Recovery of Forearm and Fine Digit Function After Chronic Spinal Cord Injury by Simultaneous Blockade of Inhibitory Matrix Chondroitin Sulfate Proteoglycan Production and the Receptor PTPσ
Publication: Journal of Neurotrauma
Volume 40, Issue Number 23-24
Abstract
Spinal cord injuries (SCI), for which there are limited effective treatments, result in enduring paralysis and hypoesthesia, in part because of the inhibitory microenvironment that develops and limits regeneration/sprouting, especially during chronic stages. Recently, we discovered that targeted enzymatic removal of the inhibitory chondroitin sulfate proteoglycan (CSPG) component of the extracellular and perineuronal net (PNN) matrix via Chondroitinase ABC (ChABC) rapidly restored robust respiratory function to the previously paralyzed hemi-diaphragm after remarkably long times post-injury (up to 1.5 years) following a cervical level 2 lateral hemi-transection. Importantly, ChABC treatment at cervical level 4 in this chronic model also elicited improvements in gross upper arm function. In the present study, we focused on arm and hand function, seeking to highlight and optimize crude as well as fine motor control of the forearm and digits at lengthy chronic stages post-injury. However, instead of using ChABC, we utilized a novel and more clinically relevant systemic combinatorial treatment strategy designed to simultaneously reduce and overcome inhibitory CSPGs. Following a 3-month upper cervical spinal hemi-lesion using adult female Sprague Dawley rats, we show that the combined treatment had a profound effect on functional recovery of the chronically paralyzed forelimb and paw, as well as on precision movements of the digits. The regenerative and immune system related events that we describe deepen our basic understanding of the crucial role of CSPG-mediated inhibition via the PTPσ receptor in constraining functional synaptic plasticity at lengthy time points following SCI, hopefully leading to clinically relevant translational benefits.
Introduction
Each year, between 250,000 and 500,000 people worldwide suffer a debilitating spinal cord injury (SCI), with over half occurring at the cervical level, leading to millions chronically paralyzed as reported by the World Health Organization.1 Damage to the high cervical spinal cord can result in paralysis of much of the body, with devastating impacts on controlled, voluntary arm, hand, and digit movements, leading to a severe decrease in individual autonomy and overall quality of life.2,3 The last several decades of research have demonstrated the crucial role of the cell and axon growth inhibitory properties of chondroitin sulfate proteoglycans (CSPGs) during normal neural development as well as their inhibition of re-growth of cells or their processes after injury.4–10 In particular, we have focused on studying the role of the CSPG family of extracellular matrix molecules in the glial scar and the perineuronal net (PNN) as critical inhibitors of functional regeneration and sprouting after SCI.8,10–14
Within a few days after injury, CSPGs increase within and adjacent to the forming glial scar.15–19 Matrix upregulation also occurs immediately following SCI as part of a tightly regulated and crucial immune response, and the upregulation persists in the extracellular space and PNN in areas rostral and caudal to the injury site in denervated distal targets.8,10–13,20 Thus, inhibitory CSPGs upregulate expansively within and proximal and distal to the lesion epicenter, leading to limited regeneration through the lesion or back into target nuclei and curtailing potential sprouting/plasticity over large distances, which could occur from spared fiber systems either ipsilateral or contralateral to the lesion.8,10–13,15,16,21,22 The regulatory effects of CSPGs on both myelin and axon regeneration as well as neuronal plasticity after an acute9,17,23–29 or sub-chronic SCI,5,17,23,26,30 and in the context of many other conditions, have been well documented.8,10,31–37
The glycosaminoglycan (GAG) side chains, especially those containing 4-O-sulfated CS-E,38,39 are known to bind with highest affinity to their receptor protein tyrosine phosphatase PTPσ (rPTPσ)38 and provide much of the inhibitory properties of CSPGs.39–41 Their effects can be greatly decreased by enzymatic digestion using the bacterial enzyme Chondroitinase ABC (ChABC).13,21,28,38,42–49 ChABC is routinely used for degrading the chondroitin sulfated GAG chains away from their resident core proteins and has been shown in various models of SCI to have pre-clinical benefits with improvements in both locomotor ability and increased axonal regeneration/sprouting toward and into previously denervated spinal sensory or motor centers.11,13,20,47,50,51 There has only been limited success when ChABC was used alone or when coupled with other strategies, such as rehabilitative training, in sub-chronic SCI models. Somewhat improved forepaw function has been reported when ChABC was combined with pellet reaching training when the treatment was given at 4 weeks after dorsal hemi-section injury.51 In another study, ChABC combined with lengthy treadmill training beginning 6 weeks after severe contusion cord lesion modestly improved locomotor behavior.50 When ChABC was locally delivered over an extended period and combined with neural precursor cells or trophic factors, limited improvements in locomotor function have been reported in clip compression lesioned SCI animals with a 6-week delay prior to treatment.30 Unlike in acute SCI, in the chronic state, dense glial scarring has formed around the lesion epicenter, and increases in CSPGs in the PNNs have maximized within deafferented spinal levels.7,11,15,52 Further, the inflammatory response has largely died down and the opportunity for neuroprotection has long past.10
We had been focusing on the return of diaphragm function at much longer chronic stages after lateral cervical level two hemi-section (LC2H). Recently, we documented some rather remarkable results showing complete and persistent return of diaphragm function at greatly protracted chronic stages (up to 1.5 years post-lesion) upon local matrix and PNN degradation with ChABC placed directly in the vicinity of the denervated phrenic motor pool.53 The robust recovery of breathing, which occurs after a near lifetime of paralysis, was surprisingly superior to anything achieved at acute or sub-chronic stages after injury using similar techniques.54 The ability to stimulate recovery of such strong, hemi-diaphragm function after enzyme-mediated matrix degradation at C4 began to manifest as soon as 1 month post-hemi-lesion at C2 and, importantly, this repair potential continued to grow over time.53 It seems likely that, in partial injury models, improvements via enzyme administration in past studies may have been limited, at least in part because not enough time had been allowed to pass prior to treatment to enable the slow process of potentially meaningful spontaneous plasticity to reach sufficient levels. Although able to breathe with the intact hemi-diaphragm, and to ambulate, feed, and drink, high cervical hemisected animals also exhibit long-lasting deficits in forelimb behavior, including reduced gross and fine movements of the ipsi-lesional forelimb and the paw. In this study, in which animals were subject to injury at C2 and treated after a lengthy time interval at C4, we noted positive changes in gross upper arm function, likely mediated by enzyme diffusion caudally toward the cervical enlargement.53,55 Although we had not optimized this treatment for forelimb recovery, we were encouraged by the incidental finding.53
The use of ChABC has not been applied clinically because of a variety of potential complications such as heat lability of a bacterial enzyme which, in addition, must be administered intraparenchymally into the injured spinal cord, which can further traumatize an already damaged central nervous system (CNS). Further, the injection field is limited to a discrete area, so positive behavioral changes are dependent on the precision of locally restricted yet functionally relevant alterations in the matrix. Several laboratories have engineered stable, longer-lived ChABC or more widespread viral vector release strategies, although the direct delivery issue remains with these novel approaches.56–60 In order to target CSPG mediated inhibition more globally, we focused our attention on the possibility of using systemic agents that could potentially diminish the abundance of CSPGs or disrupt the association with their major receptor without directly touching the spinal cord in the presence of any evolving lesion. To accomplish this, we utilized a high dose of a subcutaneously delivered rPTPσ inhibitor. This Intracellular Sigma Peptide (ISP) is a mimetic of the PTPσ wedge domain that contains a TAT (TransActivator of Transcription) domain that facilitates membrane-penetration. We have previously demonstrated that ISP works acutely in vitro and in vivo to prevent the conversion of growth cones into a dystrophic state through the excessively tight substrate adhesion mediated via the rPTPσ receptor and the CSPGs.21,61–63 In addition, we investigated the therapeutic effectiveness of a clinically approved, orally administered small molecule perineuronal net inhibitor, 4-methylumbelliferone (we call it “perineuronal net inhibitor,” [PNNi], for use in the CNS), which serves to limit a major transmembrane scaffold for PNN assembly, resulting in the PNN being unable to be stably formed or maintained.64 Therefore, in addition to using the receptor disrupting wedge peptide (ISP), we used a novel strategy in combination, which can simultaneously and expansively reduce the matrix and PNN CSPGs.64
Our chronic LC2H SCI model allows us to test the role of PNN-associated CSPGs in curtailing potential functional sprouting from the intact side of the cord at the level of the cervical enlargement or likely elsewhere within the CNS related to forearm and paw function. By strongly interrupting the rPTPσ–CSPG interaction, we were able to significantly alter the density of the CSPG component of the PNN and restore functional movements of the impaired forelimb during overground locomotor as well as cereal manipulations by the fingers during eating. The cumulative effect of ISP and PNNi further supports the crucial role of CSPG-mediated inhibition via the rPTPσ receptor in curtailing functional synaptic plasticity at lengthy chronic time points following SCI.
Methods
Ethical declaration and animal husbandry
All experiments were approved by the Institutional Care and Use Committee at Case Western Reserve University (CWRU), Cleveland. Animals were housed in groups of two or three, and exposed to a normal dark–light cycle with free access to food, water, and environmental enrichment ad libitum. The health and welfare of the animals was monitored daily by the study investigators and veterinary staff at Case Western Reserve University.
