Introduction

Injury induces innate immune activation and cascades inflammatory reactions through Toll-like receptors (TLRs) [1]. TLRs are an important family of recognizing pathogens and initiating an innate immune response to protect the host. TLR4, an important member of TLRs, initiates a cascade of intracellular events involving the Nuclear Factor-kappa B dependent production via activating myeloid differentiation factor 88 (MyD88) and then release of cytokines and chemokines such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α [2]. Peripheral nervous system (PNS) injuries can also activate TLR4 at the lesion area, which will further trigger factors in the signal pathway, causing the release of inflammatory factors in the distal nerve [3,4]. In the early inflammatory stage, cytokines including TNF-α, IL-6, and IL-1β are the three major inflammatory factors [5]. Following sciatic nerve transection, TLR4 expression is necessary for the upregulation of glial cell-derived neurotrophic factor and increased mRNA levels of interleukin-6 [6].

However, peripheral nerve injury affects innate immune reactions in the lesion site and the central nervous system (CNS) [6–8]. Research has found that after nerve injury, TLRs involved in Wallerian degeneration and are responsible for the stimulation of astrocytes and microglia that can cause induction of the proinflammatory mediators and cytokines in the spinal cord, thereby leading to the generation and maintenance of neuropathic pain [7]. Although results from different reports are not consistent regarding the contribution of TLRs to nerve regeneration and functional recovery, these studies indicated that innate immune molecules influence peripheral nerve regeneration in the CNS and PNS. As peripheral nerves derive from the CNS, we hypothesized that the expression of innate immune molecules including TLR4 and related signaling in the CNS should be remarkably increased by peripheral nerve injury, and the upregulated innate immune reaction helps initiate peripheral nerve regeneration. TLRs have been identified in the CNS neurons and glial cells including microglia, astrocytes, and oligodendrocytes [9]. But the timing and cell types of TLR4 and related molecules expression in the CNS after peripheral nerve injury remains unclear.

This study investigated the expression of TLR4 and key molecules downstream in the lumbar spinal cord on a mouse model of femoral nerve injury (FNI) by motor branch transection in an attempt to explore the relationship between injury to the peripheral nerve that innervates hind limbs and inflammation of related spinal cord as well as the involving cell type and possible function of TLR4 pathway. We found that TLR4 and the key molecules downstream were expressed in the lumbar spinal cord not only in the glial cells but on motor neurons, and TLR4 signaling in the spinal cord may be involved during the full length of the functional recovery.

Materials and methods Animals and surgical procedures

Adult female C57BL/6 mice were randomly distributed to acute (24–72 h after femoral nerve transection) and chronic (6 weeks after femoral nerve transection) surgical groups (n = 8 for each time point and n = 4 for the sham group). All the animals were treated under the instruction of the Northwest China Committee of Experimental Animal Care, and their regulations were in accordance with NIH guidelines. All efforts were made to minimize animal suffering and the number of animals used. For surgery and behavioral test, all the procedures were designed following Schachner’s lab [10]. The animals were anesthetized by intraperitoneal injections of 4% Chloral hydrate intraperitoneally (5 ml/kg). After that, 0.1% sodium pentobarbital was injected (i.p) into the mice. The right femoral nerve was exposed by a skin incision. The motor branch was cut 2–3 mm beneath the bifurcation by fine scissors. Then both cut ends of the nerve were inserted into a polyethylene tube (2 mm long, 0.38 mm inner diameter, AMT Medizintechnik, Dusseldorf, Germany) and fixed with single epineural 11–0 nylon stitches so that a 2-mm gap was present between the proximal and distal stump. The tube was then filled with PBS and the skin wound was closed with 4–0 sutures [11].

Behavior measurements

Seven days before surgery, mice were trained to walk on a horizontal plastic beam (1000 mm long, 38 mm wide) leading to their home cage. It was a strong attraction for mice to smell or see the home cage, so they would walk toward it without hesitation. After the learning phase prior to operation, once the mouse was put on the beam, it will walk steadily to the cage with no exploratory pauses. A baseline beam-walking trial was video recorded for each animal as D0 values. A rear view of the walking along the beam was captured with a WX-1 camera (Sony, Tokyo, Japan) and recorded on the storage card. Each animal was video recorded before surgery as a baseline and at different time points (1, 2, 4, 6, and 8 weeks) after nerve transaction. FNI caused quadriceps muscle dysfunction, so the external rotation of the heel became abnormal, which led to a larger angle than normal. And the heels-tail angle (HTA) was taken when the right hind limb and a maximum altitude of the contralateral swing. As the injured hind limb cannot support the body sufficiently, this angle became smaller than normal; for the foot-base angle (FBA), the video frames in which the right paw of the mouse was at the take-off position. Three or five frames were captured from the videos per mouse using Windows snipping tool and were analyzed by image-J (NIH). Two parameters (HTA, Fig. 1 A) and the FBA (Fig. 1b). For the voluntary pre-limb movements, the limb protraction ratios (PLR, Fig. 1c) were video recorded without initial training. Length measurements were performed with Image-J [11].

Fig. 1:

Time course and degree of motor recovery after femoral nerve lesion. Data plotted as individual values of HTA (a), FBA (b), and PLR (c) at different time points after nerve transection and repair. N = 8 for each time point, repeated measurement one-factor analysis of variance (ANOVA) followed by Tukey’s post hoc test. *P < 0.05.

Retrograde labeling

The animals were anesthetized with 4% Chloral hydrate for retrograde labeling of regenerated motoneurons. Five percent fluorogold (Fluorochrome, Denver, Colorado, USA, CAT 4716905) was injected into the quadriceps branch (mixed nerve containing sensory and motor axons) as a retrograde tracer [11]. After disclosing the distal end of the right femoral nerve, 10 minutes after dye application, the wound was closed. The same labeling procedure was applied to ‘non-injured’ mice as an estimate of a normal number of motoneurons projecting quadriceps branches only. After 1 week of recovery, the retrograded motoneurons can be detected at the anterior horn of the L2–L4 spinal cord.

Immunofluorescent staining

To observe the short-term and long-term changes in protein expression, acute and chronic surgical groups were detected and compared with normal mice separately. For the chronic group, 1 week after the retrograde, 10 mice (5 for surgery and 5 for the control group) were used for TLR4, MyD88, IL-1β, NeuN, GFAP, and Iba-1 immunofluorescent staining (Table 1). For the acute group, we perform the same labeling at 72 h post-surgery which was reported as a peak of immune reaction [12]. Totally 10 mice were included, 5 for surgery and 5 for coordinate control. The animals were deeply anesthetized with 4% Chloral hydrate i.p. body weight (Sigma, St. Louis, Missouri, USA) and were sacrificed by transcardial perfusion with 20 ml 0.9% saline, followed by 50 ml ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Spinal cords were removed and post-fixed in the same fixative for 2 h at 4 °C. They were then cryoprotected in 30% sucrose in 0.1 M phosphate buffer overnight at 4 °C. Alternative serial longitudinal sections of spinal cords were cut at 20 um thickness on a cryostat (CM1900; Leica, Heidelberger, Germany) and mounted on gelatin-coated slides. All subsequent immunohistochemical procedures were done at room temperature. Slides were blocked for 2 h in PBS (PBS, pH 7.4), containing 5% BSA, 5% normal goat serum (NGS), and 0.5% Triton X-100. Slides were then incubated overnight in the primary antibody listed in the dilutions in the chart below, diluted in PBS containing 1% BSA, 1% NGS, and 0.5% Triton X-100 overnight. After rinsing in PBS, slides were finally incubated with Texas Red-conjugated streptavidin (1:500; Molecular Probes, Eugene, Oregon, USA) and Alexa 488-conjugated goat anti-rabbit or anti-mouse IgG (1:500; Molecular Probes) diluted in PBS containing 0.3% Triton X-100 for 4 h. After rinsing in PBS, slides were cover-slipped with anti-fading medium containing 50% glycerin and 2.5% trimethylene diamine in PBS, and examined with a confocal laser scanning microscope (Fluoview 1000; Olympus, Tokyo, Japan) using laser beams of 543 and 488 nm with appropriate emission filters for Texas Red (590–610 nm) and Alexa 488 (510–525 nm), respectively. Digital images were captured by Fluoview application software (Olympus). To make an analysis of immunostaining, the intensity of fluorescence was quantified following the previous report [12]. Briefly, all the images were opened by Image J Software (V1.8.0.112), and dark background was chosen for fluorescence; then a proper threshold was selected for a certain labeling, and the entire image was measured automatically.

Table 1 - Antibodies for IHC Antigen Host Cat# Company Dilution TLR4 Mouse AP1504a ABGENT 1:100 Tublin-3 Rabbit 2128S CST 1:100 IL-1β Rabbit 31202S CST 1:100 NeuN Mouse MAB377 TEMECULA 1:200 MyD88 Rabbit 4283S CST 1:200 Iba-1 Rabbit 019-19741 Wako 1:1000
Western blot analysis

Tissues from the L2-L4 spinal cords in 6 control and 18 acute surgery mouse (6 for each group of 24, 48, 72 h) were removed and lysed by homogenizing in ice-cold RIPA lysis buffer containing 50 mM Tris–HCI (pH 7.5), 0.1% SDS, 0.5% sodium deoxycholate, 150 mM NaCl, 1% NP-40, and 0.1% phenylmethanesulfonyl fluoride. Clarification of the extracts was achieved by centrifugation for 10 min at 12 000 rpm and 4 °C. Protein concentration was determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, Illinois, USA). Equivalent protein samples were subjected to 10% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (0.2 m, Boehringer Mannheim). Membranes were blocked with 5% nonfat dry milk in Tris buffer, containing 0.05% v/v Tween 20 and 10 mM NaF for 1 h at room temperature, and then incubated with antibodies specific to TLR4 (1:200, ABGENT, San Diego, USA), MyD88 (1:400; CST, Danvers, USA), IL-1β (1:200, CST, Danvers, USA) or Actin (1:5000; Sigma) overnight at 4 °C. After rinsing in Tris buffer, they were incubated with peroxidase-conjugated goat anti-rabbit IgG. Signals were revealed by chemiluminescence using a SuperSignal West Pico Kit (Pierce Biotechnology). The membranes were exposed to X-ray films. The film signals were digitally scanned and quantified using Image-J software.

Statistical analyses

All statistical analyses were performed with Prism v.6.0 (GraphPad Software, Inc.). One-factor analysis of variance (ANOVA) followed by Tukey’s post hoc test, or repeated measurement ANOVA (for behavior tests) was used for analyses, and P < 0.05 was considered significant.

Results Locomotion fully recovered within 6 weeks after femoral nerve injury

Before surgery, we recorded each mouse running on a high beam as well as pencil experiments to obtain the baseline of HTA, FBA, and limb PLR (Fig. 1). In order to estimate the recovery level quantitatively, we recorded videos about mouse locomotion starting from 24 h after surgery and extended to 48 h, 72 h, and 7 d after surgery. After 7 d, we obtain the HTA, FBA, and PLR every 2 weeks until they recovered functionally. Normally this process can be finished in 8 weeks.

The HTA before surgery is 161.67° ± 2.96° average. After the injury, the angles decreased significantly and reached a peak in 1st week of about 120.10° ± 3.70°. After that, the HTA angle increased gradually until it got very close to the baseline (159.32° ± 4.34°) in the 8th week. Actually, HTA angles showed no difference between the 6th group (158.05° ± 4.41°) and the baseline, underlying the full locomotion recovery by then.

The FBA was about 52.87° ± 2.22° before the injury and also increased after surgery, peaking at 84.24° ± 2.90° in 2nd week. From 2–6 weeks post-surgery, FBA decreased more gradually than HTA. So the 6th week after surgery, the angle was 66.52° ± 2.96° which shows no difference with the baseline. In the 8th week, the angle dropped to 60.05° ± 2.75°.

The PLR is the ratio of the relative length of the intact to the lesion limb. Before surgery, the ratio was mostly 1 because both limbs were functioning normally. This ratio increased to 1.55 ± 0.08 and showed a significant difference (P < 0.05) 1 week after the transaction. Then it rose up to over 1.63 ± 0.10 in the 2nd week, which means the intact limb was 1.6 times longer than the injured one. After that, it decreased all way down to 1.22 ± 0.06 in the 4th week and got a full recovery in the 6th week after surgery (1.05 ± 0.07).

According to these behavior results, we can get to know the recovery curve after one side transaction of the femoral nerve. The locomotion dysfunction happens right after the surgery and usually peaks after 1 or 2 weeks. After that, the locomotion will recover gradually. In 4th week after surgery, the HTA recovered about 35%, 24% for FBA, and almost 68% recovery for PRL. All the data shows that in 6th week after the transection and suture, the locomotion can get a full recovery (P < 0.05). We did measure the locomotion in the 8th week, but these data show no difference from those of the 6th week. Actually, we duplicated the protocol of Dr. Schachner’s lab [13] but we got a slightly more advanced recovery timetable than theirs. They observed a thorough recovery of locomotion at 60 d. This difference is mainly because we only made a 2 mm gap between the ends of the transection but they made 3 mm. A longer absence of nerve needs more time to heal.

Femoral nerve injury-induced myeloid differentiation factor 88 and interleukin 1-β upregulation and microglia activation in the spinal cord in the acute phase

It was found that under normal conditions, IL-1β was poorly expressed in the anterior lumber cord (Fig. 2a–c). Microglia showed inactivated status as small somas with a branch of thin extensions. Few of the microglia processes can be seen which was surrounding the retrograded neurons. After 72 h, a lot of IL-1β immunostaining can be found in the spinal cord segment corresponding to the lesioned femoral nerve. And microglia turned into active status, which presented as thicker branches and enlarged cell bodies. A considerable number of overlaps of IL-1β and Iba-1 can be found in the processes of microglia and these overlaps seemed to show a more intimate and closer position to retrograde motoneurons (Fig. 2d–f). We did not check the expression of IL-1β later on because it is a kind of fast response factor that will get to its peak at 72 h after the promotion of immunoreactions and decrease ever since then [14].

Fig. 2:

Representative images of IL-1β/Iba-1 co-localization with fluorogold retrograde labeling in the anterior horn of the lumbar spinal cord. Under normal conditions (a–c), few of the microglia processes can be seen which was surrounding the retrograded neurons (c). Seventy-two hours after surgery (d–f), a lot of IL-1β immunoreactive dendrites and processes can be found at the lesioned femoral nerve (d). And microglia turned into active status, which presented thicker branches and enlarged cell bodies (e). A considerable number of overlaps of IL-1β and Iba-1 can be found on the processes of microglia and these overlaps seemed to show a more intimate and closer position to Fluorogold retrograde motoneurons (f). Fluorescence intensity analysis of IL-1β and Iba-1 staining was conducted based on immunohistochemical labeling (g). ****P < 0.0001, one-way ANOVA with Tukey’s post hoc test between the groups.

We detected the protein level of MyD88 and IL-1β at 24 h, 48 h, and 72 h after the lesion in order to confirm the change we discovered using a western blot. Bands can be observed near 35 KD For MyD88 whereas 31 KD for IL-1β (Fig. 3a). After surgery, the protein level increased starting from 24 h and showed a dramatic difference in both 48 h and 72 h groups. Compare to the level of β-actin, we can tell that these two kinds of protein have a significant trend of increase, which is consistent with our immunohistochemistry results (Fig. 3b and c).

Fig. 3:

Representative picture of the immunoblots of MyD88 and IL-1β in the lumbar spinal cord. After surgery, the level of MyD88 and IL-1β protein increased starting from 24 h and showed a dramatic increase in both 48 h and 72 h groups. ***P < 0.0005, ****P < 0.0001, one-way ANOVA with Tukey’s post hoc test between the groups at the given time points.

For the acute stage, we tested TLR4/Iba-1 co-localization with fluorogold retrograde labeling at 72 h post-surgery. We found that in intact mice (Fig. 4a–d), TLR4 had little expression on neurons or microglia. Seventy-two hours after surgery (Fig. 4e–h), we found much more red fluorescence than the control group. Most of the TLR4 was expressed around the nuclear neurons (arrowheads), showing in lined dots. These red dots can be overlapped with the branches of microglia (arrows) which surrounded the same neuron. The number of microglia which is related to retrograded neurons is not increased significantly. These results imply that, as a superior center of the femoral nerve, the lumbar spinal cord can have an immune reaction to the lesion of peripheral nerve function and express more TLR4 pathway involved molecular.

Fig. 4:

Representative images of TLR4/ Iba-1 co-localization with fluorogold retrograde labeling in the anterior horn of the lumbar spinal cord (a–D, intact mouse; e and f, surgery mouse). TLR4 had little expression on neurons or microglia in the intact spinal cord (a), while much more red fluorescence of TLR4 was located in the spinal cord of the injured group 72 h after surgery (e). Most of the TLR4 was expressed around the nuclear of the neurons (arrowheads) as lined dots. Some other red dots can be overlapped with the processes of microglia (arrows) which surrounded the same neuron. The fluorescence intensity of TLR4 and Iba-1 staining was quantified and statistical analysis was made (I). **P < 0.005, one-way ANOVA with Tukey’s post hoc test between the groups.

Spinal neurons increasingly expressed TLR4 and myeloid differentiation factor 88 till the chronic phase of femoral nerve injury

Similar to TLR4, MyD88 showed an obvious increase at 72 h as well as 6 weeks after surgery. We found a considerable amount of fluorogold retrograde motoneurons in each group of mouse tissues. In non-lesion animals, MyD88 immunostaining showed an even distribution in gray matter. Neurons were showing very ambiguous outlines and no stronger expression can be seen on the neurons compare to the intercellular matters (Fig. 5a–b). Unlikely the normal group, tissues of 72 h and 6 weeks post-surgery groups showed much stronger red fluorescent spots in somas, outlining the shape of nuclear. Most of the NeuN-immunoreactive matters are expressed in somas and can extend to axons. We can also see MyD88 immunoreactivity particles scattered in the NeuN- immunoreactive neurons as well as in the fluorogold retrograded motoneurons (Fig. 5c–f).

Fig. 5:

Representative images of MyD88/NeuN co-localization with fluorogold retrograde labeling in the anterior horn of the lumbar spinal cord. Unlikely the normal group (a and b), tissues of 72 h (c and d) and 6 weeks (e and f) after femoral nerve transection showed much stronger red fluorescent spots in somas, outlining the shape of nuclear. Immunofluorescence intensity analysis of MyD88 staining was shown in (g). *P < 0.05, **P < 0.005, ***P < 0.0005, one-way ANOVA with Tukey’s post hoc test between the groups at the given time points.

For the chronic group, we observed expressions of some key proteins in the anterior horn of the lumbar spinal cord. Under normal circumstances, TLR4 was barely expressed in the neurons or in other cells (Supplementary 1 A, Supplemental digital content 1, https://links.lww.com/WNR/A706). We can only observe some evenly spread backgrounds in the visual field. But 6 weeks after surgery, TLR4 immunoreactive substances were scattered in the cytoplasm of neurons. Some were gathered along the nuclear membrane as dots, which lined out the shape of a nuclear. The immunoreactive particles on the nuclear membrane usually showed stronger fluorescence than those in the cytoplasm. Others were distributed on the plasma of another cell type (arrowhead in Supplementary 1 F, Supplemental digital content 1, https://links.lww.com/WNR/A706). The total level of TLR4 expression increased dramatically even 6 weeks after surgery, by when was proven to have a full recovery by behavior tests.

Discussion

The innate immune reaction is regarded as the first-line defense of the nervous system and plays a crucial role immediately after the injury [15]. TLR4 and downstream signaling was reported to be involved not only in the lesion site of the peripheral nerve but also in the according spinal cord to influence the restoration process [16]. It is easy to understand TLR4 cascade will be triggered in the early stage after injury, particularly on those immune cells including monocyte/macrophages and microglia, while our observation in this study with the transection of the femoral nerve motor branch implies a long-term involvement of TLR4-MyD88 in the lumbar spinal cord during the recovery process, and the motor neurons also participate the innate immune response.

Long-term expression of TLR4-myeloid differentiation factor 88 signal in the spinal cord after peripheral nerve injury

The data from the behavioral tests showed that the transection of the motor branch of the femoral nerve induced motor function impairment of the hind limb immediately after surgery, and the function recovered in 6–8 weeks (Fig. 1) when the stumps were well treated (linked with a tube). Accordingly, the expression of TLR4 and its downstream molecules including MyD88 and proinflammatory cytokines were also found in the spinal cord during this full-length period.

As shown by western blot, MyD88, and IL-1β were upregulated in the lumbar spinal cord in the acute stage (within 72 h) after injury, compared with the intact group. And the immunofluorescent staining with retrograde labeling also indicated strong staining of MyD88 and IL-1β in both microglia and neurons (Figs. 2 and 5). To our surprise, TLR4 and MyD88 immunoreactive products were still observed in the spinal cord at 6th- week post-injury. For the CNS, injury-induced TLR signaling was found more active during the acute phase and persisted along the chronic phase [17]. For peripheral nerves, this is the first report about injury-induced TLR signaling activation in the according spinal cord lasting for such a long time.

These identical terms of motor function recovery and the expression of the TLR4-MyD88 cascade imply the role of innate immune signaling in the spinal cord may be associated with nerve restoration.

Motor neurons and microglia/macrophages in the spinal cord expressed TLR4-myeloid differentiation factor 88 signaling induced by peripheral nerve injury

TLR4 and its cascade were highly expressed in the innate immune cells like microglia and macrophages in the CNS [18] especially when stimulated by pathogen- or damage-associated molecular patterns (DAMPs) [19]. Other cell types in the CNS will also express TLRs and downstream molecules at a relatively low level [20]. Here in this study, we found that injury to the motor branch of the femoral nerve clearly induced microglia activation in the spinal cord, as shown by the obviously changed morphology of Iba-1 and IL-1β positive cells. More importantly, these TLR-expressing neurons were closely associated with activated microglia/macrophages at 72 h post-injury, indicating a crosstalk between injured neurons and microglia. The information of injury at the motor branch of the femoral nerve could be propagated to the soma of motor neurons located in the spinal cord, and the DAMPs could be released from the neurons and recruited microglia in this area to respond.

It is noted that the neuronal expression of TLR4 and MyD88 lasted for 6 weeks after FNI, indicating neuronal TLR4-MyD88 may play roles other than mediating inflammation. Previous reports documented that neurons of dorsal root ganglia MyD88 are involved in the generation of neuropathic pain [21], which may provide a similar clue to the spinal motor neurons that enhanced TLR4-MyD88 expression as their distal axons regrowing.

The connection (the immunoreaction) between the related spinal cord and the periphery nerve

There were few reports about how the periphery nerve injury affects the CNS including the spinal cord. Freria et al. reported that TLR4 may conduct a positive effect on axonal regeneration by upregulating GDNF and downregulating IL-6 while TLR2 may have the opposite function [6]. In the following report, they found that TLR2 or TLR4 does affect the nerve regeneration process through different compensatory mechanisms. These findings contribute to the concept that innate immune-related molecules influence peripheral nerve regeneration by concurrently participating in processes taking place both at the CNS and PNS [8]. Patrícia Ribeiro et al. administered lipopolysaccharide to enhance TLR4 expression in mice and studied retrograde changes in the spinal cord ventral horn following sciatic nerve crush [22]. They found that TLR4 upregulation led to synaptophysin downregulation close to spinal motoneuron cell bodies, indicating increased synaptic elimination.

Conclusion

This study showed in mice that peripheral nerve injury-induced microglia activation in the spinal cord, as well as TLR4-MyD88 signaling upregulation in the spinal neurons, and neuronal TLR4-MyD88 expression, lasted the full length of functional recovery. These findings suggest that peripheral nerve injury triggered the TLR4-MyD88 signal in the soma of spinal neurons may be involved in function and nerve restoration through neuron-glia crosstalk.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81671217) and the Key industrial chain project of Shaanxi Province (2022ZDLSF02-05).

Conflicts of interest

There are no conflicts of interest.

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