Down-Regulation of Nogo-A and PirB in Ischemic Cortex with Remote Ischemic Preconditioning in Mice

Research Article

Austin Neurol & Neurosci. 2021; 4(1): 1024.

Down-Regulation of Nogo-A and PirB in Ischemic Cortex with Remote Ischemic Preconditioning in Mice

Jiao Y and Wang J*

Departments of Neurology, The Second Affiliated Hospital of Zhengzhou University, China

*Corresponding author: Jinglan Wang, 2 Jingba Road, Zhengzhou, Henan, China

Received: March 01, 2021; Accepted: March 24, 2021; Published: March 31, 2021


Objectives: The present study aims to investigate the effect of Limb Remote Ischemic Preconditioning (LRIP) on the expression of Nogo-A and PirB in the cortex of mice with focal cerebral ischemia, and related pathways involving in axonal regeneration and neurological function recovery after cerebral ischemia.

Methods: Adult male C57/BL6 mice were divided into sham-operated (sham), transient Middle Cerebral Artery Occlusion (MCAO), LRIP and anti- PirBAb treatment group. Samples were collected 48h after cerebral ischemia. The histopathologic changes were assessed by 1,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC), and Hematoxylin and Eosin (HE) staining and TUNEL method. The expression of Nogo-A and PirB were determined by immunofluorescence, RT-PCR and Western blot respectively.

Results: TTC staining showed that LRIP treatment reduced the infarct size of mice and anti-PirBAb treatment further decline the infarct size, which was accompanied with the decline of neurological deficit score and reduction of neuronal damage. LRIP treatment also reduced the TUNEL positive cells induced by MCAO and anti-PirBAb treatment further strengthened the effect of LRIP. Except sham group, the expressions of Nogo-A and PirB in other three groups all increased with varying degrees, among which MCAO group was the highest, LRIP group was the second and the anti-PirBAb group was the lowest. The expressions of growth associated protein 43 (GAP43) showed opposite tendency.

Conclusions: LRIP plays beneficial influence on cerebral ischemia. LRIP and PirB inhibition combination has a better protective effect on nervous system after cerebral ischemia in mice.

Keywords: Limb Remote Ischemic Preconditioning; Cerebral ischemia; Nogo-A; PirB; Neuroprotection


CNS: Central Nervous System; HE: Hematoxylin and Eosin; LRIP: Limb Remote Ischemic Preconditioning; MCA: Middle Cerebral Artery; MCAO: Middle Cerebral Artery Occlusion; mNSS: Modified Neurological Severity Score; NgR: Nogo Receptor; Nogo: Neurite Outgrowth Inhibitor; rCBF: Regional Cortical Cerebral Blood Flow; TTC: 2,3,5-Triphenyl-Tetrazolium Chloride.


With the improvement of human living standard and the extension of average life expectancy, cerebrovascular disease has been becoming a severe threat to human life and health. As the most common type of cerebrovascular disease, ischemic cerebrovascular disease accounts for about 70% of all cases [1]. It has been reported that Limb Remote Ischemic Preconditioning (LRIP) could induce ischemic tolerance and reduce the nerve injury caused by ischemia [2]. In recent years, LRIP was considered as a feasible therapeutic strategy for stroke. Some relevant experiments were carried out on rat, which proved that LRIP could provide a protective effect on stroke [3-5].

Neurite Outgrowth Inhibitor-A (Nogo-A) is an important nerve growth suppressor that plays an inhibitory role in regeneration of neurons. Previous study demonstrated that the degree of functional damage after cerebral ischemia was closely related to the difficulty of neuron regeneration, which suggested Nogo-A might take part in the regulation of functional injury after cerebral ischemia. Grandpre et al. [6] indicated that inhibition of Nogo Receptors (NgR) could promote regeneration of damaged neurons. But the results of Zheng et al. [7] pointed out, that the inhibitory effect of myelin inhibitor in NgR knockout mice and wild mice was no significant difference. Therefore, maybe there is a second receptor that mediates the inhibition of myelin inhibitor exists. Earlier research found that Paired Immunoglobulin-Like Receptor B (PirB) was expressed in the central nervous system [8]. They discovered that the mRNA and protein expressions of PirB could be detected in the whole brain tissue of adult mice, and PirB protein was mainly expressed in the cerebral cortex, hippocampus, cerebellar neurons, axons, and synapses. Moreover, as the same with NgR, PirB could combine with Myelin-associated glycoprotein, Nogo-A and oligodendrocyte myelin glycoprotein to inhibit nerve regeneration [9]. Cerebral cortex is the most sensitive part of the brain to ischemia, and its structural integrity is the basis of cognitive function. Therefore, it is speculated that the up-regulated expression of Nogo-A in the cerebral cortex may be a reason for cognitive function decline after cerebral ischemia injury, which leads to decreased neuroplasticity and reduced the ability of regeneration and repair [10-12]. In this study, we investigated the expression of Nogo-A and PirB in the cortex of mice with cerebral ischemia after LRIP, and the related pathways involving in axonal regeneration and neurological function recovery after cerebral ischemia.