Behavioral training and assessments
Animals were acclimated to the laboratory room testing environment and cereals in the home cage for five consecutive days. The animals were also handled by the researchers twice a day to minimize any stress/anxiety prior to exposure to behavioral testing. The following week, the animals were acclimated to the various testing apparatuses, which included a 10-min session each day in the glass cylinder used in the cereal eating assessment. The animals were trained to eat at least three of each cereal type (sphere-shaped: i.e., Cocoa Puffs or donut-shaped: i.e., Fruit Loops) within the 10-min session. For acclimation to the forelimb locomotor testing platform, rats were placed on top of an open field platform ∼10 cm in diameter and allowed to explore for 5 min. Forelimb locomotor and cereal eating pre-training was conducted for five consecutive days. Baseline behavior performance was acquired following training and before LC2H (Fig. 1A).
Forelimb function assessments
Forelimb function was determined through assessments that involved monitoring behavior that the animals performed naturally. This included the Forelimb Locomotor Scale (FLS)65 and the Irvine, Beattie, and Breshnahan (IBB) forelimb recovery scale.64 Baseline values were taken prior to injury and at 3 months after injury just prior to daily subcutaneous injection of high dose ISP or saline alone or combined with Nutella ± PNNi oral gavage administration (for 60 days) and then weekly following treatment application (Fig. 1A). Statistical comparisons were made between treatment groups and within behavioral measurements using two-way, repeated-measures analysis of variance (ANOVA) with recommended post-hoc correction (Bonferroni) (GraphPad Prism).
Forelimb Locomotor Scale (FLS)
We used established methods detailed by Singh and colleagues to assess forelimb locomotion.65 Briefly, rats are encouraged to continuously walk on top of an elevated circular platform ∼10 cm in diameter. Rats that remained stationary for longer than 10–15 sec were enticed to move by having them follow a pencil or a piece of paper, or by lightly tapping or scratching on the side of the open field. If the animal failed to respond to these stimuli, it was picked up by the forequarters and placed in the center of the open field or opposite its previous position, which usually caused it to move. Rats were recorded during locomotion using a high-speed (60 frames/sec [FPS]) camera for offline scoring to assess forelimb walking functionality. Videos were scored in a blinded fashion in slow-motion (at least 50%) playback using the Lightworks video editing software. Recorded videos containing 3–4 min of the animal freely walking on the elevated platform were measured using an 18-point scale (0–17). Animal locomotor behaviors were assessed for impairments and assigned a score reflecting the ability of the animal to perform steps that were consistently plantar, parallel, and weight-bearing.65
IBB Forelimb Recovery Scale
These testing methods were established previously.64 Briefly, animals were placed in the 25 cm H × 20 cm W clear glass cylinder that was used for previous acclimation and training. Each rat was given one sphere-shaped (Cocoa Puff) and one donut-shaped (Fruit Loop) cereal to eat. Their manipulations of the cereals were recorded using a high-speed (60 FPS) camera for offline scoring to measure forelimb and digit abilities during eating. Videos were scored blinded in slow-motion (at least 50%) playback using the Lightworks video editing software. Recorded videos containing the animal eating one sphere-shaped cereal (i.e., Cocoa Puff) and one donut-shaped cereal (Fruit Loop) were measured using a 10-point scale (0–9).64 The animal's ability to dexterously manipulate the cereals was assessed for impairments and assigned a score reflecting the ability of the animal to grasp the cereal with subtle adjustments made using the second, third, and fourth digits and wrist movements.64
Stimulation of forelimb utilization
All animals were placed in a pool (4’ diameter) filled to a depth of ∼46 cm at a temperature of 35–38°C to match the rat's natural internal temperature. Animals were placed in the pool for 1 min. Two to four rats were placed in the pool at one time. After swimming, rats were partially dried with a cotton towel and then placed in empty cages lined with paper towels and allowed to further dry and groom themselves uninterrupted for 1 h at room temperature. The rats were then replaced back into their home cages. Swimming took place 5 days a week beginning 2 weeks after treatment application began, excluding the day prior to and the day of behavioral acquisition using the FLS and IBB assessments described (Fig. 1A).
LC2H injury and systemic treatments
SCI surgeries were performed as previously described.53 Adult female Sprague Dawley rats (280 ± 20 g; Harlan Laboratories Inc., Indianapolis, IN, USA) were anaesthetized with an intraperitoneal injection of ketamine + xylazine cocktail (70 mg kg−1/7 mg kg−1). The dorsal neck-shoulder area was shaved, cleaned, and sanitized using Betadine and 70% ethyl alcohol, and analgesics were administered through subcutaneous injection of meloxicam (1 mg kg−1). Body temperature was maintained and monitored throughout the surgery at 37 ± 1°C. A dorsal midline incision ∼3 cm in length was made over the cervical region. After the skin and paravertebral muscles were retracted, a laminectomy was performed over C2 and the rostral spinal cord was exposed (Fig. 1A). A 21G syringe needle was positioned at the midline of the spinal cord, and a left lateral durotomy and hemi-section were performed caudal to the C2 dorsal roots making certain that the needle tip extended to and was dragged along the ventral bony lamina surface. This process was repeated five times and ranged from the midline to the most lateral extent of the spinal cord. The muscle layers were sutured together with 3-0 Vicryl and the skin was closed using wound clips. The animals were given meloxicam (1 mg kg−1) and sterile saline subcutaneously for up to 5 days post-surgery along with nutritional support if their weight dropped >5% of what it had been pre-injury. The anatomical completeness of the injury was confirmed through microscopy and behavioral assessment. Post-injury animals exhibited no signs of malnutrition or infection, but those that exhibited persistent autophagy as a result of the injury before or during the systemic treatment phase were excluded from the study. Before initiating any systemic treatments, all animals were confirmed to have achieved a performance score ≤4.5 ± 1 out of 17 at 3 days post-injury (DPI) as described in the locomotor assessment, and a score of 8 ± 1 12 weeks post-injury (WPI) (Figs. 1C and 2; see Forelimb Locomotor Scale section).65 For cereal eating assessments, all animals were confirmed to have received a performance ≤0 ± 1 out of 9 at 3 DPI, and 2 ± 1 at 12 WPI (Figs. 1B and 4; see IBB Forelimb Recovery Scale section).64
Three months after LC2H, the rats were randomly assigned to begin a systemic treatment protocol that included a daily 0.5 mL subcutaneous injection of 500 μg of intracellular sigma peptide (ISP) under the skin of the back near the lesion, combined with oral gavage feeding of Nutella mixed with sunflower oil with or without the small molecule (0.2 g/mL) perineuronal net inhibitor, PNNi. Experimental animals were fed PNNi mixed with hazelnut spread thinned with sunflower oil twice daily at a dose at 2 g/kg based on weight. Control animals were injected with saline and fed hazelnut spread thinned with sunflower oil not containing PNNi. Two separate cohorts received ISP or PNNi alone. These systemic treatments were administered with the goal of simultaneously disrupting the interaction between would-be sprouting axons and the rPTPσ receptor as well as to reduce the density of inhibitory extracellular perineuronal net components. Each cohort (n = 8–10) received the treatments daily for 60 days and were tested and assessed blindly each week for arm and hand function for the duration of treatment, and continuing 4 weeks following the termination of treatments. Our assessments (see details subsequently) of forelimb/paw function were conducted using the FLS65 and the IBB forelimb recovery scale.64 Both are rating scales similar to the well-known Basso, Beattie and Bresnahan (BBB) scoring method for overground walking, but are focused on the forelimb and digits.66
Low-magnification histology of serotonergic fibers, PNNs, and confirming completeness of the cervical injury
At 24 WPI, animals received an overdose of anesthesia prior to cardiac perfusion with 4% paraformaldehyde dissolved in phosphate buffered saline (PBS). Spinal tissue containing regions of interest were dissected and post-fixed in 4% paraformaldehyde overnight. Prior to sectioning, the tissue was cryoprotected with sequential treatments first in a solution of 30% sucrose in PBS and then in a 1:1 mixture of 30% sucrose in PBS + OCT for 2 days; 30 μm coronal sections of tissue were prepared rostral to caudal from cryoprotected cervical spinal cords (C5–C8) using a Leica Cryostat. Immunofluorescence staining was performed using standard protocols. (Refer to Table 1 for a detailed list of primary antibodies.) In each case, primary antibodies were conjugated to an appropriate Alexa Fluor-containing secondary antibody for microscopic visualization. Nuclei were visualized using Hoechst a 33342 staining. Following the antibody staining, tissue sections were mounted to slides, protected with ProLong Diamond Mounting Medium, cover-slipped, and examined either with an inverted Leica SP8 confocal microscope or a Zeiss Axio Imager microscope.
