Different TLR4 expression and microglia/macrophage activation induced by hemorrhage in the rat spinal cord after compressive injury

Male Sprague–Dawley rats (approximately 220–250 g), provided by the Experimental Animal Center of the Fourth Military Medical University, were maintained with temperature (approximately 22-25°C) and light (12-h light/dark cycle) control, and had free access to water and food. All the experiments involving animals were approved by the Animal Care Committee of the Fourth Military Medical University.

A spinal cord compressive model was made, and animals were randomly divided into various groups. The numbers of animals assigned to those experiments are shown in Table 1. At 6 h, 3 days, and 14 days post injury, animals were sacrificed by intra-cardiac perfusion with 4% paraformaldehyde, and the spinal cord were removed and sectioned for histological observation. Another batch of animals was sacrificed by decapitation and their spinal cords were taken for western blotting assay. To explore the source of blood forming distal hematoma, carbon powder was injected into the compressive epicenter before compression, and hemisection in dorsal cord were made in six other rats. In order to explore the BSCB compromise, six rats were subjected to tannic acid-ferric chloride staining and immunohistochemistry for rat IgG.

Compressive injury model was performed as previously reported [20]. A 20 g metal rod was used to make the compress injury (Figure 1). The tip of the rod was covered with a plastic plate which is approximately 2.6 to 2.9 mm wide (depends on the diameter of the cord) and 0.5 mm thick. The rod was held by a metal tube which controls the position and the depth of the rod via connection with the sterotaxic apparatus but does not press the rod anytime. Before the compression injury, the rat was anesthetized with 1% sodium pentobarbital (50 mg/kg, i.p.), a 30–40 mm dorsal midline incision was made, then the spinal cord was exposed by a bilateral laminectomy of T8 vertebra (corresponding to T9 segment of the spinal cord), with the dura intact. Then the vertebral column was fixed with stabilizing forceps and the rod was placed onto the spinal cord, with the plastic plate perpendicular to the longitudinal axis of spinal cord, and the tip attached the dura surface. After that, the metal tube was moved down vertically at a speed of 0.5 mm/min with the sterotaxic apparatus to produce compression onto the spinal cord by the weight of the rod. After 5 min, the plastic plate was lowered to the bottom of the vertebral canal to achieve a full compression of the cord. The rod was remained at this position for 1 min, followed by a slow withdraw within 1 min. The wound was closed by suturing muscles and skin over the vertebral column. The rats were kept in cages with soft bedding, and manual evacuation of urinary bladder was performed twice daily.

Rats were sacrificed at 6 h, 3 days, or 14 days post injury by an overdose of sodium pentobarbital (100 mg/kg) and perfused intra-cardially with 100 mL of warm normal saline followed by 400 mL 4% cold paraformaldehyde in phosphate buffer (pH 7.4). After perfusion, a 2-cm-long spinal cord segment, with the injured site in the middle, was removed and put into 25% sucrose in phosphate buffer at 4°C until it sank to the bottom of the container (at least 12 h). Then serial 20 μm frozen sagittal sections were cut with a cryostat and mounted on slides in eight sets for hematoxylin and eosin (H-E) staining and immunohistochemistry.

For H-E staining, sections were rinsed in distilled water and were stained in hematoxylin solution for 5 min. After washing with running tap water for 5 min, the sections were differentiated in 1% acid-alcohol for 30 s and were washed again with tap water for 1 min. Then the sections were put into Eosin for 30 s, and were dehydrated through 70%, 80%, 90%, and 100% alcohol for 2 min each. After two changes of xylene, sections were covered with xylene-based mounting medium. Then the stained sections were observed to figure out the lesion site and hematoma.

For immunohistochemistry, sections were rinsed with 0.01 M phosphate buffer saline (PBS) and then blocked with 1% bovine serum albumin (Sigma) in PBS containing 0.5% Triton X-100 for 30 min at room temperature. Then the sections were incubated with primary antibody overnight and fluorescence conjugated second antibody for 2 h. To see the serum protein extravasation in the injured spinal cord, rat immunoglobulin G (IgG) was detected by immunohistochemistry. Briefly, spinal sections were directly incubated with Alexa Fluor 488 goat anti-rat IgG for 2 h. Antibodies against IBa-1 (1:500, rabbit polyclonal, Wako, Tokyo, Osaka, Japan), ED1 (1:400, mouse monoclonal, Serotec, Raleigh, NC, USA), TLR4 (1: 200, mouse monoclonal, Abcam, Cambridge, MA, USA), reactive endothelial cell antigen (RECA) (1:500, mouse monoclonal, Abcam, Cambridge, MA, USA) were used. Fluorescence conjugated secondary antibodies were purchased from Molecular Probes, Oregon, USA. Omission of the primary antibody served as the negative control. The sections were observed under an Olympus FV1000 laser scanning confocal microscope.

At 3 days post compression SCI, animals were deeply anesthetized and sacrificed by decapitation (n = 4). Spinal cord tissues of 3 cm, with epicenter of the injury in the middle, were removed rapidly. Then the spinal cord was divided into three parts equally as rostral, central, and caudal segments, 1 cm of each. All the spinal cord segments were stored in liquid nitrogen and then processed for extraction of protein. Briefly, tissue samples were homogenized with 0.5 mL of ice-cold lysis buffer (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM DTT, 20 μg/mL aprotinin, 1 mM PMSF, and 2 mM sodium orthovanadate). The homogenates were centrifuged at 13,000 rpm for 10 min at 4°C and supernatant were removed. The protein concentration was determined using Bradford method, a detergent-compatible protein assay with a bovine serum albumin as standard. Samples were boiled at 100°C for 10 min and then were electrophoresed on 10-15% SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore, Bedford, MA, USA). The filter membranes were blocked with 5% BSA for 1.5 h at room temperature and incubated with the primary antibody (NF-κB p50, 1:5,000, Epitomics, CA, USA; phospho-IκB, 1:10,000, Epitomics, CA, USA; Homeglobin-alpha, 1:2,000, Epitomics, CA, USA, β-actin, 1:5,000, Cwbiotec, Beijing, China) for approximately 16 to 24 h at 4°C. The membrane was then washed with TBST buffer and incubated with the secondary antibody conjugated with horseradish peroxidase (1:5,000; Cwbiotec, Beijing, China) for 1 h at room temperature and visualized in ECL solution. The density of specific bands was measured with Image J (NIH, USA) software.

Statistical analysis of the density of the specific bands was made with software SPSS 16.0. Density of band NF-κB p50 and phosphor-IκB were compared with that of Hemoglobin-alpha, respectively, then the ratio of NF-κB p50/hemoglobin-alpha, phosphor-IκB/ hemoglobin-alpha of lesion segments, and far-away hematoma segments were analyzed by paired T test (segments of the same spinal cord was paired, n = 4); P value at 0.05 was considered significant.

In order to find out the source of the blood that formed hematoma distal to the lesion site, carbon powder injection experiment was designed. Immediately after the compressive injury was completed, 1 μL PBS containing 0.1 g/mL fine carbon powder was injected slowly with a microsyringe into the lesion site, at a depth of approximately 1 mm. After injection, the tip of syringe was kept for 2 min and was withdrawn carefully. The whole process was completed within 10 min.

In the case that bleeding from the lesion may expand to the distal area, transection between the lesion site and the distal area could be a barrier to impede formation of distal hematoma. To prove this hypothesis, dorsal hemisection was made in another group of animal immediately after compression. At sites approximately 0.3 to 0.4 mm rostral and caudal to lesion center, dorsal hemisection was made with a sharp blade at a depth of approximately 1 mm.

All these animals were killed by intra-cardiac perfusion with 4% paraformaldehyde, and the spinal cord was sectioned for histological analyses.

BSCB may determine the innate immune reaction. Here in this study, tannic acid-ferric chloride staining plus reactive endothelial cell antigen (RECA) immunohistochemistry were used to show vascular integrity. Firstly, RECA immunolabeling was performed in the spinal section at 6 h post injury, to show the existence of blood vessels in both hemorrhage foci, since RECA is biomarker of endothelial cells. Another batch of animals was killed at 6 h post injury by perfusion with tannic acid and ferric chloride to cover the endothelial cells of the blood vessel. The spinal sections from these animals will be immunolabeled by RECA later. The broken down blood vessels may not be well perfused with tannic acid-ferric chloride, so the RECA immunoreactivity would be positive; otherwise, the endothelial cells of the intact blood vessels would not be labeled with RECA.

Animals were anesthetized with an overdose of sodium pentobarbital and were perfused through intracardially with saline (37°C) followed by 4% paraformaldehyde, 2% tannic acid, and 0.5% glutaraldehyde in 0.01 M PBS (37°C). Then 250 mL ferric chloride solution (3%, 37°C) was perfused immediately and spinal cord was removed promptly after the perfusion.

As shown by H-E staining, hemorrhage in the spinal cord formed two distinct foci, the compressive lesion area and the far-away hematoma. The hematoma usually appeared in the caudal, dorsal part of the cord, approximately 1.5 cm away from the epicenter of the injury, which could be even seen grossly (Figure 2A). Distal hematoma was spindle shaped with clear limitation to spinal cord parenchyma. At 6 h post injury, the far-away hematoma formed, while it developed continuously to a larger hematoma till 3 days post injury. Later at 14 days post injury, the spindle-shaped hematoma became a cavity (Figure 2B-G).

Carbon powder injected into the epicenter of the injury indicated that the far-away hematoma may originate from the lesion site, because in the far-away hematoma, carbon powder which was injected to the lesion site immediately after the compression was found 6 h post injury (Figure 3B-D). Moreover, in the compression injured spinal cord with hemi-transection in the dorsal part at rostral and caudal sides, there was no far-away hematoma formed even at 3 days post compression (Figure 3A).

Immunohistochemistry showed that TLR4 immunoreactivity and activated microglia/macrophage could be seen in both lesion site and far-away hematoma, but differently in time points and distribution.

At 6 h post injury, the immunolabeling of TLR4 and IBa-1 was similar in the lesion site and the hematoma. Some of the IBa-1 positive cells appeared bigger than those in the hematoma, and TLR4 immunolabeling could be found but only in several cells in the epicenter (data not shown).

At 3 days post injury, a large number of microglia/macrophage appeared in the epicenter, and most of them were activated, indicating by ED1 immunoreactivity and the shape of the cells (Figure 4B). While in the area adjacent to the hematoma far away from the lesion site, most of the IBa-1 positive cells are not ED1 labeled, and their cell body remained small and the processes were thin (Figure 4C).

Coming to the TLR4 immunoreactivity, similar profile as microglia/macrophage activity was found. Namely, at 3 days post injury, TLR4 immunolabeling were seen in the center of the compressive lesion, and most of the labeling was co-located with round microglia/macrophage, indicating by confocal microscopy. At the same time point, little TLR4 could be seen in the area of hematoma. Apart from the area around, there were few IBa-1 positive cells inside the hematoma. The distribution of IBa-1 immunoreactivity was clearly limited according to the border of the hematoma (Figure 5).

TLR4 immunoreactivity was increased in the hematoma far away from the epicenter of the lesion at 7 days (not shown) and 14 days, which is nearly parallel to the activation of microglia/macrophage. At 14 days, strong TLR4 labeling was seen within the hematoma but not in the adjacent area (Figure 6C, G, K). This profile of TLR4 immunolabeling and microglia/macrophage was similar to that of epicenter at 3 days post injury. Notably, it was clearly shown by confocal microscopy that TLR4 immunoreactivity was located inside the IBa-1 positive cells which were extremely activated as their soma became large and round like huge bubbles (Figure 6D, H, L).

With immunofluorescent labeling, one can see abundant capase-3 positive cells in the lesion site at 3 days, but very few cells in the far-away hematoma at the same time point. However, the number of capase-3 immunoreactive cells markedly increased in the area around hematoma at 14 days post injury (Figure 7).

In order to compare the inflammatory signal downstream TLR4, we detected the NF-κB p50, phosphorylated inhibitor of kappa B (p-IκB) protein levels of the spinal segment of lesion site and the segment of hematoma at 3 days post injury, based on hemoglobin-α level which was used to represent red blood cells. Repetitive western blotting assay showed that ratio of NF-κB p50/hemoglobin-αlevel was higher in the lesion segment than that in the hematoma segment. The ratio of p-IκB /hemoglobin-α was consistent, statistical analysis showed the differences were both significant (Figure 8).

Immunohistochemistry for RECA showed that there were blood vessels in lesion site and the far-away hematoma at 6 h post injury. However, spinal cord of animals that were perfused with tannic acid-ferric chloride before sacrifice, RECA positive labeling could only be found in the lesion area, but not in the far-away hematoma, indicated the integrity of blood-vessels in the area of hematoma (Figure 9).

Shown by immunohistochemistry, rat IgG immunoreactive labeling could be seen in the lesion site and around area of the injured spinal cord at 3 days after compression. However, there was very little IgG immunoreactivity in the far-away hematoma (Figure 10).