echo Time (TE) = 8.142ms,field ofvision (FOV) =27×27mm ². SPM8 (STASTISTICAL PARAMETTIC MAPPING) statistical analysis software package (http:// www.fil.ion.ucl.ac.uk/SPM /) of Matlab 2014b (MathWorks, USA) platform was used for data preprocessing and data analysis. The data is first converted to Nifti format using Bru < 2 > -nii toolbox, then time-layer correction (SLICE TIMING), head-movement correction (Realign) are performed using SPM8 software, then non-brain tissue is stripped off using MRIcroN software, and the origin of the image is reset to better register the individual brain image with the template brain. A normalization (Normalize) and smoothing (smooths) process is then performed. The motor cortex on the same side (left side) of the focus is selected as a region of interest (Region ofinterest, ROI), the functional connection (Functional Connectivity, FC) of the motor cortex on the affected side and each voxel of the whole brain is calculated, and statistical analysis is performed after Fisher' sZ transformation is performed. Different groups of rats were subjected to the two-sample t-test in SPM8 and the results were presented in a standard rat brain template. P <0.001 considers the difference statistically significant.

Assessing cerebral infarction volume;

The rat brain was rapidly removed and frozen at-20℃for 20min. Subsequently, the frozen brain was coronally sectioned to a thickness of 2mm for a total of 5 slices. . Brain tissue sections were stained with 2%2,3, 5-triphenyltetrazolium chloride (2, 3,5-trihenyl tetrazolium chloride, TTC), incubated in the dark at 37 ℃ for 20min, and then fixed with 4% paraformaldehyde. The infarct size was calculated using Image J software.

Western blot；

Brain tissue and cells were collected and specimens were lysed with RIPA lysate. Protein concentration was determined using BCA kit. After electrophoresis of the tissue samples (80. Mu.g) and the cell samples (30. Mu.g), they were transferred to PVDF membrane and blocked. After the end of the blocking, the PVDF membrane was taken out and placed in TBST for 5min X3 times, and then placed in an antibody incubation box separately, shaking table at 4℃overnight. The next day, PVDF membrane was placed in secondary antibody dilution solution and incubated on a shaking table at room temperature for 1 h. Imaging and picture acquisition was performed using the us UVP gel imaging system. The grey values of the bands were quantitatively analyzed using Image J software.

H & E and Nissl staining;

Nissl staining, hematoxylin-eosin (H & E) staining and immunofluorescence staining of tissue specimens were fixed in 4% paraformaldehyde for 24H, dehydrated with alcohol gradient, transparent xylene, paraffin embedded, and brain sections were performed with coronal plane from front to back, paraffin sections of 5 μm were prepared, guaranteeing five sections each. H & E staining and Nissl staining were performed according to the kit instructions.

Immunofluorescence staining;

Paraffin sections were dewaxed to water, sections were antigen thermally repaired, 10% goat serum blocking solution containing 0.3% triton X-100 was added dropwise, incubated for 1h at room temperature, and incubated overnight at 4 ℃ with the indicated primary antibodies. PC12 cells were washed with PBS, fixed with 4% paraformaldehyde, 0.3% Triton X-100 was infiltrated with PBS for 15min, and incubated with 10% goat serum albumin for 1h at room temperature. Next, the slides were incubated overnight at 4 ℃ against the first antibody. The following day, secondary antibodies were incubated, nuclei counterstained, and blocked. . Images were acquired with an inverted fluorescence microscope at 400 x field of view.

Fluorescent quantitative PCR (qRT-PCR);

Cell and brain tissue RNAs were extracted using TRIzol reagent and reverse transcribed into cdnas using reverse transcription kit. Gene expression levels were detected using SYBR Green kit and LightCycler 480 real-time fluorescent quantitation. The reaction conditions are as follows: pre-denaturation, 5min 1 cycle at 95 ℃; amplification was performed for 10s at 95℃denaturation, 10s at 60℃annealing, and 10s at 72℃extension, for 45 cycles. U6 (for miRNA) and beta-actin (for mRNA) references. Tables 1 and 2 show the primer sequences designed for this study. The relative expression level of the gene was analyzed by the method ^[73] of 2 ^-△△CT. The delta CT calculation formula is as follows: ΔΔct= (CT _{Target gene} -CT _{Reference gene} ) _{Experimental group} -(CT _{Target gene} -CT _{Reference gene} ) _{Control group} . Statistical methods were applied to compare the results between groups for statistical differences.

A transmission electron microscope;

Brain tissue was cut into 1mm ³, fixed with 2.5% glutaraldehyde for 2 hours, then samples were fixed with 1% osmium acid for 1 hour, dehydrated, and embedded. Then, ultrathin sections of 50nm are prepared, and dyeing is carried out by adopting a uranyl acetate-lead citrate double dyeing method. And (5) observing by using a transmission electron microscope, and acquiring an image.

Golgi staining, sholl analysis and dendritic spine density measurement;

The FD rapid golgi staining kit performs golgi staining. The extracted brain was soaked in a golgi dye solution for 2 weeks in the dark at room temperature. Immersing and washing 3 times with distilled water, immersing the tissue in 80% glacial acetic acid overnight, washing with distilled water after the tissue becomes soft, and placing in 30% sucrose. . The tissue was cut into slices with a thickness of 100 μm using an oscillating microtome, and the slices were attached to gelatin slides. Treating tissue slide with concentrated ammonia water for 15min, washing with distilled water for 1min, treating with fixing solution for 15min, washing with distilled water for 3min, air drying, and sealing with glycerol gelatin. Analysis of results was performed using Image J, neuronJ, shollAnalysis and NeuronStudio software.

Statistical analysis;

Experimental data were statistically analyzed using SPSS 22.0 software and expressed as mean ± Standard Error (SEM). The comparison between groups was performed by one-wayANOVA, the comparison between the variance individuals was performed by LSD method, and the variance individuals were tested by Dunnet's T3. P <0.05 considered the difference statistically significant.

Claims (4)

1. The neuron protection and function recovery method after focal ischemic cerebral apoplexy and the verification experiment method are characterized in that: the neuron protection and function recovery method is to inhibit nerve injury by regulating miR-212-5p/PLXNA2 so as to promote nerve protection and function recovery after ischemic cerebral apoplexy.

2. The method for protecting neurons and recovering functions after focal ischemic cerebral apoplexy and the verification experiment method according to claim 1, wherein the method is characterized in that: miR-212-5p in brain tissue of cerebral apoplexy animal model targets its downstream PLXNA gene to inhibit nerve injury, and qRT-PCR is used for detecting intracellular levels of pro-inflammatory cytokines and anti-inflammatory cytokines in brain tissue, and quantitative analysis is carried out on the phenotype change of cortical ischemia semi-dark band microglial cells after transient cerebral ischemia so as to verify neuroprotection and functional recovery.

3. The method for protecting neurons and recovering functions after focal ischemic cerebral apoplexy and the verification experiment method according to claim 2, wherein the method is characterized in that: the verification experiment method is that on the 3 rd day after operation, microglial exosomes are collected from ischemic penumbra of a cerebral apoplexy animal model, and separated exosome samples are identified by using Westernblot analysis of NTA, TEM and exosome surface markers.

4. The method for protecting neurons and recovering functions after focal ischemic cerebral apoplexy and the verification experiment method according to claim 2, wherein the method is characterized in that: the verification experiment method is a behavioural test to investigate whether miR-212-5p can improve the exercise function of MCAO/R animals, and specifically a step walking test is used.