HIF‐1α promotes astrocytic production of macrophage migration inhibitory factor following spinal cord injury

Abstract Background Macrophage migration inhibitory factor (MIF) is an important mediator of neuropathology in various central nervous system (CNS) diseases. However, little is known about its inducers for production from the nerve cells, as well as the underlying regulatory mechanism. Injury‐induced HIF‐1α has been shown to exacerbate neuroinflammation by activating multiple downstream target molecules. It is postulated that HIF‐1α is involved in the regulation of MIF following spinal cord injury (SCI). Methods SCI model of Sprague–Dawley rats was established by cord contusion at T8–T10. The dynamic changes of HIF‐1α and MIF protein levels at lesion site of rat spinal cord were determined by Western blot. The specific cell types of HIF‐1α and MIF expression were examined by immunostaining. Primary astrocytes were isolated from the spinal cord, cultured and stimulated with various agonist or inhibitor of HIF‐1α for analysis of HIF‐1α‐mediated expression of MIF. Luciferase report assay was used to determine the relationship between HIF‐1α and MIF. The Basso, Beattie, and Bresnahan (BBB) locomotor scale was used to assess the locomotor function following SCI. Results The protein levels of HIF‐1α and MIF at lesion site were significantly elevated by SCI. Immunofluorescence demonstrated that both HIF‐1α and MIF were abundantly expressed in the astrocytes of the spinal cord. By using various agonists or inhibitors of HIF‐1α, it was shown that HIF‐1α sufficiently induced astrocytic production of MIF. Mechanistically, HIF‐1α promoted MIF expression through interaction with MIF promoter. Inhibition of HIF‐1α activity using specific inhibitor markedly reduced the protein levels of MIF at lesion site following SCI, which in turn favored for the functional recovery. Conclusion SCI‐induced activation of HIF‐1α is able to promote MIF production from astrocytes. Our results have provided new clues for SCI‐induced production of DAMPs, which may be helpful for clinical treatment of neuroinflammation.


| BACKG ROU N D
Traumatic injury to the spinal cord always results in the loss of locomotor function, which is worsened by, to some extent, a complex cascade of secondary injury events. 1 At the early acute injury phase, the neurons, astrocytes, and oligodendrocytes at lesion site suffer to damage, in conjunction with the activated immune cells and the disrupted vasculature and the blood-spinal cord barrier (BSCB). 2 Immediate after that, a more insidious and deleterious secondary tissue damage is programmed to arrive and persists for several days or longer, characterized by ischemia, cellular necrosis, formation of glial scar, and cystic cavities. 3The overwhelming inflammatory response evoked by damage-associated molecular patterns (DAMPs) is recognized as a culprit in initiation of the pathogenesis, as genetic deletion or pharmacological inhibition of endogenous DAMPs or the according receptors has been shown to improve the locomotor function. 4Injury-induced DAMPs are derived from active secretion by immune-related cells or passive release from necrotic cells. 5,6However, the specific factor(s) that drives the production of DAMPs from resident nerve or immune cells, as well as the underlying mechanism following SCI remains unclear.
Astrocytes are the abundant glial cells that widely distribute in the central nervous system (CNS), associating with health and disease. 7,8They have a myriad of well-documented roles in support of neuronal homeostasis including formation of the blood-brain barrier, secretion of the neurotrophic factors and control of the microenvironment. 9,10In response to stimuli, astrocytes can undergo morphological, molecular and functional remodeling termed reactivity, which can exert a protective or detrimental influence on surrounding neurons. 11,12The reactive astrocytes are also considered as active player in the CNS inflammation by expressing a wide variety of receptors including pattern recognition receptors (PRRs) and cytokine receptors, 13,14 through which astrocytes can respond to multiple cues in the CNS microenvironment and produce various pro-inflammatory cytokines and chemokines. 15,168][19][20][21] Accumulative evidence has emphasized that the reactive astrocytes per se are major source of DAMPs following SCI, which interact with diverse cell types and contribute to crosstalk between immune and neural systems. 22,23So far, less information is available regarding the regulatory mechanism of DAMPs production from astrocytes following SCI.
Macrophage migration inhibitory factor (MIF), a critical proinflammatory cytokines, is constitutively or inducibly expressed by a variety of cell types in most tissues, such as macrophages, fibroblasts, insulin secreting β-cells of the pancreas, pituitary cells, and endothelial cells. 24,25It has been shown to function in organ development, tumorigenesis, tissue injury, and inflammatory activation. 25 the spinal cord, expression of MIF is upregulated within neurons, microglia, and astrocytes after injury. 22,26The overproduced cytokine activates microglia and astrocytic inflammation, resulting in a worsened lesion milieu. 26,27Deletion or inhibition of MIF significantly attenuates local neuronal death and promotes functional recovery after SCI. 28,29Thus, the elevated extent of MIF at the lesion site is proportional with the prognosis of CNS damage severity. 30chanistically, MIF performs proinflammatory function through binding to CD74 and/or CXCR2/4 receptors, and activates intracellular MAPKs and NF-κB signal pathway. 31However, the regulators responsible for the astrocytic production of MIF following SCI remain elusive.
Hypoxia induced by SCI always triggers many cellular responses. 32,338][39] Lines of evidence demonstrate that functions of HIF-1α and HIF-2α vary depending on the cell type of tissue or tumor. 40llowing SCI, a local hypoxic environment can induce overexpression of HIF-1α, which in turn triggers a cascade of downstream signals.4][45][46] Given that HIF-1α acts as a universal switch in the regulation of inflammation, it is conceivable that HIF-1α possibly promotes astrocytic production of MIF following rat SCI.In the  present study, we analyzed the relations between HIF-1α activation and MIF protein level in the astrocytes after rat spinal cord contusion.We further investigated the mechanism of hypoxia-mediated MIF expression in astrocytes, and observed the effects of HIF-1α suppression on the hindlimb motor functional recovery.Our results provided a prospective strategy for clinical treatment of SCI-induced neuroinflammation.

