Immunohistochemical labeling of ongoing axonal degeneration 10 days following cervical contusion spinal cord injury in the rat

doi:10.21203/rs.3.rs-4829525/v1

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Immunohistochemical labeling of ongoing axonal degeneration 10 days following cervical contusion spinal cord injury in the rat

https://doi.org/10.21203/rs.3.rs-4829525/v1

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Study Design:

Experimental Animal Study

Objective

To continue validating an antibody which targets an epitope of neurofilament light chain (NF-L) only available during neurodegeneration and to utilize the antibody to describe the pattern of axonal degeneration 10 days post-unilateral C4 contusion in the rat.

Setting:

University of Florida

Methods

Sprague Dawley rats received either a unilateral 150kdyn C4 contusion (n = 6; n = 3 females, n = 3 males) or a laminectomy control surgery (n = 5; n = 3 males, n = 2 females). Ten days following SCI or laminectomy, spinal cords and brainstems were processed for immunohistochemistry. Serial spinal cord and brainstem cross-sections were stained with the degeneration-specific NF-L antibody (MCA-6H63) and dual labeled with either an antibody against the C-terminus portion of neurofilament light chain (NF-L-Ct), to label healthy axons, or an antibody against amyloid precursor protein (APP), considered the current “gold standard” for identifying degenerating axons. The pattern of ongoing axonal degeneration was assessed.

Results

Spinal cord and brainstem cross-sections from injured rats had punctate MCA-6H63 positive fibers with pathological appearance, loss of anti-NF-L-Ct co-labeling, and frequent colocalization with APP. Immunopositive fibers were abundant rostral and caudal to the lesion in white matter tracts that would be disrupted by the unilateral C4 contusion. This pattern of staining was not observed in control tissue.

Conclusions

The MCA-6H63 antibody labels degenerating axons following SCI and offers a promising tool to quantify axonal degeneration.

Biological sciences/Neuroscience/Diseases of the nervous system/Neurodegeneration

Health sciences/Neurology/Neurological disorders/Spinal cord diseases

Axonal degeneration begins immediately after spinal cord injury (SCI)(1). The distal portion of the axon, which loses connection to the cell body, undergoes Wallerian degeneration, characterized by the formation of axonal spheroids, or irregular swellings of the axon which create a “beads on a string” appearance, followed by fragmentation, and ultimately degeneration of the entire distal portion of the axon(2). The proximal portion of the axon, which remains connected to the cell body, undergoes axonal dieback or retraction, during which the axon undergoes prolonged retrograde degeneration with the formation of a retraction bulb followed by stabilization of the axon, and typically a failed attempt to regenerate(2). SCI causes axonal shearing, compression, and severing that results in an initial wave of axonal degeneration(3). A subsequent cascade of factors such as ischemia, excitotoxicity, oxidative stress, and inflammation, composing the secondary injury, leads to the collateral damage of additional axons and more extensive degeneration of axons which may have otherwise been spared(4). Mitigating the secondary injury cascade is a target for neuroprotective strategies(5). The axonal degeneration that results from both the primary and secondary injury has profound impacts on patients’ functional ability, quality and longevity of life, and responsiveness to rehabilitation strategies. Axonal degeneration can lead to sensorimotor impairment, chronic pain, spasticity, autonomic dysfunction, bowel and bladder dysfunction, and respiratory failure.