Materials and Methods

Experimental animals

A total of 120 adults C57/BL6 mice (male, weighing 20-25 grams) were obtained from the central animal facility of Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All mice were reared at a temperature of 23±2°C in a 50±5% humiditycontrolled room with a 12-hour light-dark cycle with a standard diet. All the experiments were performed strictly according to ’Guide for the Care and Use of Laboratory Animals’ published by the National Institutes of Health, USA20. After fed in our laboratory for one week, the mice were randomly divided into following groups using a lottery-drawing box: 1) Sham-operated group (sham); 2) Middle Cerebral Artery Occlusion (MCAO); 3) LRIP + MCAO (LRIP); 4) LRIP + MCAO + anti PirB antibody (anti-PirBAb) treatment group. All outcome evaluations were carried out by investigators blinded to groups. The research protocols were approved by the Institutional Animal Ethics Committee of Zhengzhou University (Zhengzhou, China).

Limb remote ischemic preconditioning

Noninvasive LRIP was performed as described previously [2,13,14]. LRIP was conducted on both hindlimbs of mice anesthetized with 1-3% isoflurane by non-invasive occlusion of hind limb blood flow with a gauge bandage, in contrast to an invasive, direct femoral artery occlusion. The two hind limbs were simultaneously tied with a bandage to occlude blood circulation for 10min and then released for 10min to allow for reperfusion. The occlusion/reperfusion cycle was repeated three times.

MCAO model

MCAO was performed as described previously [15]. Briefly, mice were anesthetized with 1.5~2% isoflurane. Then, the mice were placed with a supine position, fur was sterilized with 70% ethanol. With the aid of a dissecting microscope, a skin incision was made on the right side of the head from the anterior of the ear toward the corner of the eye horizontally and from the corner of the eye vertically 5mm. Using a fine battery-powered drill (Dremel), a small hole was made 2mm in diameter on the skull bone to remove dura and expose the MCA. The MCA was then cauterized using an electronic coagulator (Codman & Shurtleff), and the incised muscle layer and skin were sutured. In the sham operation group, the same craniotomy was performed, but the middle cerebral artery and its branches were not electro coagulated. In the LRIP group, the mice underwent distal ischemic preconditioning before MCAO surgery. In anti-PirBAb group, mice were subjected to the same protocol as LRIP group, however, after 2h of ischemia, mice were given an intracranial injection of anti-PirB antibody.

In the experiment, Regional Cortical Cerebral Blood Flow (rCBF) was monitored by laser Doppler flowmetry (Periflux System 6000, Olympus, Germany) in the ipsilateral cortex and rectal temperature was monitored and maintained at 37oC (90303B, Spacelabs Healthcare, Snoqualmie, WA, USA). The rCBF values were obtained 5min before and after MCAO, and 5min after the reperfusion of CIP. Mice in the MCAO group (after recovery from anesthesia) that had rCBF value above 40% of baseline were excluded from the study.

To minimize pain and infections after operation, mice were given daily subcutaneous injections of Rimadyl® (2.5mg/kg, Pfizer) and Baytril® (5mg/kg, Bayer) for consecutive 2 days after surgery.

Infarct size measurement

2,3,5-Triphenyl-2H-Tetrazolium Chloride (TTC) staining was used for measuring the infarct size after 48h of reperfusion when the mice were euthanized. Infarct volume was determined using ImageJ analysis by an observer blinded to the experimental group assignment. We evaluated the infarction area ratio using the following method: infarction area ratio = (white infarct area × thickness)/(whole slice area × thickness) ×100%. The detailed protocol has been described previously [16].

Behavioral testing

Modified Neurological Severity Score (mNSS) were examined by 2 investigators who were blinded to the experimental groups to assess sensorimotor as described in a previous study [17]. Neurological function is graded on a scale of 0 to 18 (normal score, 0; maximal deficit score, 18), and the higher score represents the greater severity of the injury.

Hematoxylin and Eosin (HE) staining

The areas of brain injury were evaluated by HE staining. Briefly, brain tissues were cut into 5μm thick paraffin-embedded sections, and then the sections were dried at 50°C for at least 30min. Routine hematoxylin and eosin staining was used to evaluate brain injury areas. The sections were processed as described previously [18].

Tunel staining

Terminal deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) was used to identify dying cells with damaged DNA in each group according to the instructions of TUNEL kit (Roche, USA). Positively stained cells were counted using ImageJ software.


The mice in each group (n=5) were sacrificed under anesthesia with 1.5~2% isoflurane. The expressions of Nogo-A and PirB in the cerebral ischemia of mice were also detected by immunofluorescence method. The dilution ratio of anti-Nogo-A antibody was 1:1000 (Santa Cruz, USA) and the dilution ratio of anti-PirB antibody was 1:200 (Santa Cruz, USA). Results were observed and obtained by inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Western blotting

The mice in each group (n=5) were sacrificed under anesthesia with 1.5~2% isoflurane. The cerebral cortex of mice was collected on ice and stored at -80°C immediately after the mice were euthanized. Samples were homogenized in RIPA lysis buffer (Catalog No. R0020, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and centrifuged at 12000 x g for 15min at 4°C. Then, the total proteins in the supernatant were collected and the concentration of protein was determined using bicinchoninic acid protein assay kit (Catalog No. BCA02, Beijing Dingguo Changsheng Biotechnology Co., Ltd., BeiJing, China). Followed by electrophoresing and transferring, the proteins in Nitrocellulose (NC) membrane (Millipore, USA) were incubated with anti-Nogo-A antibody (1:1000, ab62024, Abcam, USA), anti-PirBantibody (1:10000, ab170909, Abcam, USA), antigrowth associated protein 43 (GAP43) antibody (1:1000, ab75810, Abcam, USA) and anti-β-actin antibody (1:5000, ab8227, Abcam, USA) respectively overnight at 4oC. After washing with TBS-T, the membrane was incubated with secondary antibodies for 1h at room temperature. The immunoreactivity was visualized with Super ECL Plus detection reagent (PE0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) using Bio-Rad Gel Doc XR+ (Hercules, USA). Band intensity was analyzed using ImageJ software (Olympus, Germany). The ratios of gray scale values of target proteins to internal control (β-actin) were used to measure the relative amount of proteins.

Quantitative RT-PCR analysis

The mRNA expressions of Nogo-A and PirB in the mice cortex were evaluated by RT-PCR. Briefly, the mice (n=5) were sacrificed to obtain right cerebral cortex from each group at the time point. Total RNA was extracted with the Trigol reagent (DH353-2, Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). Primed RNA (1μg) was reverse transcribed with PrimeScriptTM RT Master Mix (Code No. RR036Q, Takara, Otsu, Shiga, Japan) according to the manufacturer’s instructions. The reverse-transcription condition was 37°C for 15min followed by a final termination step at 85°C for 5s. cDNA was amplified by PCR using 7500 Real-Time PCR System (Applied Biosystems, CA, USA) with the following conditions: initial denaturation at 95°C for 30s (1 cycle), denaturation at 95oC for 5s, annealing for 60°C for 35s (40 cycles), dissociation stage. The relative mNRA expressions of Nogo-A and PirB were calculated by the grayscale ratios of DNA bands of both Nogo-A and PirB in relation to the internal control (β-actin). Primers sequences were as follows: Nogo-A–forward: 5’ - A A G G T G A G T C A C G C C A A A C T G - 3’, reverse: 5’ - C T T T C G G T T G C T G A G G T A - 3’; PirB–forward: 5’ - G A C T T A T G C C C A G G T G A A A C C - 3’, reverse: 5’ - A G A T T C G G C A G C C T G A T T G T T - 3’; GAP43- forward: 5’ - A C C T A A G G A A A G T G C C C G A C - 3’, reverse: 5’ - G C A T C G G T A G T A G C A G A G C C -3’; β-actin–forward: 5’ - C A G T G C C A G C C T C G T C T C A T - 3’, reverse: 5’ - A G G G G C C A T C C A C A G T C T T C - 3’.

Statistics analysis

Experimental data were presented as Mean ± Standard Deviation. SPSS Statistics for Windows version 18.0 (SPSS Inc., Chicago, USA) was used for statistical analysis. Significant differences were analyzed using one-way ANOVA. Statistical significance was accepted for P values of less than 0.05.


Infarct size in 4 groups

We first performed TTC staining to determine the area of cerebral infarction. The results of TTC staining showed that the infarct size in MCAO group was significantly larger than that in sham operation group (P<0.001). MCAO group had the largest infarct size. LRIP treatment could reduce the size of infarct induced by MCAO (P<0.001) and anti-PirBAb treatment further decline the infarct size (P<0.05, Figure 1A).