Antigen | Host species | Dilution | Vendor | Catalog number |
---|---|---|---|---|
GFAP | Chicken | 1/1000 | EnCor Biotechnology | CPCA-GFAP |
Biotinylated-WFA lectin | - | 1/500 | Vector Laboratories | B-1355 |
Cat 301 (ACAN) | Mouse | 1/1000 | Millipore | MAB5284 |
SERT | Rabbit | 1/1000 | EnCor Biotechnology | RPCA-SERT |
Iba1 | Rabbit | 1/1000 | EnCor Biotechnology | RPCA-IBA1 |
Neuro Filament (NF) | Rabbit | 1/1000 | EnCor Biotechnology | RPCA-NF-H |
5 HT | Rabbit | 1/1000 | Immuno Star | 20080 |
Strepavidin; Alexa Fluor 555 | - | 1/500 | Invitrogen (Life Technologies) | S32355 |
Mouse; Alexa Fluor 488 | Donkey | 1/500 | Invitrogen (Life Technologies) | A21202 |
Chicken; Alexa Fluor 488 | Donkey | 1/500 | Jackson Immuno Research | 703-545-155 |
Rabbit; Alexa Fluor 488 | Donkey | 1/500 | Invitrogen (Life Technologies) | A21206 |
Mouse; Alexa Fluor 555 | Donkey | 1/500 | Invitrogen (Life Technologies) | A31570 |
Chicken; Alexa Fluor 555 | Goat | 1/500 | Invitrogen (Life Technologies) | A21437 |
Hoechst 33342, trihydrochloride trihydrate | - | 1/3000 | Invitrogen (Life Technologies) | A21437 |
Quantification of Wisteria floribunda agglutinin (WFA)+ aggregates
Tissues from three separate animals per treatment condition were evaluated. For a given animal, 12–15 representative images were captured (∼ 5 images per section per anatomical segment). The number of WFA bright inclusions were totaled using the Cell Counter Plugin of the FIJI Image Analysis Software. In Figure 8P–R, each dot represents the number of inclusions counted in a single image.
Lesion area and volume
Sixteen spinal cords were selected for analysis from the total available using a random number generator. Sections were obtained using a cryostat at 30 μm/section spanning a ∼15-mm-long cervical segment containing the LC2H. Spinal cord lesion area and volume were analyzed through fluorescent Nissl staining (ThermoFischer) performed on cross-sectioned tissue of each spinal cord at 500-μm intervals. The coverage of the lesion as well as the location was determined using a Zeiss Axio Imager microscope and quantified blindly using National Institutes of Health (NIH) ImageJ software analysis of three to four sections per animal that included the lesion epicenter by using monochromatic image and background readings subtracted at the defined area of interest. In all subjects, the left half of the cervical spinal cord was completely severed from the midline to the lateral-most extent of the tissue, and there were no deviations (Fig, 1A).
Statistical analysis
For statistical analysis, divergences where p < 0.05 were considered significant. Sample size for the in vivo behavioral studies was based on a power calculation in which α = 0.05 and β = 0.80, resulting in n = 8. Power analysis was conducted prior to all experiments to ensure that numbers (n) of animals per group were sufficient. The data are presented as mean ± standard error of the mean (SEM). To ensure a normal distribution, data were subjected to the Shapiro–Wilk test for normalcy prior to analysis. Statistical analysis was performed using a one-way ANOVA with post-hoc Bonferroni correction (GraphPad Prism). At the time of processing and analyses of all experiments and assessments, investigators were blinded as to the treatment group of each animal. The functional and behavioral recordings from every animal in each group were analyzed without exclusion based on the outcome.
Results
All treatments improve gross function following a chronic incomplete cervical SCI
All rats displayed a baseline pre-injury FLS performance level at a score of 17 of the (left) forelimb during walking behavior, or consistent plantar stepping of the forepaw, predominant parallel placement. and continuous toe clearing (Figs. 1C and 2A, B). At 3 DPI all rats exhibited an FLS score of ∼4, which improved slightly at 1 WPI to ∼7 (Figs. 1C and 2A).65 These findings demonstrate the consistency of the targeted removal of descending supraspinal motor inputs upon inducing the lateral transection across all of the treatment groups immediately following SCI. Unsurprisingly, by 12 WPI, all rats had reached a pre-treatment baseline FLS score of ∼8, indicating that the animals displayed some spontaneous recovery of function that was consistent prior to treatment and not statistically different between groups (Figs. 1C and 2A, B). An FLS score of 8 denotes the animals' ability to perform partial weight-supported steps but only on the dorsal surface of the impaired limb/paw (Figs. 1C and 2A, B and Supplementary Video 1).65
Interestingly, the control group never gained further FLS assayed function beyond that demonstrated in the pre-treatment assessment, and therefore, any recovery observed is likely a result of the administered treatment (Figs. 2B, C and 3, and Supplementary Video 2). The various treatments caused an initial increase in performance as early as the 1st week after their onset (Fig. 2). There was a significant difference between the FLS score at 14 WPI in the animals treated with PNNi alone (Fig. 2A), whereas the combination treatment group revealed a significance in recovery beginning at 16 WPI, which then improved slowly during the drug administration phase (Fig. 2A). At 24 WPI, the control rats maintained an average FLS score of ∼8, whereas rats that received ISP alone, PNNi alone, or both in combination displayed significant improvements in forelimb recovery during walking compared with the control group (Figs. 2 and 3). There was little evidence of synergy between the two treatments. At 24 WPI, animals treated with ISP alone or PNNi alone reached an average FLS score of ∼11, which functionally translates to continuous plantar stepping but with poor wrist control and no toe clearance (Figs. 2 and 3, and Supplementary Videos 3 and 4). The combination- treated rats were slightly better, receiving an average final score of ∼12, indicating continuous weight bearing and plantar stepping that was predominantly parallel but without toe clearance (Supplementary Video 5). However, the best-responding animals in this group could achieve levels as high as 17, which means that during active locomotion, the forelimb stepping performance displayed proper toe clearing and parallel placement of the forepaw, indicating wrist control recovery that was functionality comparable to baseline walking behavior (Fig. 2C).
Combination treatment best improves skilled forelimb and digit function after a partial chronic cervical SCI
When assessing for cereal-eating ability, all rats displayed a baseline pre-injury performance level at a score of 9 when both the soon to be injured (left) and uninjured (right) were measured. Normal performance is described as almost always maintaining properly shaped paws (conforming to that of the cereal) during grasping of the cereal with a fully flexed elbow, extensive distal limb movements, subtle cereal adjustments, and volar supported manipulations.64 At all weekly behavioral testing time points before and during systemic treatment administration, the uninjured forelimb displayed consistent normal function during cereal eating. At 3 DPI and 1 WPI, all rats exhibited an IBB score of ∼0.5 and ∼2, respectively (Figs. 1B and 5). By 12 WPI, all rats reached a pre-treatment baseline IBB score of ∼2, which translates to cereal-eating behavior that uses the non-volar surface of the injured forelimb, with a predominant fixed forepaw position that is clubbed into a fist-like flexed state, while some animals display an extended forepaw phenotype (Figs. 1B, 4A, B, and 5).64 Unlike the FLS data previously detailed, the IBB assay did not reveal a significant effect of treatment until week 4. Further, the significant behavioral recovery observed was only demonstrated in the animals that received both PNNi and ISP in combination (Figs. 4C and 5).
Our behavioral results using the IBB assessment to measure forelimb and paw cereal manipulations further demonstrated that the combination treatment group eventually developed the ability to use their once-paralyzed paw/fingers remarkably better than controls. The control animals recovered paw/finger function only minimally over time (compared with normal animals) to an IBB score between 2 and 3, where the affected paw may remain partially clubbed and mostly rests on the ground and where, at best, some crude, exaggerated cereal adjustments are made (Figs. 5 and Supplementary Video 6).64 The paw does not exhibit adaptability and fails to conform to the shape of the piece of cereal. Although animals receiving either ISP or PNNi alone did not show a similar effect of meaningful forelimb functional recovery as we showed in the FLS assessment, these animals did show a strong trend toward significance by the end of the study compared with the control group (Figs. 4 and 5). On the contrary, the combination of ISP and PNNi led to clear improvements in paw/finger function during cereal eating.64 On average, at 24 WPI and with the combinatorial treatment, the once- paralyzed animals recovered to an IBB score of between 5 and 6 with extensive contact manipulatory movements (Figs. 4 and 5, and Supplementary Video 7).64 Our most encouraging finding was that the best-responding animals, especially when eating a donut-shaped cereal such as a Fruit Loop, could achieve a remarkable score of 7 (9 is the highest possible score)64 where sometimes normal, shape-adapting grasping can occur. In summary, these behavioral data confirm the hypothesis that modulating CSPGs in the scar and especially in the PNN as well as in their receptor simultaneously is advantageous to the recovery of precision digit function long after SCI.