Axonal degeneration is typically assessed histologically using important outcome measures such as axonal pathology, axonal counts, and stains identifying actively degenerating axons. Identification of axons with pathological features such as axonal spheroids or retraction bulbs provides insight into the process of axonal degeneration and can allow for low-throughput assessments of the extent of axonal degeneration(2,6,7). Immunohistochemical stains for epitopes associated with healthy axons can be quantified to assess axonal loss compared to a control group(1,8). Axonal loss as an outcome measure has improved understanding of how different lesion models impact axonal degeneration and the importance of axonal sparing(1,9). In a recent publication, we described novel findings relevant to studies of axonal damage and degeneration. We epitope mapped the two commercial monoclonal NF-L antibodies used in the Uman Diagnostics NF-Light™ ELISA, the Quanterix Simoa™, and similar assays(8). These assays efficiently detect elevations of NF-L in the blood and CSF in the setting of neurodegeneration and have become widely used in a rapidly growing variety of experimental and clinical studies(10,11). We showed that both Uman antibodies bind neighboring epitopes in the center of the α-helical “Coil2” segment of NF-L, amino acids 311–362 of the human sequence(8). Surprisingly both antibodies did not stain axons in control CNS sections apart from a few very rare profiles which we interpreted as axons undergoing spontaneous degeneration. However, abundant Uman antibody staining was observed in white matter tracts of rats given SCI 1–5 days previously. The immunopositive material was typically swollen, irregular in shape, discontinuous, and in some cases sinusoidal, as expected for degenerating or degenerated axonal material(8). As part of this study, we found that several NF-L antibodies binding to the non-helical C-terminal “tail” segment of NF-L, here referred to as NF-L-ct reagents, behaved in a complementary fashion, binding healthy axons but for the most part, not the Uman antibody positive material. Control spinal cord sections exposed to proteases revealed robust axonal immunopositivity with Uman-type antibodies and concomitant loss of binding with NF-L-ct antibodies(8). We concluded that during axonal degeneration, proteases degrade neurofilaments revealing the Uman type epitopes while also destroying NF-L-ct epitopes. We noted little overlap between the two staining profiles suggesting that the transition from NF-L-Uman positive NF-L-ct negative to NF-L-Uman positive NF-L-ct negative is rapid. We made a novel panel of antibodies against amino acids 311–362 of human NF-L which we showed all stained in the degeneration-specific fashion exactly as described for the Uman reagents. One of these, MCA-6H63, was utilized in the present study.

In the current work, we studied rats with a mid-cervical contusion injury to accomplish two major goals. The first was to continue the validation of the MCA-6H63 antibody for its ability to specifically identify degenerating axons after SCI. This was accomplished through comparison to traditional methods of identifying degenerating axons such as assessment of axonal pathology, loss of neurofilament C-terminus immunopositivity, and labeling with an antibody against amyloid precursor protein (APP). The second goal was to utilize the MCA-6H63 antibody to assess the pattern of degenerating axons at a subacute time point (10 days post-injury), relevant for ongoing secondary injury and the antibody’s potential use as an outcome measure in neuroprotective studies.

Experimental Animals

All procedures were approved by the University of Florida’s Animal Care and Use Committee and in compliance with the National Institute of Health guidelines. A total of 11 Sprague Dawley rats (n = 6 male, n = 5 female; Sprague Dawley® SD, Envigo Indianapolis, IN) were used in this experiment. Rodents were housed on a 12:12 light-dark cycle with ad libitum access to food and water.

Laminectomy and Spinal Cord Injury Surgeries

Rats (12 weeks old and 218 ± 32g) were anesthetized with ketamine (80mg/kg) and xylazine (10 mg/kg; IP). Contusion spinal cord injuries were performed as follows (n = 6, 3 males, 3 females) as previously described(12,13). The skin was incised above the cervical spinal cord to expose the muscles over C2-C6. The muscles were reflected and the vertebrae were exposed using the curette. A laminectomy was performed on the right side of the fourth cervical vertebrae (C4). In the contusion group, stabilization clamps were placed on C3 and C6 and the Infinite Horizons impactor (Precision Systems and Instrumentation, Lexington, KY) with a 2.5 mm tip was used to deliver a 150kdyne contusion to the right side of the spinal cord. The stabilization clamps were removed and the overlying muscle layers were sutured with sterile 4 − 0 vicryl. The skin was closed with 9mm wound clips. Post-operative medications including three days of buprenorphine (0.03mg/kg every 12 hours) and 48 hours of carprofen (5mg/kg, q.d), baytril (5mg/kg,q.d.), and lactated ringer’s solution (10mL/day, q.d) were administered. Rats were monitored daily for signs of distress, dehydration, and weight loss with appropriate veterinary care given as needed. A group of rats (n = 5, 3 males, 2 females) received only a laminectomy, with removal of the overlying vertebrae with no injury to the spinal cord.

Immunohistochemistry

Ten days post-contusion SCI or laminectomy surgery rats were injected with 1mL of intraperitoneal Beuthanesia-Special-D. Rats were transcardially perfused with saline followed by 4% paraformaldehyde. The spinal cords were harvested and then post-fixed in 4% paraformaldehyde overnight. The tissue was then cryoprotected with 30% sucrose until the tissue sank, embedded into Optimal Cutting Temperature media, frozen, and stored at -80º. Spinal cords were cryosectioned in 20µm thick sections into a seven-section series to generate slides with sections 120µm apart. One set per animal was used for cresyl violet staining as per previously described methods(13). Another set was used for immunofluorescent staining with the degeneration-specific mouse monoclonal antibody to NF-L Degenotag™ peptide (EnCor Biotechnology MCA-6H63; 1:5000) and rabbit polyclonal antibody to NF-L-ct (EnCor Biotechnology RPCA-NF-L-ct; 1:5000). MCA-6H63 binds to an epitope centered on amino acids 311–315 of the human NF-L sequence, while RPCA-NF-L-ct was raised against amino acids 515–543 of the rat NF-L sequence. A slide with sections spanning caudal to the injury (approximately C8) through the injury and including the caudal medulla was dual stained with MCA-6H63 and rabbit polyclonal antibody to amyloid precursor protein (clone Y188; Abcam, ab32136; 1:200). Secondary antibodies were goat anti-Mouse AF488 (Invitrogen; 1:1000) and goat anti-rabbit AF 594 (Invitrogen; 1:1000). Injured spinal tissue was incubated with isotype and concentration matched control Ig instead of exposure to primary antibody to provide another negative control. Importantly, the MCA-6H63 antibody staining is not compatible with antigen retrieval methods that can cause degradation of neurofilament polypeptide and produce positive labeling not due to the injury or disease state.

Imaging

Microscopy was conducted using the Keyence microscope (BZ-X700, Keyence Corporation of America, Itasca, IL). Before imaging, all sections in each animal were screened and assessed for the presence and pattern of staining. Representative images were selected for their resemblance with the composite pattern of staining observed across animals in each group (injured vs laminectomy control). Whole section images were created by stitching 10X images using Adobe Photoshop Photomerge. The intensity of fluorescent images was increased using levels to enhance visibility. Cresyl violet sections were white balanced. All adjustments made to images were applied to the entire image and standardized across images in both experimental and control groups. To create a 3D reconstruction of the lesion, serial 2X images of cresyl violet stained sections were captured using the Keyance microscope. Adobe Illustrator was then used to trace the section, the white/grey matter junction, and the lesion. The traced images were converted into 3D images and stacked. Sections with sufficient damage, such as rips or folds, which prevented accurate tracing, were eliminated (13 out of 78 total sections were eliminated). These sections were skipped during the creation of the 3D reconstruction. No more than 2 consecutive sections were eliminated. The example 3D reconstruction was chosen based on the best cresyl violet staining with the minimal number of excluded sections to depict the most accurate reconstruction.

Lesion Histology After Unilateral C4 Contusion

Cresyl violet staining allowed for the assessment of lesion histopathology. Within the lesion (solid black line Fig. 1A) there was destruction of normal tissue architecture, loss of neurons, and the presence of blood, cellular debris, and vacuoles. The lesion extended contralesionally in 5 of 6 rats, primarily in the center of the spinal cord and the dorsal white matter (Fig. 1A). The lesion extended rostrally and caudally in the ipsilesional white matter approximately one to two spinal segments. A three-dimensional reconstruction of the lesion depicts the area of injury (Fig. 1B and C).

MCA-6H63 Antibody Stains Degenerating Axons

Spinal cord sections stained with MCA-6H63 had abundant small and irregularly shaped punctate immunopositive profiles in the white matter rostral and caudal to the lesion. This pattern of staining was consistent in all rats in the contusion group (n = 6/6; Fig. 1D). and was not observed in the laminectomy-only group (n = 5/5; Fig. 1E). Spinally injured sections incubated with an isotype and concentration matched control Ig did not have positive staining rostral or caudal to SCI (Fig. 1F). MCA-6H63 positive fibers were also observed in the ventral roots (Fig. 1G). MCA-6H63 positive axons in the ventral roots had a beaded, pathological appearance demonstrated by the irregular width, shape, and contour of the axon (Fig. 1G and I). Axons stained with NF-L-ct had more regular width, shape, and contour indicative of healthy axons (Fig. 3F and G). As noted in our previous study(8), axons labeled with the MCA-6H63 antibody were generally not positive for NF-L-ct (Fig. EG, F, I). Cross sections of injured spinal cords dual stained with APP and MCA-6H63 (rostral dorsal white matter, Fig. 1J-L and caudal ventral white matter, Fig. 1N-P) revealed frequent colocalization (examples: white arrows). However, there were fibers interspersed which were only positive for MCA-6H63 or APP.

Pattern of Axonal Degeneration 10 Days Following C4 Contusion

Rostral to the lesion (Fig. 2A-H), staining was most abundant in the ventrolateral (n = 6/6) and dorsal (n = 6/6) white matter ipsilateral to the lesion. Dorsal white matter staining appeared most robust in the dorsal fasciculus with some staining located in the region of the dorsal corticospinal tract. Dorsal white matter staining was bilateral in most rats (n = 5/6). The rat without bilateral contralesional dorsal column staining did not have significant extension of the lesion across midline. Immunopositive fibers were also present in the contralesional ventrolateral white matter (n = 6/6) but were more sparse than the ipsilesional staining. Caudal to the lesion (Fig. 2I-P), ipsilesional immunopositive fibers were robust in the ventral and lateral white matter (n = 6/6). Contralesional staining was observed in the ventral, ventrolateral, and lateral white matter (n = 6/6) but was most abundant in the ventral white matter (n = 5/6). One rat that did not exhibit robust staining in the contralesional ventral white matter did not have contralesional extension of the lesion across midline on cresyl violet lesion analysis. Immunopositive fibers were observed in the dorsal column caudal to the lesion (n = 6/6). However, dorsal column staining was very sparse and mostly in sections proximal to the lesion (n = 6/6).

MCA-6H63 positive fibers were also observed in the dorsal (Fig. 3A, B, E) and ventral (Fig. 3A, C, D, E) medulla. Staining in the medulla was sparser than in the cervical spinal cord. The most abundant staining in the medulla was in the ipsilesional ventral medulla (n = 6/6). Sparse contralesional staining was also observed in the ventral medulla (n = 6/6; Fig. 3C). Sparse immunopositive fibers in the ipsilesional dorsal medulla (Fig. 3B) were observed in all six rats although were rarely observed in one of those rats. Contralesional staining in the dorsal medulla was rare but was observed at least intermittently, in all 6 rats.

The MCA-6H63 antibody, an anti-NF-L-Coil2 antibody, specifically labeled degenerating axons in white matter tracts disrupted by a unilateral C4 contusion spinal cord injury and revealed abundant continued degeneration rostral and caudal to the lesion epicenter 10 days post-injury. The unilateral C4 contusion (150 kydne) used here produces a moderate injury with substantial spinal pathology including loss of normal tissue architecture, vacuolization, cyst formation, and cell loss(1,4). The lesion extends rostrally, caudally, and contralaterally from the epicenter. Axons disrupted by the lesion, both due to primary and secondary injury, degenerate and lead to functional deficits. Studies in rats after SCI assessing differences in axonal loss during subacute and chronic time points suggest continued axonal loss into the chronic period after injury(1). However, traditional methods of labeling actively degenerating axons such as the Marchi method(14), the modified silver stain(15), fluorojade(16), and antibodies against APP(17,18) have limitations which have impaired reliable and reproducible study of actively degenerating axons. As a result, there are conflicting data regarding the timing of axonal degeneration after SCI and challenges using ongoing axonal degeneration as an outcome measure in SCI studies.

In the current study, robust MCA-6H63 staining was present rostral and caudal to the lesion in axonal tracts which would be expected to be disrupted by the unilateral C4 contusion. Immunopositive fibers had a swollen and irregularly shaped punctate appearance consistent with pathological axons in cross-section. MCA-6H63 positive axons in ventral roots demonstrated the beaded appearance indicative of an axon undergoing Wallerian degeneration(2) whereas the MCA-6H63 negative, NF-L-ct positive axons had a healthier appearance with a more regular shape and contour. As found in our previous study(8), axons labeled with MCA-6H63 typically did not co-label with the NF-L-ct. The presence of axonal pathology and loss of NF-L-ct staining confirms that the MCA-6H63 positive axons in the spinal cord and ventral roots were degenerating. The absence of MCA-6H63 staining in all rats in the laminectomy group supports the specificity of the MCA-6H63 antibody for degenerating axons by indicating that the MCA-6H63 antibody is not staining normal, healthy axons. Additionally, immunopositivity was not present in injured spinal cord cross sections rostral or caudal to the lesion in sections incubated with concentration matched, isotype control Ig instead of primary antibody. This supports that MCA-6H63 staining was not non-specific Ig binding, autofluorescence, or artifact.

To further support that the MCA-6H63 antibody labels specifically degenerating axons, sections were dual labeled with the MCA-6H63 antibody and an antibody against APP, considered the gold standard for labeling degenerating axons after traumatic brain injury(19). APP is produced in the cell body and transported along the axon in vesicles through rapid anterograde transport. After axonal injury, disruptions in axonal transport result in APP accumulation in axonal spheroids and retraction bulbs(17,18). Colocalization of APP and MCA-6H63 staining was present in spinal cord cross-sections 10 days following injury. However, there were frequent axonal profiles that were only positive for APP or MCA-6H63. This finding is consistent with previous work determining that primary and secondary injury result in a spectrum of axonal pathology and that impaired axonal transport which leads to accumulation of APP is an independent process to the development of neurofilament pathology(20,21). In some cases, impaired axonal transport and APP accumulation may occur in the absence or precedence of axonal degeneration. APP accumulation has been suggested as a marker for where secondary axotomy will occur(22). Conversely, severe or rapid axonal degeneration may occur in the absence of APP accumulation(21,23). APP also accumulates in the retraction bulb and axonal spheroids(18), whereas neurofilament pathology is not confined to these regions of the axon(21). Additionally, the ability of APP to label the portion of the axon undergoing Wallerian degeneration is unclear. In the current study, APP-positive axons were found in the same regions of white matter as MCA-6H63 positive axons, and, as in Li et al(24), were found in both tracts expected to undergo Wallerian degeneration and axonal dieback. However, other studies have not observed evidence of APP accumulations in axons with pathology indicative of Wallerian degeneration(22,25,26). The MCA-6H63 antibody may be a more sensitive and specific marker for axonal degeneration while APP may be a better indicator of total axonal pathology. However, additional research is necessary to determine the full interpretability of these stains.

Ten days following a unilateral C4 contusion, abundant MCA-6H63-positive fibers were present in putative ascending and descending tracts both rostral and caudal to the lesion. Continued degeneration observed even 10 days post-injury highlights the continued importance of neuroprotective therapeutics even in the subacute period after injury. MCA-6H63-positive fibers were generally most abundant in axonal tracts where axons were likely undergoing Wallerian degeneration. For example, caudal to the lesion, MCA-6H63 positive fibers were most abundant in the lateral and ventral white matter, which are the putative locations of descending tracts such as the rubrospinal, reticulospinal, and vestibulospinal tracts(27,28). Rostral to the lesion, MCA-6H63 positive fibers were abundant in the ventrolateral white matter, the putative locations of the spinothalamic tract and ventral spinocerebellar tract, and the dorsal fasciculi. These tracts are ascending tracts that carry sensory information to the brain or cerebellum(27,28). MCA-6H63 positive fibers in putative ascending sensory and spinocerebellar tracts were also visualized in the medulla(29). Wallerian degeneration causes the eventual loss of the entire distal portion of the axon and is thus a farther-reaching process than axonal dieback(2). Interestingly, staining in the dorsal corticospinal tract was largely absent caudal to the lesion where Wallerian degeneration would be expected to occur. Robust anti-NFL-Coil2 staining was present caudal to the lesion in the dorsal corticospinal tract was present 3 days following a midline C4 contusion. Because dorsal corticospinal tract staining was present rostral to the lesion and lesion analysis with cresyl violet confirmed a significant impact of the lesion to the dorsal corticospinal tracts, it is unlikely that the lack of staining at 10 days represents a difference in lesion model. It is possible that Wallerian degeneration in the dorsal corticospinal tracts occurs at an earlier time point than 10 days post-injury, a finding which would have implications on the necessary timing of neuroprotective interventions aimed at improving motor function.

Staining was also visualized in white matter tracts likely undergoing axonal dieback. Rostral to lesion there were MCA-6H63 immunopostive axons in the dorsal corticospinal tract. Caudal to the lesion, MCA-6H63 staining was present in the ventrolateral white matter in the putative spinothalamic tract and spinocerebellar tracts. However, staining in the dorsal fasciculi caudal to the lesion was rare and when observed was very sparse and in segments closer to the lesion. There is evidence that by one-week post-injury, proximal portions of ascending sensory axons in the dorsal white matter stabilize and discontinue axonal dieback whereas dieback in the corticospinal tracts may continue for a longer duration of time and more extensive distance(30). Although axonal regeneration typically fails endogenously in the central nervous system, one area of SCI research focuses on improving axonal regeneration. Understanding the timing and extent of axonal dieback in different white matter tracts and the discovery of neuroprotective mechanisms to ameliorate dieback could assist in improving regenerative ability by decreasing the distance in which axons would need to regrow.

The current study demonstrated that in the rat contusion SCI model, the MCA-6H63 antibody can be utilized as a specific immunohistochemical marker for degenerating axons undergoing either axonal dieback or Wallerian degeneration, important components of the pathophysiology following SCI. Importantly, the MCA-6H63 antibody appears to not suffer from many of the key limitations of traditional methods to identify degenerating axons. The results of the current study support that the MCA-6H63 antibody is specific and is not accompanied by off-target labeling or artifact. Further work assessing the impact of SCI on axonal degeneration will be important to understanding the pathophysiology of axonal degeneration after cervical SCI and the optimal timing of neuroprotective interventions for improved sensory, motor, and respiratory outcomes. The present study suggests that the MCA-6H63 antibody could be a useful tool in these future studies and as an outcome measure for ongoing axonal degeneration in SCI research.

DATA AVAILABILITY SATEMENT

The data supporting the described findings can be made available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTIONS

AFF: design of the experiment, data acquisition, analysis, and interpretation; initial draft of the manuscript. SR: design of the study, data interpretation, and manuscript editing. VEB: data acquisition and review of the manuscript draft. MDS: data acquisition and review of the manuscript draft. MJ: data acquisition and review of the manuscript draft. GS: Data acquisition and interpretation; manuscript editing. DDF: conception and design of the study, interpretation of the data, revisions, and final approval of the manuscript. All authors read and approved of the final manuscript.

FUNDING

Support for this work was provided by the National Institutes of Health: R01 HL139708-01A1 (DDF), R01 HL153140-01 (DDF). SR was supported by 1K99NS133388-01A (SR). AFF was supported by 5T32HD043730-19 and the Dr. Frank M Davis MD Chairman Emeritus Grant.

ETHICAL APPROVAL

All procedures described in this manuscript involving rats and tissue were approved by the University of Florida Institutional Animal Care and Use Committee and in strict accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals.

COMPETING INTERESTS

Gerry Shaw is owner, founder and CEO of EnCor Biotechnology Inc. which supplied certain commercial reagents used in this report. He may therefore benefit from sales or equity growth.

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Immunohistochemical labeling of ongoing axonal degeneration 10 days following cervical contusion spinal cord injury in the rat

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