| Animals
Adult male Sprague-Dawley (SD) rats weighing 180-220 g were provided by the Center of Experimental Animals, Nantong University.All the rats were housed in standard cages (five rats in each cage) under identical conditions maintained at 22 ± 2°C on a 12-12 h light-dark cycle and allowed ad libitum to feed and water.All animal protocols were approved by the Animal Care and Use Committee of Nantong University and the Animal Care Ethics Committee of Jiangsu Province.

| Establishment of the SCI model and drug treatment
The number of animals subjected to surgical treatment was six per group in triplicate.The contusion SCI rat model was prepared as previously described. 47In short, all animals were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg).The fur of the surgical area on the back was shaved and the skin was disinfected with chlorhexidine solution.A midline incision was made over the thoracic vertebrae, followed by a T9 laminectomy with the dura remaining intact.Using the IH-0400 Impactor device (Precision Systems and Instrumentation), animals received a 150-kilodyne contusion injury on the exposed spinal cord segment.The impact rod was removed immediately, and the wound was irrigated.The muscle layers and the skin were then sutured using silk threads.The procedure was visually checked by the formation of hematoma and by paralysis of the hindlimbs after animal awakening from the anesthesia.For drug delivery, a total of 8 μL of 8 mM HIF-1α inhibitor LW6 (MCE) was slowly injected intrathecally, prior to the incision suture.The rats were undergone the proper care following surgery.

| Cell culture and treatment
Astrocytes were prepared from the spinal cord of neonatal (1 day after birth) SD rats.The method of isolating and culturing astrocytes was according to the previous description. 29Briefly, the spinal cord segments were enzymatically dissociated in 0.25% trypsin (Gibco-BRL) for 15 min at 37°C, and the suspension was then centrifuged at 1200 rpm for 5 min.Finally, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified 5% CO 2 incubator at 37°C.A monolayer of astrocytes was obtained 7-10 days after the plating.Microglia and other non-astrocytes were separated from the bottom of the dishes by shaking and removed by changing the medium.Prior to the experiment, the second or third passage cells were kept quiescence in the medium containing 5% FBS for 4 days.Astrocyte phenotype was evaluated by positive staining of astrocytic marker glial fibrillary acid protein (GFAP).For treatment under hypoxic conditions, dish or plates were placed into a modified incubator chamber (Billups Rothenberg, Del Mar, CA), filled with 1% O 2 , 5% CO 2 , and 94% N 2 (called hypoxic conditions) to culture.For CoCl 2 or DMOG treatments, astrocytes were incubated with 0.5 mM CoCl 2 or 1.5 mM DMOG at 37°C for desired time under normoxic condition.

| Immunohistochemistry
The spinal segments or the cultured cells were harvested and fixed.obtained by using Leica TCS SP5 confocal laser scanning microscope system (Leica).

| Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was prepared with Trizol (Thermo Fisher Scientific) from cultured cells as mentioned above.The first-strand cDNA was synthesized using HisScript II Q Select RT SuperMix for qPCR (R223-01; Vazyme) in a 20 μL reaction system that contained 2 μg total RNA.The PCR reactions were performed in a final volume of 20 μL according to protocol of ChamQTM SYBR qPCR Master Mix kit (Q711-02; Vazyme, Nanjing, China).The primers used in the reactions were designed based on the genome sequences with sense primer 5′-GGT CAC ACC GCA CTT AAC AC-3′ and anti-sense primer 5′-CGC TCG TGC CAC TAA AAG TC-3′ for MIF.The LightCycler96 software (Roche) was used for PCR reactions and the data analysis.

| ELISA
Tissue samples were sonicated using the lysis buffer with protease inhibitor PMSF as mentioned above.The lysis was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected for MIF (Elabscience) ELISA assay.The concentration of MIF was expressed as ng/mg for the lysate of the cord tissues.Plates were read with a multifunctional enzyme marker (Biotek Synergy2) at a 450 nm wavelength.

| Subcellular fractionation
After the cell stimulation with DMOG (1.5 mM) for 4 h, the extraction of cytosolic and nuclear protein from astrocytes was isolated according to the protocol of Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime).Briefly, the cells were washed with ice-

| Luciferase assays
The sequence of MIF promoter was amplified by PCR using prim- Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol.

| Behavioral tests
The hindlimb locomotor function recovery was evaluated using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.After intrathecal injection of 8 μL of DMSO or 8 μL of 8 mM LW6 at 0, 7, 14, and 21 days, three well-trained investigators blind to the study were invited to observe the behavior of rats for 5 min.The BBB score ranged from 0 to 21 according to the rating scale.Every rat had a BBB score of 21 before surgery, and 0 to 1 after a successful SCI.

| Statistical analysis
Statistical analysis used GraphPad Prism 8 software.All data are presented as mean ± SEM.The D'Agostino-Pearson omnibus normality test was used to assess data distribution.Comparisons between two groups with normal distribution were analyzed by two-tailed unpaired Student's t test or the Mann-Whitney test when the distribution was not parametric.Differences between multiple groups were analyzed using one-way or two-way analysis of variance, followed by Tukey's or Sidak's post hoc test.P-value <0.05 was considered statistically significant and was denoted in the figures as P < 0.05.

| SCI significantly induces astrocytic expression of HIF-1α
To understand the potential effect of hypoxia on SCI-induced dynamic changes of MIF expression, the protein levels of HIF-1α and HIF-2α at lesion sites were determined at 0, 1, 4, and 7 days following spinal cord contusion.Western blot demonstrated that the expression of HIF-1α, but not of HIF-2α, was significantly induced at 1 day after SCI (Figure 1A-D).Meanwhile, the protein levels of MIF were also elevated at 4 and 7 days (Figure 1B,D), indicating SCI-induced hypoxia and overproduced DAMPs around the lesion milieu.
To examine specific cell types that were induced to express HIF-1α in the injured cord, tissue sections were prepared from 0.5 cm segments harvested around the epicenter of contusion at 1d following SCI.Immunostaining revealed that HIF-1α protein colocalized with GFAP-positive astrocytes (Figure 1E-H The data indicate that the astrocytes, together with neurons, are the hypoxia-sensitive cell types following SCI. To observe whether SCI-induced HIF-1α expression had a colocalization with those of MIF, immunostaining was performed on the cord at lesion sites following SCI.Results revealed that HIF-1α and MIF positive signals were well colocalized in the cord tissues, and significantly induced by SCI at 4 and 7 days after SCI (Figure 2A-P).
The data indicate that the SCI-induced HIF-1α at the lesion sites may be involved in regulation of MIF expression.

| Hypoxia promotes astrocytic expression of MIF
To validate the inducible roles of hypoxia on the production of MIF in astrocytes, the primary astrocytes isolated from spinal cord were cultured with purity over 92% (Figure 3A).The cells were incubated in an anoxic incubator with 1% oxygen for different times, and then collected for analysis.Western blot assay showed that the protein level of HIF-1α was significantly increased from 1 h with a peak at 2 h following hypoxia treatment, but that of HIF-2α was unaffected (Figure 3B-D).The expression of MIF at transcriptional and translational level was determined following cell hypoxia for 8 to 48 h.The hypoxia stress promoted MIF transcription within astrocytes at 24 h, as examined by PCR assay.ELISA measurement of both cell lysates and supernatants demonstrated that astrocytes were induced to produce MIF in response to hypoxia for 24, 36, and 48 h (Figure 3E-H).These data indicate that hypoxia promotes MIF production from astrocytes.

| Activation of HIF-1α facilitates the astrocytic production of MIF
To elucidate whether hypoxia-activated HIF-1α participates in the astrocytic production of MIF, cobalt chloride (CoCl 2 ), the chemical inducer of HIF-1α, was applied to stimulate astrocytes.As was expected, cell exposure to 0.5 mM CoCl 2 significantly promoted the transcription and translation of MIF in the astrocytes, by activation of HIF-1α, but not of HIF-2α (Figure 4A-E).Similarly, cell treatment with 1.5 mM dimethyloxalylglycine (DMOG), another HIF-1α activator, also resulted in the induction of MIF in the astrocytes, together with activation of HIF-1α (Figure 4F-J).The data indicate that activation of HIF-1α facilitates MIF production from astrocytes.

| HIF-1α selective inhibitor serves to attenuate the astrocytic production of MIF
To clarify whether inhibition of HIF-1α can attenuate the astrocytic production of MIF, a selective inhibitor LW6 at final concentration of 20 μM was added to culture medium of astrocytes in the presence of 1.5 mM DMOG.PCR results showed that the transcription of MIF in the astrocytes was markedly inhibited by LW6 at 24 h (Figure 5A).Also, the protein levels of MIF were decreased after treatment of astrocytes with LW6 in the presence of DMOG (Figure 5B,C).The data indicate that inhibition of HIF-1α is able to suppress hypoxiamediated astrocytic production of MIF.

| HIF-1α regulates MIF production by interaction with the promoter of MIF
Previous studies have shown that HIF-1α regulates expression of downstream genes through entering into the cellular nuclei and binding to the promoter of the target genes.To shed light on the regulatory mechanism of HIF-1α on the astrocytic MIF, the subcellular distribution of HIF-1α was firstly examined following astrocyte treatment with 1.5 mM DMOG for 4 h.Immunoblot analysis for the cytosolic and nuclear fraction showed that HIF-1α was predominantly distributed in the nucleus of astrocytes rather than in the cytoplasm in response to DMOG stimulation (Figure 6A,B).Immunostaining further confirmed the nuclear translocation of HIF-1α following DMOG-induced hypoxia (Figure 6C,D).In addition, luciferase assays were performed to detect the activity of luciferase reporter gene in the MIF-promoter constructs.The results revealed that overexpression of HIF-1α significantly increased the activity of luciferase in MIF-promoter constructs, whereas in MIF-promoter-Mut did not (Figure 6E).The data indicate that HIF-1α regulates MIF expression by interaction with MIF promoter in the astrocytes.

| Inhibition of HIF-1α significantly attenuates the astrocytic production of MIF following SCI
To examine the effects of HIF-1α inhibition on the production of MIF following SCI, a total of 8 μL of 8 mM LW6 were intrathecally injected at lesion sites.ELISA assay for the 0.5 cm cord segments demonstrated that the protein levels of MIF were significantly reduced at lesion site following LW6 administration at 4 days (Figure 7A).inhibition-mediated reduction of MIF following SCI was thus expected to have protective roles on the functional recovery.To evaluate the effects of LW6 on the contused spinal cord of rats, behavioral tests were performed during 3 weeks to assess hind-limb motor function.BBB scores showed that administration of 8 μL of 8 mM LW6 at the lesion sites of the cord efficiently promoted the improvement of the locomotor function of rats in comparison with the control (Figure 7D).The data indicate that suppression of HIF-1α is able to reduce the production of MIF in astrocytes, which in turn contributes to the locomotor functional recovery following SCI.

| DISCUSS ION
9][50][51] Various DAMPs, including several heat-shock proteins, HMGB1, hyaluron fragments, ATP, uric acid, heparin sulfate, and the DNA materials, are recognized to aggravate neuropathology following CNS injury or disorder. 52,53MIF is recently defined as one of DAMPs that mediates multiple neurological diseases and severity of SCI. 24Distinct to other DAMPs, the inducers of MIF production in the injured CNS have not been fully elucidated.We have previously shown that injury-activated thrombin is able to contribute to the MIF production following SCI. 22In the present study, we revealed that HIF-1α also served as an enhancer of MIF expression in astrocytes, suggesting that SCI-induced production of MIF is promoted by multiple mediators.Whether other DAMPs are governed by HIF-1α following SCI deserves further study.
Hypoxia is associated with neurological disorders, such as trauma, ischemic stroke, Alzheimer's disease (AD) and Parkinson's disease (PD). 33,54Once the CNS, the most sensitive tissue to the supply of oxygen, is damaged, HIF-1α is immediately induced by multiple cell types to regulate the transcription of various target genes that mediate oxidative stress, inflammation, cell death and adaptive response. 36,55,56For example, within the earliest 24-48 h after ischemia, the accumulation of HIF-1α triggers a series of brain events related to pentose phosphate pathway inhibition and promotes neuronal apoptosis by upregulating the expression of downstream genes in neurons such as NKCC1 (Na + -dependent chloride transporter 1) in rat. 42,57,58Notably, hypoxia can activate HIF-1α within 1-3 h, whereas HIF-1α-mediated expression of target molecules, as well as related pathophysiological effects needs to take more time.
Several controversy debates regarding to the roles of HIF-1α following CNS insults, however, come from that a blockade of HIF-1α activation can lead to increased brain damage and decreased neurogenesis during the postischemic stages both in vitro and in vivo. 59,602][63][64] Thus, HIF-1α functions in neuropathology dependent on spatio-temporal injury modes, which drives differential downstream targets.As for HIF-1α-mediated inflammation in the pathological CNS, HIF-1α is found to promote the production of chemokines and proinflammatory cytokines including MCP-1, MCP-5, TNFα, and IL-1β in hypoxic astrocytes, which aggravate the neuroinflammation after acute ischemic stroke. 65,66In the present study, we showed that SCI-induced HIF-1α was able to facilitate proinflammatory MIF -1α, hypoxia, inflammation, MIF, spinal cord F I G U R E 1 Determination of HIF-1α and MIF expression at the lesion sites following SCI.(A) Western blot analysis of HIF-1α, HIF-2α and MIF protein following SCI at 0, 1, 4, and 7 days, respectively.Quantities were normalized to an endogenous β-Actin.(B-D) Quantification data as shown in (A).(E-T) Colocalization of HIF-1α with GFAP-positive astrocytes, NeuN-positive neurons, IBA-positive microglia, and MBP-positive oligodendrocytes at 0d (uninjured) and 1d (injured).The rectangle indicates the magnified region.Arrowheads indicate the colocalization signals.Experiments were performed at least in triplicate.Data are expressed as mean ± SEM, *p < 0.05.Scale bars, 500 μm, and 50 μm in magnification.

F I G U R E 2
Analysis of HIF-1α colocalization with MIF in the cord tissues.Sections were prepared at 0, 1, 4, and 7 days following SCI.The rectangle indicates the magnified region.Arrowheads indicate the colocalization signals.Scale bars, 500 μm in (A, E, I, M); 50 μm in magnifications.
1 mM PMSF, and 0.1 mM β-mercaptoethanol.Protein concentration of each specimen was detected by the BCA kit (Beyotime) to maintain the equivalent loads.Protein extracts were boiled for 5 min, and electrophoretically separated on 10% SDS-PAGE before electro-transferred to PVDF membranes.The membranes were F I G U R E 3 Effects of hypoxia on the astrocytic expression of MIF.(A) Primary cultured astrocytes were stained with GFAP and Hoechst 33342, showing purity over 92%.(B) Western blot analysis of HIF-1α and HIF-2α protein levels in the astrocytes induced by hypoxia for 0-24 h.(C, D) Quantification data as shown in (B).Quantities were normalized to endogenous β-Actin.(E) RT-PCR analysis of mif in the astrocytes induced by hypoxia for 8, 16, and 24 h.Quantities were normalized to endogenous gapdh.(F) Western blot analysis of MIF protein levels in the astrocytes induced by hypoxia for 24, 36, and 48 h, respectively.(G) Quantification data as shown in (F).(H) ELISA determination of MIF in the supernatants of cultured astrocytes exposed to hypoxia for 24, 36, and 48 h.Experiments were performed in triplicates.Error bars represent the SEM (*p < 0.05).
cold PBS, followed by lysis in 200 μL cytoplasmic protein extraction agent A with 1 mM PMSF on ice for 15 min after a high-speed vortex for 5 s.Then, the cytoplasmic protein extraction agent B was added, and the mixture was vortexed for 5 s prior to incubation on ice for 1 min.Samples were centrifuged at 16,000 g at 4°C for 5 min, and the supernatant (cytosolic fraction) was immediately collected.The pellet (nuclear fraction) was resuspended in nuclear protein extraction agent supplemented with 1 mM PMSF.The samples were vortexed thoroughly at high speed for 15-20 times at 30 min, followed by a centrifugation at 16,000 g for 10 min.Then, the supernatants containing the nuclear extracts were collected.Proteins of nuclear and cytosolic extracts were determined by Western blot.Both GAPDH and Histone H3 were used as internal control of cytoplasmic and nuclear proteins, respectively.
ers: 5′-TTT CTC TAT CGA TAG GTA CCT TCA GGT ACC TGG GGA GTC C-3′; 5′-CTT AGA TCG CAG ATC TCG AGA GCT CAG GAC CGC CCC AAG CCG G-3′.The sequence at length of 2040 bp was cloned into pGV238 reporter vector to form a reporter plasmid (MIF-wt), and a control plasmid (MIF-mut) was constructed by mutation of A-T at predicted HIF-1α binding elements.Cells grown to 70% confluence were transfected in triplicate with pGV238-MIF-wt, pcDNA-HIF-1α or pGV238-MIF-mut.After 48 h of transfection, cells were collected and the luciferase activity was measured using the F I G U R E 5 HIF-1α inhibitor attenuates MIF production of astrocytes.(A) RT-PCR analysis of mif in the astrocytes stimulated by 1.5 mM DMOG in the presence or absence of 20 μM LW6 for 24 h.Quantities were normalized to endogenous gapdh.(B) Western blot analysis of MIF protein levels in the astrocytes treated by 20 μM LW6 in the presence of 1.5 mM DMOG.(C) Quantification data as shown in (B).Quantities were normalized to the endogenous β-Actin.Experiments were performed in triplicates.Error bars represent the SEM (*p < 0.05).| 3809 HAO et al.
Immunofluorescence showed that the expression of MIF in the astrocytes was markedly decreased by the LW6 treatment at 4 days (Figure 7B,C).As massive release of MIF can result in the aggravation of neuroinflammation and severe loss of locomotor function, HIF-1α F I G U R E 6 HIF-1α promotes astrocytic expression of MIF through interaction with the promoter.(A) Western blot analysis of HIF-1α protein levels in the cytoplasm and nucleus following astrocytes treatment with 1.5 mM DMOG for 4 h.Quantities were normalized to endogenous GAPDH (cytoplasm) and Histone H3 (nucleus).(B) Quantification data as shown in (A).(C) Colocalization of HIF-1α with GFAP and DAPI in the astrocytes treated by 1.5 mM DMOG for 4 h.Arrowheads indicate the colocalization signals.(D) Quantification data as shown in (C).(E) Assay of HIF-1α overexpression on the activity of luciferase in MIF-promoter constructs.Scale bar, 50 μm in (C).Experiments were performed in triplicates.Error bars represent the standard deviation (*p < 0.05).

F
I G U R E 7 HIF-1α inhibitor attenuates MIF production at lesion site of the cord and evaluation of rat hindlimb motor function following SCI.(A) ELISA assay of MIF at lesion sites following intrathecal injection with 8 μL of 8 mM LW6 for 0, 1, 4, and 7 days, respectively.(B) Immunostaining showed colocalization of MIF with S100β-positive astrocytes following injection of 8 μL of 8 mM LW6 for 0 and 4 days.Rectangles indicate the magnified region.Arrowheads indicate the positive signals.(C) Quantification data as shown in (B).(D) BBB score of hindlimb at 0, 7, 14 and 21 days following injection of 8 μL of 8 mM LW6 at lesion sites, n = 6.Scale bars, 500 μm in (a′), (e′), and (i′); 50 μm in magnifications.Experiments were performed in triplicates.Error bars represent the standard deviation (*p < 0.05).