Histological analyses reveal reductions in WFA+ matrix and PNNs, and sprouting of serotonin (5-HT) axons in the spinal cord of treated animals
At the end of our behavioral study (24 WPI), we visualized WFA immunoreactivity using WFA staining, focusing on the ventral horn gray matter at spinal levels caudal to the LC2H near the forelimb muscle innervating motor pools for all experimental groups. In control rats that were chronically injured at C2 and received no systemic treatment, there was an upregulation in the accumulation of WFA in the extracellular matrix (ECM) compartment and in PNNs surrounding cells in the cervical enlargement, compared with animals that received ISP with or without oral PNNi (Fig. 6). Incidentally, because WFA-lectin binds numerous glycoproteins, we verified our WFA staining with a well-known aggrecan (ACAN)-specific antibody, CAT301. We observed a nearly 1:1 correlation between the staining patterns revealed by CAT301 and WFA, indicating that the WFA staining does represent CSPGs, at least in part (Fig. S1). Although this does not rule out the possibility that the WFA-Lectin has bound other glycoproteins, we can definitively report that CSPGs are contained within the perineuronal net and visualized by WFA. This increased WFA signal was observed throughout the entire cervical region (C5–C8) and was seen on both the injured and uninjured sides suggesting, as have others,13,15,19,67,68 that much of the spinal column distal to the lesion responds to a very localized SCI by upregulating the production of CSPGs over a wide distance.
Additionally, the increased inhibitory matrix persists for months (24 WPI) after the initial spinal insult has occurred (Fig. 6). Therefore, in the chronic stage of SCI, CSPGs within the ECM and PNNs are ideal targets for restoration of forelimb movements. Along with the increase in the PNN and extracellular matrix, there was an obvious decrease in the presence of 5-HT+ fibers within the cervical ventral horn gray matter (Fig. 6A, B). Together, these data suggest that the observed behavioral deficits following chronic LC2H correlate with increased PNN-CSPGs and loss of descending motor input, including serotonergic axon innervation onto downstream targets. We then compared the WFA matrix and 5-HT+ fiber densities in animals that received ISP ± PNNi to the control animals described. In animals receiving daily monotherapy with either ISP or PNNi, a significant decrease of CSPGs in the ECM and PNNs on the injured (left) and uninjured side of the spinal cord was observed (Fig. 6C). A similar (but not beyond that which occurred in the single-treatment animals) reduction in the inhibitory extracellular matrix happened again when both systemic treatments were administered together during the chronic SCI phase (Fig. 6C).
In addition, we measured the serotonergic axon innervation density in the same region. We found that animals that received a systemic treatment daily, either alone or in combination, exhibited a significant increase in 5HT+ fibers within the cervical enlargement compared with animals with unperturbed PNNs (Fig. 6B). Further, at this level of examination, we did not observe a difference in the serotonergic innervation on the contralateral, uninjured side (Fig. 6B). To complement these quantification studies, we examined serotonergic axon innervation at C5 at a higher resolution within the ventral horn gray matter in animals that received daily treatment with PNNi + ISP as compared with controls. We were encouraged to see that these high-magnification studies confirmed our quantified results. We verified decreased serotonergic innervation in the ipsilateral, injured side regardless of treatment condition when compared with the contralateral, uninjured side (compare Fig. 7A and C to 7B and D). Comparing the ipsilateral side of drug-treated versus the vehicle-treated animals, we were able to visualize enhanced serotonergic axon sprouting in the injured, ventral horn resulting from combination treatment with PNNi + ISP. In our previous studies showing the therapeutic efficacy of ISP acutely after severe contusive spinal cord injury,7 we demonstrated a rather unique pattern of sprouting of serotonergic axons that occurred in unusually shaped, dense clusters. Upon examination of 5-HT axons caudal to the chronic injury site in our combination-treated animals, we rarely observed this type of clustered axonal sprouting.7,55,69–72
Identification of a novel WFA-Binding Aggregate in the chronically injured cord
Our efforts to elucidate the neuronal mechanism of recovery from chronic injury led us to a secondary, never-before-identified phenomenon that was tightly correlated with behavioral improvements. Throughout the cervical enlargement, from the level of injury at C2–C9, we noted numerous, roughly spherical, aggregations of matrix. These plaque-like structures, which we never observed in the cervical cords of age-matched, uninjured animals, stained intensely with WFA-lectin and presented largely within the lesioned white matter where they were localized to a small subset of descending tracts: (1) the dorsomedial corticospinal tract (CST), (2) the rubrospinal or possibly the lateral CST, and (3) the ventral medial reticulospinal tract (Fig. 8A–C). These structures ranged in size from ∼25_40 μm in diameter within the lateral and ventral medial tracts (Fig. 8D–F) to 5–10 μm within the dorsomedial CST (Fig, 8G–I). Although these structures were reliably identified using WFA-lectin, they may be composed of only select CSPGs. For example, they could not be visualized using a CAT-301, aggrecan-specific antibody (data not shown).
The precise mechanisms of action of PNNi plus ISP remains unknown. The recovery we observed from treatment with this combination may involve the generalized loss of extracellular CSPGs and overall increase in 5-HT sprouting visualized in Figures 6 and 7. However, these observations cannot account for the superior recovery of digital dexterity resulting from combination treatment over the individual compounds reported in Figure 4. Remarkably, in the ISP + PNNi combination group, we noted substantial clearing of these WFA+ aggregates exclusively from the midline CST (Fig. 8J, M, and P). Surprisingly, these structures were not cleared from the other motor pathways in the chronically injured spinal cord (Fig. 8K, L, N, O, Q, and R). Although we do not yet understand what these structures are or from where and when they emanate or why their enhanced clearance is restricted to the midline CST, it is striking that their removal strongly correlated with recovered forelimb function and digital dexterity.
Matrix aggregates are internalized by microglia in the dorsomedial CST
Having identified these pathological WFA+ structures and correlated their selective removal to precision forelimb and fine digit motor recovery, we sought a deeper understanding of the neural cells with which they were most closely associated. Using immunofluorescence, we interrogated the microenvironment of the chronically injured cervical cord in animals treated with PNNi plus ISP compared with controls. We examined the spatial relationship of aggregates to microglia using allograft inflammatory factor 1 (IBA1, Fig. 9A–F) as well as to reactive astrocytes using glial fibrillary acidic protein (GFAP, Fig. 9G, H). Although aggregates presented within fields of reactive astrocytes, it did not appear as though these cell types were actively engaged in the generation, clearance, or encapsulation of these structures. In contrast, in the combination-treated animals, microglia were intimately associated with them. In the dorsomedial CST only, microglia/macrophages appear to have internalized the WFA+ spheroids, suggesting that clearance may involve inflammatory cell-mediated phagocytosis. Note the co-localization of WFA and IBA1 identified by white arrows and the insert in Figure 9A. In total, these data suggest the possibility that matrix aggregates containing CSPGs represent a newly identified, pathological structure that presents in the chronically injured spinal cord. Additionally, the combined use of PNNi and ISP may alter the physiological state of the microglia which, in turn, facilitates phagocytic removal of the aggregates.
Discussion
We assessed improvements of forelimb behavior in a hemi-section model of chronic cervical SCI and have revealed that treatments with PNNi and ISP alone but especially in combination can stimulate a good measure of recovery. Further, these functional improvements in the forelimb persisted for at least 4 weeks after the daily treatment(s) had ended. When given for 2 months following a chronic LC2H, ISP or PNNi administered alone were able to improve proximal and distal forelimb use during more crude locomotion. Moreover, we assessed fine motor behaviors during cereal eating and noted rather remarkably improved digit function when we administered our systemic treatments together.
It is quite interesting that recovery of forearm function begins to occur relatively rapidly following systemic treatment in our chronic injury model. Surprisingly, behavioral improvements occur much more quickly than they do after similar treatments during the acute stage.14 This time frame mimics that which we have shown previously in our ChABC-treated chronic LC2H SCI respiratory model suggesting, again, that sprouting likely had already occurred but was somehow being masked by the CSPG component of the PNN.53 Thus, in our 12-week cervical hemi-lesion model, certain sub-populations of the contralateral descending supraspinal tracts with small numbers of axons that re-decussate within the spinal cord caudal to the level of injury,72 or intervening interneurons with similar properties of plasticity,73 may be very slowly sprouting new processes that re-cross the midline to the denervated side ipsilateral to the lesion even in the presence of the PNN.74–78 Of course, we are not ruling out the possibility that this process may be augmented during the further 2 months of treatment. Various motor systems underlying forelimb and paw movement, including the serotonergic, propriospinal, and even the cortico-spinal neurons have an innate propensity to sprout on their own, although at a very diminished rate.44,72,73,78–81 These new collaterals could produce latent, or inactive synapses which, over time, could be beneficial, harmless, or possibly even disadvantageous to recovery.53,82 Our results demonstrate that the potential activity of such sprouting can be supportive to functional recovery but is largely kept dormant by CSPGs within the PNN in the vicinity of the relevant motor pools.
There is a question as to what is the mechanism by which the PNN may curtail synaptic activity and perhaps help to create such silent synapses. rPTPσ might be more directly involved with the formation, maintenance, or function of these new connections. Increases in PNN density and changes in the chondroitin sulfation pattern to a more growth-inhibitory state occur during normal aging83 and after SCI.16,84 It is well established that the PNN plays important roles not only in regulating various morphological aspects of axonal sprouting during the development of critical periods4,16,18 as well as after injury in the adult,7,15,17,41,47,49,52,85 but also in the control of synaptic activity.84,86–89 Interestingly, in a model of Alzheimer's disease, the PNN in the cortex encroaches into synaptic clefts, which shrinks the perimeter of ECM barriers around the PNN holes, thereby reducing the diameter of the portals for communication between pre- and post-synaptic elements.90,91 However, although the function dampening consequnces of peri-neuronal net invasion of the synapse has been suggested, the mechanism of this possible physiological barrier property has not yet been fully elucidated.91,92 Latent synapses are known to occur throughout the CNS.93 One of the most interesting and well-known latent descending motor projections is the crossed phrenic pathway.77,94–96 It is already present but can be revived in the adult in seconds after an LC2H injury by creating anoxic stress after lesioning the contralateral phrenic nerve53,77 as well as by modulating the leukocyte common antigen-related (LAR) family receptors.97,98 It can also be reawakened rather rapidly long after cord injury by administration of chondroitinase.49,53 Axons of the corticospinal system are known to re-cross the midline of the spinal cord rather extensively during normal development, but such ipsilateral CST projections are largely eliminated.99 It is possible that there might be a would-be pruned cohort of double decussated CST axons that actually persists in the adult but is difficult to label because it exists in a withered, quiescent state. Perhaps, in addition to other proposed mechanisms that can lead to synaptic dysfunction, such connections of sprouted or dormant axons in the cervical enlargement could be made inactive via the matrix-invading phenomenon which, in turn, might be reversed by PNNi removal of the net.94–96,100–103
There is a question as to what might be the mechanism by which ISP modulation of rPTPσ mediates functional recovery. In addition to its role in curtailing axonal sprouting and/or regeneration via excessively strong adhesion to CSPG substrates,7,104,105 there is also abundant evidence, although mostly in vitro, that rPTPσ can function in neural circuit assembly by tightening the bond among its multiple ligands at developing synapses.25,106–111 Thus, ISP-mediated disruption of the receptor could loosen its connections with a variety of binding partners and allow for axonal sprouting, albeit at the expense of synaptic reformation.97,98 Although we did not quantify synaptic densities in the reinnervated portions of the combination-treated spinal cord denervated by the hemi-lesion, we have reported an increase, not a decrease, in penumbral synapses in a functionally beneficial ISP-treated mouse model of malignant stroke.105 Importantly, the notion that rPTPσ as well as the other LAR family receptors are critical organizers during synapse formation has become controversial because of work that showed that synaptic connectivity in the hippocampus was completely unaffected by in-vivo deletion of all three LAR-rPTPs beginning in the neonate.89 However, more recently, clear in-vivo evidence has been presented for a role of rPTPδ in the assembly and maintenance of climbing fiber synapses on Purkinje cells in the developing cerebellum.112 Therefore, although the precise mechanism by which ISP leads to functionally beneficial sprouting remains elusive, the manner in which it does this may be indirect and downstream of rPTPσ signaling involving the hypersecretion of specific inhibitory matrix degrading proteases (see subsequent discussion).
Recently, an independent study confirmed the benefits of subcutaneous peptide treatment showing that 500 μg/day of ISP given acutely after lateral crush greatly improved hindlimb locomotor, sensory, and bladder function.113 Indeed, with the use of the well-characterized BBB locomotor rating scale, the study's contusion-injured animals (that stabilized at a control baseline score of 6) on average recovered by a remarkable 9 full points to a level of 15.113 Importantly, although bladder function (but not walking) showed a dose response when cord-injured rats were given ISP at a maximum of 44 μg/day,7 the much higher dose of 500 μg/day provides the first evidence for a dose response by ISP in promoting enhancements in locomotor behavior, at least after acute SCI. Similarly, ISP has been shown to have some therapeutic effects after acute T8 cord hemi-section in the adult rat when assessed by an inherently objective automated gait analysis system.62,114 ISP has also now been shown to be effective in promoting regeneration and functional recovery after SCI when delivered via a plasma exosome- based biological scaffold115 as well as by a self-assembling peptide hydrogel.116 The regenerative potential of blocking the rPTPσ receptor by ISP has, in addition, been demonstrated in models of multiple sclerosis (MS),8 ischemic and hemorrhagic stroke,104,105 ischemic heart attack,117,118 and peripheral nerve injury.119,120 The high dose of 500 μg/day of ISP is what we chose to use, and was effective in our chronic study. However, the most optimal dosing regimen and the best route of delivery, as well as the optimal amount of peptide that is most functionally beneficial have not yet been established. Also, it is important to stress that as a standalone treatment, ISP is not especially effective in cases of extremely severe cord injury, as our laboratory has recently observed in ongoing studies using a more complete model of thoracic spinal cord injury (data not published).
Hyaluronan (HA) is the major scaffold for PNN assembly, and if HA synthesis is blocked, then the PNN is not able to structure itself properly.113,121,122 In addition to the use of the wedge peptide ISP, we employed a novel strategy in combination, which can substantially reduce the PNN CSPGs in the CNS via systemic delivery of an already clinically approved small molecule proteoglycan synthesis inhibitor called 4-methylumbelliferone (4MU) or hymecromone.121–123 Referred to as PNNi in our study, we repurposed this small molecule in our model of chronic SCI. One of the major challenges for translation of SCI-regenerative strategies from rodent to human is the enormous relative difference in size of the human, which necessitates careful considerations of concentrations, toxicity, and routes of delivery to optimize the amount and time of residence of the therapeutic drug within the CNS. Oral delivery of a small molecule inhibitor that is already clinically approved is a preferred strategy for drug delivery. A novel therapeutic approach to overcome the inhibitory effects of the PNN involves curtailing the production of HA. PNNi specifically inhibits HA synthesis by acting as a competitive substrate for hyaluronan synthase, an enzyme critical for HA production.122 Preliminary findings have shown that inhibiting HA synthesis with PNNi resulted in significant decreases in the hyaluronan link protein as well as in the amount of CSPGs in an in-vitro model as well as in the brain and spinal cord. In vivo, the reduction of the CSPG component of the PNN was especially prominent in the ventral horn around motor neurons.124,125 We therefore hypothesized that PNNi, especially when administered together with ISP, could be an obvious dual therapy, because the combined strategy would decrease the ligand as well as modulating the receptor globally, and could be an effective, clinically viable treatment method following chronic partial cervical lesions. Indeed, we confirmed that chronic disruption of proper PNN assembly in the two cohorts of animals that received 4-MU (± ISP) significantly reduced the presence of WFA+ CSPGs within the cervical enlargement. Importantly, the reduction in CSPGs was inversely correlated with the increased density of serotonergic axons observed within the ventral gray matter of the same region. PNNi as well as ISP are non-invasive treatments with low toxicity that can be stopped at any time to reverse the receptor blockade and HA-synthesis-inhibiting effects of the combination, and allow for the PNN to be re-established. This can help avoid potential long-term adverse side effects and help stabilize newly formed synapses.
Although reduction of CSPGs in the 4-MU-treated animals was expected, there was a question as to why there was also a reduction of CSPG matrix, again where 5-HT axon densities had increased, in the animals treated with ISP only. A variety of motile cell types, including neurons, use tightly regulated release of proteases to remodel the surrounding extracellular matrix along their potential routes of migration.69,126–128 We have described, in vitro and in vivo, an interesting downstream event in both dorsal root ganglion and serotonergic neurons that occurs as a consequence of rPTPσ modulation by ISP, resulting in the locally enhanced release of the matrix-degrading enzyme, cathepsin B. Cathepsin B is a proteolytic enzyme that is normally produced, albeit in limited amounts, by growing neurons during crucial periods of extracellular matrix remodeling, and is also augmented in neurons within the spinal cord gray matter after injury.129,130 The induced exaggerated secretion of cathepsin B, likely at the leading edge of ISP-treated axon growth cones, helps them to navigate within or past an inhibitory CSPG barrier.69,129,130 We have shown that this phenomenon of enzyme hyper-secretion, which could synergize with PNNi, is protease specific and occurs not only in neurons but also in multiple migratory cell types that bear the rPTPσ receptor as they encounter the CSPGs. Thus, in oligodendrocyte progenitor cells in models of MS as well as subventricular zone (SVZ) derived neural progenitor cells in an ischemic model of stroke, ISP specifically stimulated release of matrix metalloproteinase (MMP)2. The protease encouraged digestion of glial-scar-associated CSPGs which, in turn, restored stem cell migrations and their differentiation through the penumbra and well into the lesion core, resulting in functional improvements.8,69,104
Descending serotonergic fibers are known to influence, indirectly or directly, the excitability of spinal cord motoneurons, and it is well established that 5-HT plays an important role in modulating locomotion.131–133 Many studies have shown that functional recovery following a SCI is potently influenced by increased numbers of serotonergic axons at spinal levels caudal to the initial injury.7,53,69 Whether the increased 5-HT fiber density, that we have described here, plays a role in restoring fine digit control is not known, but there are several studies that have shown a role for 5-HT in the modulation of certain aspects of distal flexor function.70,71
Although we did not observe enhanced dorsal CST sprouting or frank regeneration through the lesion (the CST is a tract well known for its role in voluntary control of distal musculature)134–138 we did observe a rather dramatic change in the inflammatory reaction to the large plaque-like CSPG deposits only in the lesioned white matter of the dorsomedial CST. It is now known that ISP can promote a more M2-like, reparative immune response after SCI.139 Conceivably, this immune-system-related CSPG clearing phenomenon in the dorsal white matter might be occurring at some level far more broadly, even within the gray matter distal to the lesion, suggesting that such activated microglia could also be playing a role in the local elimination of the PNN in the spinal cord leading to increased synaptic plasticity from the undamaged portions of the CST as well as the serotonergic system. However, whether this drug-induced, phagocytic reaction in the degenerating portions of the dorsal CST is a reflection of a wound-healing contingent of microglia throughout the entire corticospinal system is unknown. Therefore, the anatomical substrate (whether it be newly sprouted or latent) that underlies such robust functional recovery chronically as well as the origin and biological impact of the WFA+ plaques must await further studies.
Transparency, Rigor, and Reproducibility Summary
The study was registered after the study began. The study is registered at BioRxiv (https://www.biorxiv.org/content/10.1101/2022.08.01.502398v).1 The analysis plan was not formally pre-registered, but the team member with primary responsibility for the analysis certifies that the analysis plan was pre-specified.2 A sample size of eight subjects per group was planned based on an expected effect size calculated to yield 88–96% power to detect significant behavioral recovery using repeated measures ANOVA with a p value of <0.05.3 Sixty animals were considered for inclusion; 10 animals per group (4 groups) for SCI, behavior, and histological analysis; 32 animals received each treatment; 10 animals died from post-operational complications; and 11 animals had incomplete assessments and were excluded from the study.4 Subjects were randomly assigned to groups using a random number generator.5 Investigators who administered the therapeutic intervention were blinded to group assignment by use of an identically appearing placebo treatment. Investigators who conducted the outcome assessments were blinded to group assignment by restricting contact with investigators who administered the therapeutic intervention.6 Timing of administration relative to injury was 12 WPI. Purity of pharmacological reagents was 99% based on certificate of analysis. Controls used included naïve, uninjured animals and vehicle-only injured animals. Specificity and anatomical accuracy of lesions used for experimental manipulations were verified using fluorescent Nissl.7 All the materials used for the therapeutic intervention came from a single batch prepared on May 4, 2020 at Case Western Reserve University School of Medicine's Neuroscience Department.8 The experimental injury model is an established standard in the field. The primary outcome measure is an established standard in the field. Validation is included (https://doi.org/10.3389/fneur.2014.00116; https://doi.org/10.1016/j.jneumeth.2014.01.001).9 The statistical tests used were based on the assumptions of normal distributions and the sample sizes reflect the number of independent measurements. Non-independent measurements have been addressed using two-way, repeated measures ANOVA.10 Correction for multiple comparisons was performed using Bonferroni correction.11 Planned/ongoing external validation studies have been pre-registered at BioRxiv.12 There is no analytical code associated with this study.13 All materials used to conduct the study are available to qualified investigators via direct request from the corresponding author, and purchase from the GoldBio vendor.14 The authors agree to publish the manuscript using the Mary Ann Liebert Inc. “Open Access” option under the appropriate license.15
Acknowledgments
We thank Drs. S. Fischer and K Brock for their dedicated veterinary assistance. We further thank Dr. W. Alilain, as well as Drs. P. Popovich, D. McTigue, and M. Basso (The Ohio State University Spinal Cord Injury Course) for surgical and behavioral technical training.
Supplementary Material
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References
1. Sekhon LHS, Fehlings MG. Epidemiology, demographics, and pathophysiology of acute spinal cord injury. Spine (Phila Pa 1976) 2001;26(Supplement):S2–S12;
2. Kumar R, Lim J, Mekary RA, et al. Traumatic spinal injury: global epidemiology and worldwide volume. World Neurosurg 2018;113:e345–e363;
3. Anderson KD. Targeting recovery: priorities of the spinal cord-injured population. J Neurotrauma 2004;21(10):1371–1383;
4. Snow DM, Steindler DA, Silver J. Molecular and cellular characterization of the glial roof plate of the spinal cord and optic tectum: a possible role for a proteoglycan in the development of an axon barrier. Dev Biol 1990;138(2):359–376;
5. Brittis PA, Canning DR, Silver J. Chondroitin sulfate as a regulator of neuronal patterning in the retina. Science (1979) 1992;255(5045):733–736;
6. Brooks JM, Su J, Levy C, et al. A molecular mechanism regulating the timing of corticogeniculate innervation. Cell Rep 2013;5(3):573–581;
7. Lang BT, Cregg JM, DePaul MA, et al. Modulation of the proteoglycan receptor PTPσ promotes recovery after spinal cord injury. Nature 2015;518(7539):404–408;
8. Luo F, Tran AP, Xin L, et al. Modulation of proteoglycan receptor PTPσ enhances MMP-2 activity to promote recovery from multiple sclerosis. Nat Commun 2018;9(1):4126;
9. Kazanis I, ffrench-Constant C. Extracellular matrix and the neural stem cell niche. Dev Neurobiol 2011;71(11):1006–1017;
10. Tran AP, Warren PM, Silver J. The biology of regeneration failure and success after spinal cord injury. Physiol Rev 2018;98(2):881–917;
11. Massey JM, Hubscher CH, Wagoner MR, et al. Chondroitinase ABC digestion of the perineuronal net promotes functional collateral sprouting in the cuneate nucleus after cervical spinal cord injury. J Neurosci 2006;26(16):4406–4414;
12. Busch SA, Silver J. The role of extracellular matrix in CNS regeneration. Curr Opin Neurobiol 2007;17(1):120–127;
13. Alilain WJ, Horn KP, Hu H, et al. Functional regeneration of respiratory pathways after spinal cord injury. Nature 2011;475(7355):196–200;
14. Li L, Zheng H, Ma X, et al. Inhibition of astrocytic carbohydrate sulfotransferase 15 promotes nerve repair after spinal cord injury via mitigation of CSPG mediated axonal inhibition. Cell Mol Neurobiol 2023;
15. Andrews EM, Richards RJ, Yin FQ, et al. Alterations in chondroitin sulfate proteoglycan expression occur both at and far from the site of spinal contusion injury. Exp Neurol 2012;235(1):174–187;
16. Lipachev N, Arnst N, Melnikova A, et al. Quantitative changes in perineuronal nets in development and posttraumatic condition. J Mol Histol 2019;50(3):203–216;
17. Silver DJ, Silver J. Contributions of chondroitin sulfate proteoglycans to neurodevelopment, injury, and cancer. Curr Opin Neurobiol 2014;27:171–178;
18. Dyck SM, Karimi-Abdolrezaee S. Chondroitin sulfate proteoglycans: Key modulators in the developing and pathologic central nervous system. Exp Neurol 2015;269:169–187;
19. Hryciw T, Geremia NM, Walker MA, et al. Anti-chondroitin sulfate proteoglycan strategies in spinal cord injury: temporal and spatial considerations explain the balance between neuroplasticity and neuroprotection. J Neurotrauma 2018;35(16):1958–1969;
20. Grycz K, Głowacka A, Ji B, et al. Regulation of perineuronal net components in the synaptic bouton vicinity on lumbar α-motoneurons in the rat after spinalization and locomotor training: new insights from spatio-temporal changes in gene, protein expression and WFA labeling. Exp Neurol 2022;354:114098;
21. Sakamoto K, Ozaki T, Ko Y-C, et al. Glycan sulfation patterns define autophagy flux at axon tip via PTPRσ-cortactin axis. Nat Chem Biol 2019;15(7):699–709;
22. Grimpe B, Silver J. A novel DNA enzyme reduces glycosaminoglycan chains in the glial scar and allows microtransplanted dorsal root ganglia axons to regenerate beyond lesions in the spinal cord. J Neurosci 2004;24(6):1393–1397;
23. Yamaguchi Y. Lecticans: organizers of the brain extracellular matrix. Cell Mol Life Sci 2000;57(2):276–289;
24. Lau LW, Cua R, Keough MB, et al. Pathophysiology of the brain extracellular matrix: a new target for remyelination. Nat Rev Neurosci 2013;14(10):722–729;
25. Dyck SM, Alizadeh A, Santhosh KT, et al. Chondroitin sulfate proteoglycans negatively modulate spinal cord neural precursor cells by signaling through LAR and RPTPσ and modulation of the Rho/ROCK pathway. Stem Cells 2015;33(8):2550–2563;
26. Sherman LS, Back SA. A ‘GAG’ reflex prevents repair of the damaged CNS. Trends Neurosci 2008;31(1):44–52;
27. Hartmann U, Maurer P. Proteoglycans in the nervous system — the quest for functional roles in vivo. Matrix Biol 2001;20(1):23–35;
28. Karimi-Abdolrezaee S, Schut D, Wang J, et al. Chondroitinase and growth factors enhance activation and oligodendrocyte differentiation of endogenous neural precursor cells after spinal cord injury. PLoS One 2012;7(5):e37589;
29. Khalil AS, Hellenbrand D, Reichl K, et al. A localized materials-based strategy to non-virally deliver chondroitinase ABC mRNA improves hindlimb function in a rat spinal cord injury model. Adv Healthc Mater 2022;11(19):2200206;
30. Karimi-Abdolrezaee S, Eftekharpour E, Wang J, et al. Synergistic effects of transplanted adult neural stem/progenitor cells, chondroitinase, and growth factors promote functional repair and plasticity of the chronically injured spinal cord. J Neurosci 2010;30(5):1657–1676;
31. Gandhi T, Liu C-C, Adeyelu TT, et al. Behavioral regulation by perineuronal nets in the prefrontal cortex of the CNTNAP2 mouse model of autism spectrum disorder. Front Behav Neurosci 2023;17;
32. Nogueira-Rodrigues J, Leite SC, Pinto-Costa R, et al. Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection. Dev Cell 2022;57(4):440-450.e7;
33. Shafqat A, Albalkhi I, Magableh HM, et al. Tackling the glial scar in spinal cord regeneration: new discoveries and future directions. Front Cell Neurosci 2023;17;
34. Carter LM, Starkey ML, Akrimi SF, et al. The yellow fluorescent protein (YFP-H) mouse reveals neuroprotection as a novel mechanism underlying chondroitinase ABC-mediated repair after spinal cord injury. J Neurosci 2008;28(52):14107–14120;
35. Ouchida J, Ozaki T, Segi N, et al. Glypican-2 defines age-dependent axonal response to chondroitin sulfate. Exp Neurol 2023;366:114444;
36. Michel-Flutot P, Lane MA, Lepore AC, et al. Therapeutic strategies targeting respiratory recovery after spinal cord injury: from preclinical development to clinical translation. Cells 2023;12(11):1519;
37. Petrosyan HA, Alessi V, Lasek K, et al. AAV vector mediated delivery of NG2 function neutralizing antibody and neurotrophin NT-3 improves synaptic transmission, locomotion, and urinary tract function after spinal cord contusion injury in adult rats. J Neurosci 2023;43(9):1492–1508;
38. Miller GM, Hsieh-Wilson LC. Sugar-dependent modulation of neuronal development, regeneration, and plasticity by chondroitin sulfate proteoglycans. Exp Neurol 2015;274:115–125;
39. Pearson CS, Mencio CP, Barber AC, et al. Identification of a critical sulfation in chondroitin that inhibits axonal regeneration. Elife 2018;7:e37139;
40. Wang H, Katagiri Y, McCann TE, et al. Chondroitin-4-sulfation negatively regulates axonal guidance and growth. J Cell Sci 2008;121(18):3083–3091;
41. Hussein RK, Mencio CP, Katagiri Y, et al. Role of chondroitin sulfation following spinal cord injury. Front Cell Neurosci 2020;14:208;
42. García-Alías G, Barkhuysen S, Buckle M, et al. Chondroitinase ABC treatment opens a window of opportunity for task-specific rehabilitation. Nat Neurosci 2009;12(9):1145–1151;
43. Silver J, Miller JH. Regeneration beyond the glial scar. Nat Rev Neurosci 2004;5(2):146–156;
44. Cafferty WBJ, Bradbury EJ, Lidierth M, et al. Chondroitinase ABC-mediated plasticity of spinal sensory function. J Neurosci 2008;28(46):11998–12009;
45. Ramer LM, Ramer MS, Bradbury EJ. Restoring function after spinal cord injury: towards clinical translation of experimental strategies. Lancet Neurol 2014;13(12):1241–1256;
46. Sakamoto K, Kadomatsu K. Mechanisms of axon regeneration: the significance of proteoglycans. Biochim Biophys Acta Gen Subj 2017;1861(10):2435–2441;
47. Brown JM, Xia J, Zhuang B, et al. A sulfated carbohydrate epitope inhibits axon regeneration after injury. Proc Natl Acad Sci 2012;109(13):4768–4773;
48. Sakamoto K, Ozaki T, Kadomatsu K. Axonal regeneration by glycosaminoglycan. Front Cell Dev Biol 2021;9;
49. Massey JM, Amps J, Viapiano MS, et al. Increased chondroitin sulfate proteoglycan expression in denervated brainstem targets following spinal cord injury creates a barrier to axonal regeneration overcome by chondroitinase ABC and neurotrophin-3. Exp Neurol 2008;209(2):426–445;
50. Shinozaki M, Iwanami A, Fujiyoshi K, et al. Combined treatment with chondroitinase ABC and treadmill rehabilitation for chronic severe spinal cord injury in adult rats. Neurosci Res 2016;113:37–47;
51. Wang D, Ichiyama RM, Zhao R, et al. Chondroitinase combined with rehabilitation promotes recovery of forelimb function in rats with chronic spinal cord injury. J Neurosci 2011;31(25):9332–9344;
52. Tom VJ, Steinmetz MP, Miller JH, et al. Studies on the development and behavior of the dystrophic growth cone, the hallmark of regeneration failure, in an in vitro model of the glial scar and after spinal cord injury. J Neurosci 2004;24(29):6531–6539;
53. Warren PM, Steiger SC, Dick TE, et al. Rapid and robust restoration of breathing long after spinal cord injury. Nat Commun 2018;9(1):4843;
54. Warren PM, Kissane RWP, Egginton S, et al. Oxygen transport kinetics underpin rapid and robust diaphragm recovery following chronic spinal cord injury. J Physiol 2021;599(4):1199–1224;
55. Warren PM, Alilain WJ. Plasticity induced recovery of breathing occurs at chronic stages after cervical contusion. J Neurotrauma 2019;36(12):1985–1999;
56. Hettiaratchi MH, O'Meara MJ, O'Meara TR, et al. Reengineering biocatalysts: Computational redesign of chondroitinase ABC improves efficacy and stability. Sci Adv 2020;6(34);
57. Hettiaratchi MH, O'Meara MJ, Teal CJ, et al. Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains. J Control Release 2019;297:14–25;
58. Lee H, McKeon RJ, Bellamkonda R V. Sustained delivery of thermostabilized chABC enhances axonal sprouting and functional recovery after spinal cord injury. Proc Natl Acad Sci 2010;107(8):3340–3345;
59. Bartus K, James ND, Didangelos A, et al. Large-scale chondroitin sulfate proteoglycan digestion with chondroitinase gene therapy leads to reduced pathology and modulates macrophage phenotype following spinal cord contusion injury. J Neurosci 2014;34(14):4822–4836;
60. Burnside ER, de Winter F, Didangelos A, et al. Immune-evasive gene switch enables regulated delivery of chondroitinase after spinal cord injury. Brain 2018;141(8):2362–2381;
61. Shen Y, Tenney AP, Busch SA, et al. PTPσ is a receptor for chondroitin sulfate proteoglycan, an inhibitor of neural regeneration. Science (1979) 2009;326(5952):592–596;
62. Ham TR, Farrag M, Soltisz AM, et al. Automated gait analysis detects improvements after intracellular σ peptide administration in a rat hemisection model of spinal cord injury. Ann Biomed Eng 2019;47(3):744–753;
63. Fisher D, Xing B, Dill J, et al. Leukocyte common antigen-related phosphatase is a functional receptor for chondroitin sulfate proteoglycan axon growth inhibitors. J Neurosci 2011;31(40):14051–14066;
64. Irvine K-A, Ferguson AR, Mitchell KD, et al. The Irvine, Beatties, and Bresnahan (IBB) Forelimb Recovery Scale: an assessment of reliability and validity. Front Neurol 2014;5:116;
65. Singh A, Krisa L, Frederick KL, et al. Forelimb locomotor rating scale for behavioral assessment of recovery after unilateral cervical spinal cord injury in rats. J Neurosci Methods 2014;226:124–131;
66. Basso DM, Beattie MS, Breshanhan JC. A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma 1995;12(1):1–21;
67. Lemons ML, Howland DR, Anderson DK. Chondroitin sulfate proteoglycan immunoreactivity increases following spinal cord injury and transplantation. Exp Neurol 1999;160(1):51–65;
68. Hunanyan AS, Garcia-Alias G, Alessi V, et al. Role of chondroitin sulfate proteoglycans in axonal conduction in mammalian spinal cord. J Neurosci 2010;30(23):7761–7769;
69. Tran AP, Sundar S, Yu M, et al. Modulation of receptor protein tyrosine phosphatase sigma increases chondroitin sulfate proteoglycan degradation through cathepsin B secretion to enhance axon outgrowth. J Neurosci 2018;38(23):5399–5414;
70. Vitrac C, Benoit-Marand M. Monoaminergic modulation of motor cortex function. Front Neural Circuits 2017;11;
71. Seo NJ, Fischer HW, Bogey RA, et al. Effect of a serotonin antagonist on delay in grip muscle relaxation for persons with chronic hemiparetic stroke. Clin Neurophysiol 2011;122(4):796–802;
72. Bareyre FM, Kerschensteiner M, Raineteau O, et al. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat Neurosci 2004;7(3):269–277;
73. Fenrich KK, Rose PK. Spinal interneuron axons spontaneously regenerate after spinal cord injury in the adult feline. J Neurosci 2009;29(39):12145–12158;
74. Porter WT. The path of the respiratory impulse from the bulb to the phrenic nuclei. J Physiol 1895;17(6):455–485;
75. Friedli L, Rosenzweig ES, Barraud Q, et al. Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates. Sci Transl Med 2015;7(302);
76. Lane MA, White TE, Coutts MA, et al. Cervical prephrenic interneurons in the normal and lesioned spinal cord of the adult rat. J Comp Neurol 2008;511(5):692–709;
77. Alilain WJ, Goshgarian HG. Glutamate receptor plasticity and activity-regulated cytoskeletal associated protein regulation in the phrenic motor nucleus may mediate spontaneous recovery of the hemidiaphragm following chronic cervical spinal cord injury. Exp Neurol 2008;212(2):348–357;
78. Hawthorne AL, Hu H, Kundu B, et al. The unusual response of serotonergic neurons after CNS injury: lack of axonal dieback and enhanced sprouting within the inhibitory environment of the glial scar. J Neurosci 2011;31(15):5605–5616;
79. Ishida A, Kobayashi K, Ueda Y, et al. Dynamic interaction between cortico-brainstem pathways during training-induced recovery in stroke model rats. J Neurosci 2019;39(37):7306–7320;
80. García-Alías G, Truong K, Shah PK, et al. Plasticity of subcortical pathways promote recovery of skilled hand function in rats after corticospinal and rubrospinal tract injuries. Exp Neurol 2015;266:112–119;
81. Asboth L, Friedli L, Beauparlant J, et al. Cortico–reticulo–spinal circuit reorganization enables functional recovery after severe spinal cord contusion. Nat Neurosci 2018;21(4):576–588;
82. Ueno M, Ueno-Nakamura Y, Niehaus J, et al. Silencing spinal interneurons inhibits immune suppressive autonomic reflexes caused by spinal cord injury. Nat Neurosci 2016;19(6):784–787;
83. Foscarin S, Raha-Chowdhury R, Fawcett JW, et al. Brain ageing changes proteoglycan sulfation, rendering perineuronal nets more inhibitory. Aging 2017;9(6):1607–1622;
84. Carulli D, Verhaagen J. An extracellular perspective on cns maturation: perineuronal nets and the control of plasticity. Int J Mol Sci 2021;22(5):2434;
85. Goussev S, Hsu J-YC, Lin Y, et al. Differential temporal expression of matrix metalloproteinases after spinal cord injury: relationship to revascularization and wound healing. J Neurosurg Spine 2003;99(2):188–197;
86. Carceller H, Gramuntell Y, Klimczak P, et al. Perineuronal nets: subtle structures with large implications. Neuroscientist 2022;107385842211063;
87. Gottschling C, Wegrzyn D, Denecke B, et al. Elimination of the four extracellular matrix molecules tenascin-C, tenascin-R, brevican and neurocan alters the ratio of excitatory and inhibitory synapses. Sci Rep 2019;9(1):13939;
88. John U, Patro N, Patro I. Perineuronal nets: Cruise from a honeycomb to the safety nets. Brain Res Bull 2022;190:179–194;
89. Sclip A, Südhof TC. LAR receptor phospho-tyrosine phosphatases regulate NMDA-receptor responses. Elife 2020;9:e53406;
90. Stoyanov S, Sun W, Düsedau HP, et al. Attenuation of the extracellular matrix restores microglial activity during the early stage of amyloidosis. Glia 2021;69(1):182–200;
91. Tsien RY. Very long-term memories may be stored in the pattern of holes in the perineuronal net. Proc Natl Acad Sci 2013;110(30):12456–12461;
92. Lev-Ram V, Lemieux SP, Deerinck TJ, et al. Do perineuronal nets stabilize the engram of a synaptic circuit? BioRxiv 2023.
93. Atwood HL. Silent synapses in neural plasticity: current evidence. Learn Mem 1999;6(6):542–571;
94. Goshgarian HG. Invited Review: The crossed phrenic phenomenon: a model for plasticity in the respiratory pathways following spinal cord injury. J Appl Physiol 2003;94(2):795–810;
95. Goshgarian HG. The crossed phrenic phenomenon and recovery of function following spinal cord injury. Respir Physiol Neurobiol 2009;169(2):85–93;
96. Lewis LJ, Brookhart JM. Significance of the crossed phrenic phenomenon. Am J Physiol 1951;166(2):241–254;
97. Urban MW, Ghosh B, Block CG, et al. Protein tyrosine phosphatase σ inhibitory peptide promotes recovery of diaphragm function and sprouting of bulbospinal respiratory axons after cervical spinal cord injury. J Neurotrauma 2020;37(3):572–579;
98. Cheng L, Sami A, Ghosh B, et al. LAR inhibitory peptide promotes recovery of diaphragm function and multiple forms of respiratory neural circuit plasticity after cervical spinal cord injury. Neurobiol Dis 2021;147:105153;
99. Martin JH. The corticospinal system: from development to motor control. Neuroscientist 2005;11(2):161–173;
100. Chen B, Li Y, Yu B, et al. Reactivation of dormant relay pathways in injured spinal cord by KCC2 manipulations. Cell 2018;174(6):1599;
101. Weng Y-L, An R, Cassin J, et al. An intrinsic epigenetic barrier for functional axon regeneration. Neuron 2017;94(2):337-346.e6;
102. Buttry JL, Goshgarian HG. Injection of WGA-Alexa 488 into the ipsilateral hemidiaphragm of acutely and chronically C2 hemisected rats reveals activity-dependent synaptic plasticity in the respiratory motor pathways. Exp Neurol 2014;261:440–450;
103. Liu JA, Tam KW, Chen YL, et al. Transplanting human neural stem cells with ≈50% reduction of SOX9 gene dosage promotes tissue repair and functional recovery from severe spinal cord injury. Adv Sci 2023;10(20):e2205804;
104. Luo F, Wang J, Zhang Z, et al. Inhibition of CSPG receptor PTPσ promotes migration of newly born neuroblasts, axonal sprouting, and recovery from stroke. Cell Rep 2022;40(4):111137;
105. Yao M, Fang J, Li J, et al. Modulation of the proteoglycan receptor PTPσ promotes white matter integrity and functional recovery after intracerebral hemorrhage stroke in mice. J Neuroinflammation 2022;19(1):207;
106. Um JW, Ko J. LAR-RPTPs: synaptic adhesion molecules that shape synapse development. Trends Cell Biol 2013;23(10):465–475;
107. Takahashi H, Craig AM. Protein tyrosine phosphatases PTPδ, PTPσ, and LAR: presynaptic hubs for synapse organization. Trends Neurosci 2013;36(9):522–534;
108. Kim HY, Um JW, Ko J. Proper synaptic adhesion signaling in the control of neural circuit architecture and brain function. Prog Neurobiol 2021;200:101983;
109. Woo J, Kwon S-K, Choi S, et al. Trans-synaptic adhesion between NGL-3 and LAR regulates the formation of excitatory synapses. Nat Neurosci 2009;12(4):428–437;
110. Yim YS, Kwon Y, Nam J, et al. Slitrks control excitatory and inhibitory synapse formation with LAR receptor protein tyrosine phosphatases. Proc Natl Acad Sci 2013;110(10):4057–4062;
111. Woo J, Kwon S-K, Kim E. The NGL family of leucine-rich repeat-containing synaptic adhesion molecules. Mol Cell Neurosci 2009;42(1):1–10;
112. Okuno Y, Sakoori K, Matsuyama K, et al. PTPδ is a presynaptic organizer for the formation and maintenance of climbing fiber to Purkinje cell synapses in the developing cerebellum. Front Mol Neurosci 2023;16;
113. Rink S, Arnold D, Wöhler A, et al. Recovery after spinal cord injury by modulation of the proteoglycan receptor PTPσ. Exp Neurol 2018;309:148–159;
114. Ham TR, Pukale DD, Hamrangsekachaee M, et al. Subcutaneous priming of protein-functionalized chitosan scaffolds improves function following spinal cord injury. Mater Sci Eng C Mater Biol Appl 2020;110:110656;
115. Ran N, Li W, Zhang R, et al. Autologous exosome facilitates load and target delivery of bioactive peptides to repair spinal cord injury. Bioact Mater 2022;25:766-782;
116. Sun X, Liu H, Tan Z, et al. Remodeling microenvironment for endogenous repair through precise modulation of chondroitin sulfate proteoglycans following spinal cord injury. Small 2023;19(6):2205012;
117. Gardner RT, Wang L, Lang BT, et al. Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac sympathetic innervation and prevents arrhythmias. Nat Commun 2015;6(1):6235;
118. Sepe JJ, Gardner RT, Blake MR, et al. Therapeutics that promote sympathetic reinnervation modulate the inflammatory response after myocardial infarction. JACC Basic Transl Sci 2022;7(9):915–930;
119. Li H, Wong C, Li W, et al. Enhanced regeneration and functional recovery after spinal root avulsion by manipulation of the proteoglycan receptor PTPσ. Sci Rep 2015;5:14923;
120. Lv S-Q, Wu W. ISP and PAP4 peptides promote motor functional recovery after peripheral nerve injury. Neural Regen Res 2021;16(8):1598;
121. Duncan JA, Foster R, Kwok JCF. The potential of memory enhancement through modulation of perineuronal nets. Br J Pharmacol 2019;176(18):3611–3621;
122. Nagy N, Kuipers HF, Frymoyer AR, et al. 4-methylumbelliferone treatment and hyaluronan inhibition as a therapeutic strategy in inflammation, autoimmunity, and cancer. Front Immunol 2015; 23;6:123;
123. Caon I, Bartolini B, