Expression Pattern of Ngb in Astrocytes after Spinal Cord Injury and the Clinical Significance

In the current study, we evaluated the expression pattern of neuroglobin (Ngb) in the astrocytes after spinal cord injury (SCI) and explore the clinical significance. For this purpose, a total of 48 SpragueDawley rats were divided into the SCI group (n = 40) and Sham group (n = 8). Rats in the SCI group were used to prepare the SCI models by using the modified Allen’s method, followed by the HE staining to observe the post-SCI pathological changes and immunofluorescent staining to observe the dynamic changes of Ngb in astrocytes after SCI. Then, oxidative stress injury models were constructed on the astrocytes in the spinal cord of rats by using peroxide in different concentrations (0, 50, 100, 150, 200 and 400μmol/L), and at 6 and 12 h after treatment, the vitality of astrocytes that were treated by peroxide in different concentrations was determined using the MTT method, while the ability of astrocytes to generate radical oxygen species (ROS) was determined by using the flow cytometry. The mRNA expression of Ngb after the oxidative stress injury in astrocytes was measured by using the realtime quantitative PCR. Results of HE staining demonstrated that rats with SCI presented with the gradual transition from acute injury into the glial scar, a natural repair, while the results of immunofluorescent staining indicated that after SCI, expression of Ngb in the astrocytes experienced an increase followed by a decrease, and the peak level was attained at 14 d after SCI. Following the treatment of H2O2 at different concentrations (50, 100, 150, 200 and 400μmol/L) for 6 and 12 h, the vitality of astrocytes in the model groups was significantly lower than that in the control groups (all P < 0.05). As the concentration of H2O2 increased (50, 100, 150, 200, 400μmol/L) and exposure to H2O2 prolonged (6, 12 h), mRNA expression was firstly increased but then decreased in astrocytes in a timedose dependent pattern (all P < 0.05). After SCI, the expression of Ngb in the astrocytes of spine was upregulated, suggesting that Ngb may be involved in the anti-oxidative stress injury in astrocytes after SCI, thereby playing as an endogenous protector of cells.


Introduction
Spinal cord injury (SCI) is divided into the primary and secondary types, where primary SCI presents mainly with the neuronal necrosis, rupture of nerve fibers, damaged blood-spinal cord barrier and vascular injury (1), while the secondary SCI preliminarily with the cell apoptosis and progressive axonal loss, mainly including the ischemia and hypoxia in microcirculation, oxidative stress injury, excitatory amino acid poisoning and inflammatory responses (2). Research has shown that oxidative stress injury is critical to the development and progression of secondary SCI (3). Accumulated radical oxygen specimens (ROS) can cause oxidative stress injury to the spinal cord tissue, mainly involving the lipid peroxidation of the membrane, damage to the nucleic acid and proteins, inhibition of enzyme system in oxidation respiratory chain, nerve fiber degeneration and delineation, eventually exacerbating the dysfunction and damage of spinal cord functions (4)(5)(6)(7). Astrocytes are a group of cells that are mostly distributed and richest in the central nerve system, and they, as reported, could protect neurons by secreting the neurotrophic factors, alleviating the oxidative stress injury and pre-adapting to the ischemia (8). Following SCI, the formation of an astrocytic scar could mitigate the nervous inflammatory responses and inhibit the delineation of axons and nerve fibers to protect the neurons from secondary injury (9). Anderson et al. reported that astrocytic scar is conducive to post-SCI axonal regeneration (10). It has been reported that the post-SCI activation of astrocytes is a potential target for protecting the spinal cord function and repair (11,12). Ngb is an oxygencarrying globin in the cytoplasm that is mainly distributed in the nerve system, retinal cells and endocrine tissues (13). In brain injury, cerebral malaria and autoimmune encephalitis, Ngb is not only expressed in neurons but also in the reactive astrocytes in the injured regions and is associated with the formation of astrocytic scar (14,15). The post-SCI protective effect of Ngb on the neurons has been frequently reported in the current literatures, but the role of Ngb in astrocytes has not yet been reported. From January 2020 to February 2021, we explored the changes in the expression pattern of Ngb in the astrocytes in SCI animal models and the role of Ngb in protecting the astrocytes from the oxidative stress injury in the cell models.

Preparation of SCI models
The 48 SD rats were divided into two groups randomlythe Sham group (n = 8) and the SCI group (n = 40). Modified Allen's method was used to prepare the SCI models on rats (16). In brief, rats, after being anesthetized by 10% chloral hydrate (350 mg/kg), were fixed on the operating table to remove the hair. With the junction between the floating rib and the 13th thoracic vertebra as a mark, an incision in the length of 3 cm was made along the linea mediana posterior to expose the paravertebral soft tissues, and the T10 spinal cord was exposed by removing the spinal processes and vertebral plate. A 10 g bar was used to hit the T10 spinal dura mater by falling freely from the height at 25 mm, and 10 s later, the moderate injury was made to the spinal cord. The incision was then sutured, while rats were fed in the separated cages. Criteria for the successful construction of SCI model: bleeding of the spinal cord, distended spinal dura mater in amaranth, the tail of rats in spasmodic movement, low limbs and soma of rats in retracting movement, rats in delayed paralysis. This study had been approved by the Ethical Committee for Laboratory Animals of Yan'an University.

Sample collection of the spinal cord and HE staining
At 1, 3, 7, 14 and 28 days after surgery, 8 rats and 5 rats were selected randomly from the SCI group and the sham group, respectively. Following anesthesia, the heart of the rat was exposed, and normal saline in the volume of 250 mL was perfused through the intubation via the ascending aorta. Perfusion was terminated when colorless, clear fluid effused from the auricula dextra, and paraformaldehyde was then perfused. Subsequently, the injured spinal cord was taken out from the vertebrae and fixed in 4% paraformaldehyde for paraffin-embedding and slicing into the sections in thickness of 4 μm for HE staining. At 1 h after SCI, the spinal cord was taken out, presenting with continuous interruption, severe degeneration and necrosis on the surface and surrounding the injured site, while rats in the sham group showed the integrated spinal cord, with no evident bleeding or necrosis.

Collection and identification of the spinal astrocytes
The neonatal Sprague-Dawley rats aged 1 or 2 days were immersed in 75% ethanol and then sacrificed to obtain the spinal cord on a super clean bench, where the spinal cord and vessels were dissected and cut into pieces in the cold PBS. After being digested in 0.25% trypsin at room temperature for 15 min, tissue samples were placed into the 12% FBS-DMEM to prepare the cell suspension. The cell suspension was then filtrated by using the 200-mesh filter, centrifuged, resuspended and seeded onto the T25 culture flask, where the fibroblasts were removed by the method of differential adhesion, and the medium was refreshed at 24 h initially, then every 2 to 3 days. After being cultured for 6 -8 days, cells in 90% of confluence were shaken horizontally at 160 r/min and 37°C for 16-18 h to remove the oligodendrocytes and microglial cells. When cells were subcultured to the third generation (about 21 days), a few cells were selected for identification of astrocytes and purity via the GFAP-and DAPI-labeled fluorescent staining. Within the stained vision, we could see the closely arranged protrusions of the GFAP-positive cells, and GFAP-and DAPI-positive cells took up more than 90% of the total cells. Thus, astrocytes in the third generation were selected for the following experiments.

GFAP and Ngb immunofluorescent staining
Astrocytes in the 3rd generation were seeded into the gelatin-coated 24-well plate at a density of 1×105/mL, and at a confluence of 80% or so, cells were rinsed three times in the cold PBS and then fixed in 4% paraformaldehyde for 30 min at 4°C, followed by 3 washes in PBS. Following procedures were performed as the method mentioned above to observe the expression of GFAP and Ngb in the astrocytes.

Detection of astrocyte viability
In this part, an MTT assay was performed. In brief, astrocytes in the 3rd generation were seeded on a 96well plate at a density of 5×104/mL, with 200 μL in each well, and cultured in a CO 2 thermostat incubator for 72 h. At a confluence of 80% or so, cells were treated by H 2 O 2 at different concentrations (0, 50, 100, 150, 200 and 400 μmol/L) for different times (6 or 12 h) for later MTT assay. The absorbance of each well at a wavelength of 490 nm was also determined using the full-wave microplate reader, and cell viability were calculated by using the following formula: Cell viability = (A treatment group -A reference) / (A control group -A reference) × 100%.

Generation of endogenous ROS in astrocytes
Flow cytometry was adopted in this part. AS in the third generation was seeded in a 6-well plate at a density of 1.25×105/mL, with 2 mL in each well. Following 72 hours of culture, cells were treated with H 2 O 2 in varying concentrations (50, 100, 200 and 400 μmol/L) for different times (6 and 12 h) and then digested in trypsin. Thereafter, cells were resuspended to prepare the cell suspension, where a DCFH-DA probe was added and the final concentration was adjusted to 10 μmol/L. After 1 hour of incubation at 37°C, cells were rinsed in the serum-free medium and centrifuged. Cells in the sediment were resuspended in 2% FBS-DMEM and subjected to the measurement in a flow cytometer.

Detection of mRNA expression of Ngb, SOD 2 and CAT
Real-time quantitative PCR was adopted in this study. AS, after being treated by H 2 O 2 in varying concentrations (50, 100, 200 and 400 μmol/L) for different times (6 and 12 h), were subjected to the extraction of total mRNA by using the TRIzol reagent. With 1 μg total RNA, cDNA was prepared by using the reverse transcription kit and then diluted to the concentration of 5 ng/μL, followed by the fluorescent quantitative PCR with SYBR Green PCR Master Mix and corresponding primers: Ngb, forward primer 5'-GGCCATCCAAGAGAGGTGAT-3' and reverse primer 5'-CCATGCCTCCTCACTACCAA-3'; SOD 2 , forward primer 5'-CTGCTGGGGATTGATGTGTG-3' and reverse primer 5'-CTACAAAACACCCACCACGGCAT-3'; CAT, forward primer 5'-GCGGATTCCTGAGAGAGTGG-3' and reverse primer 5'-GAATCGGACGGCAATAGGAG-3'. The reaction system was set as follow: 95°C for 2 min, 95°C for 5 s, 60°C for 30 s, 72°C for the 30s, 72°C for 5 min, a total of 35 cycles. Relative expression of the targeted gene was calculated by the method of 2 -ΔΔCt .

Statistical analysis
SPSS 20.0 software was used to perform the statistical analysis. Measurement data were expressed by mean ± standard deviation (SD), and the data in normal distribution were compared by using the oneway analysis of variance, followed by LSD-t test for pairwise comparison. Comparison between two groups was performed by using t-test for independent samples. P < 0.05 suggested that the difference had statistical significance.

Results and discussion Expression of GFAP and Ngb in AS after SCI
In the sham group, Ngb was low expressed in the spine, while GFAP-positive cells were distributed sporadically; in the white matter, AS were small, with thin and straight protrusions but fewer branches. On the margin of the white matter, a few cells were coexpressing GFAP and Ngb in the cytoplasm, with weak signals. At 1 d after the operation, the spinal structure was destroyed massively, and surrounding the injured site, the quantity of Ngb-positive cells increased. As compared to the sham group, coexpression of GFAP and Ngb was upregulated slightly. At 3 d after the operation, Ngb was upregulated continuously and co-expression of GFAP and Ngb surrounding the injured site also increased evidently. Upregulation of GFAP was observed 1 week after the operation, mainly at the margin of the surrounding site. Similarly, co-expression of GFAP and Ngb also increased, significantly higher than the region closing to the non-injured site. At the 14th and 28th days after the operation, the most evident increase in the expression of GFAP was seen, and the peak level was attained at the 28th day, with a significant augment in the number of protrusion of AS cells forming the compact network and the boundary between the lumen and cavity, while in the surrounding region, the peak level of co-expression of GFAP and Ngb was observed at the 14th day and began to decline from the 28th day.

Changes in the viability of AS cells under the treatment of H 2 O 2 in varying concentrations and different time
After  (Table 1). Note: * P < 0.01 vs. the cells that were not exposed to H2O2 at 6 h; # P < 0.001 vs. the cells that were not exposed to H2O2 at 12 h.

Generation of ROS induced by H 2 O 2 for 6 and 12 h
H 2 O 2 could induce the generation of ROS in AS. Within 6 h, ROS generation increased gradually as the H 2 O 2 concentration increased, with the right-skewed peak level (F = 262.33, P < 0.01). Following exposure to H 2 O 2 for 12 h, ROS generation in AS increased firstly and then decreased (F =297.30, P<0.01), and the peak level was attained in AS treated by H 2 O 2 in the concentration of 100 μmol/L (Figure 1 and 2).

Changes in the mRNA expression of Ngb, SOD 2 and CAT under the induction of H 2 O 2 at different time points
Following exposure to H 2 O 2 at concentration between 50 and 400 μmol/L for 6 or 12 h, mRNA expression of Ngb increased firstly but then decreased (F6h=67.48, P < 0.01; F12h=51.47, P < 0.01), and the peak level was attained at 6 or 12 h in concentration of 100 μmol/L (t = 4.72; P = 0.042). Similarly, mRNA expression of CAT and SOD 2 also increased firstly but then decreased (CAT:F6h=95.31, P < 0.001;F12h = 101.20, P < 0.001, SOD 2 :F6h=94.73, P < 0.001;F 12h=41.54, P < 0.01), while after exposure to H 2 O 2 at concentration of 100 μmol/L for 12 h, the expression of SOD 2 was higher than the exposure for 6 h (t = 6.78, P = 0.032). The peak level of mRNA expression of Ngb, CAT and SOD 2 was attained in concentration of 50 μmol/L or 100 μmol/L, followed by continuous decreases, the lowest level was shown in concentration of 400 μmol/L, and such changes were in a dose-time-dependent manner (all P < 0.05; Figure  3A, B and C). Oxidative stress injury refers to an imbalanced status of pro-oxidant and antioxidant, resulting in the excessive generation of free radicals, the tendency to oxidation, infiltration of neutrophils, increases in the protease secretion and massive generation of oxidative intermediate (17). Bermudez et al. (18)reported that oxidative stress exists continuously in the acute or chronic phase of SCI. Amri et al. (19) also found that H 2 O 2 in the concentration of 100 μmol/L can trigger the atrophy of AS and collapse in the glial network, with a decrease in cell density, while the treatment of Ngb could reverse changes above and exert the antiapoptotic effect via reducing the generation of ROS and downregulating the expression of proinflammatory factors (19).
CNS in mammals lacks the regeneration ability, and in rodent animals, the spinal function could be restored naturally to some extent after the acute phase of SCI besides the complete transection spinal cord injury (20). For cell response to the CNS injury, human beings evolve seemingly to isolate the minor injury and prevent the infection in the injured site, instead of repairing the large or irreparable injury, which coincides with the behaviors of AS (21,22). After SCI, neurons, as compared to the AS, are more susceptible to the attack of ROS, while AS could protect the neurons from the oxidative stress injury, which may correlate with the abundant content of antioxidants (like glutathione and vitamin E) and the natural selection in cell evolution (23).
Current research has shown that SCI could trigger the proliferation and activation of AS, and the activated AS can further inhibit the expansion of the SCI region, eliminate the cell debris and remodel the injured site (24). Nathan et al. (25) reported that the spinal environment is the key that alters the fortune of AS. Okada et al. (9) confirmed that during the recovery from SCI, the knockout of gene inhibiting the activation of Socs3 in AS could enhance the abilities of AS in migration, isolating the injured site and restricting the dissemination of inflammation. In this study, we confirmed that in the natural recovery from SCI, SCI could enhance the expression of Ngb in AS. In the early stage of SCI, the upregulation of Ngb may associate with the increase in the oxygen supply to the injured site. Ever since the 7th day after SCI, co-expression of GFAP and Ngb is upregulated, and the peak level is attained at the 14th day when the formation rate of glial scar is also the fastest, which coincides with the function of AS. Thus, Ngb may facilitate the activation of AS, thereby promoting the natural recovery of SCI. However, during the 1 st day to the 28th day after SCI, Ngb expression presents with an increase and then a decrease, suggesting that in addition to the activation of AS, Ngb also plays a role in anti-oxidation and -stress injury, which coincides with the findings of Amri et al. (19) and Fabrizius et al. (26).
ROS is an indicator reflecting the level of oxidative stress. Results of this study indicate that after 6 or 12 h of treatment by H 2 O 2 in varying concentrations, spinal cells presented with the oxidative stress injury in varying degrees, with significant declines in the viability of AS in a dose-dependent manner. Results of real-time PCR showed that H 2 O 2 -induced oxidative stress injury in AS could upregulate the expression of SOD 2 , CAT and Ngb, and with the increase in the concentration of H 2 O 2 and prolongation of exposure, the mRNA levels of SOD 2 and CAT, the endogenous anti-oxidative stress enzymes, also had an increase and then a decrease. Moreover, the mRNA expression of Ngb also showed expression patterns similar to the expression of SOD 2 and CAT, and the peak level was attained at a concentration of 100 μmol/L suggests that Ngb has a similar effect and is involved in the anti-oxidative stress injury together with SOD 2 and CAT. Douiri et al. (27) treated the H 2 O 2 -injured AS with the exogenous Ngb and found that Ngb could upregulate the mRNA expression of SOD 2 and CAT evidently, thereby antagonizing the oxidative stress injury, while inhibiting the activity of endogenous antioxidase system in AS could curb the protective effect of endogenous molecules on cell survival.
The results of this study confirmed that SCI could upregulate the expression of Ngb in AS and that Ngb may be involved in the antioxidative stress injury to AS after SCI, thereby advancing the natural recovery of cells from SCI. These findings lay the theoretical foundation for the involvement of astrocytes and neuroglobin in the treatment of SCI.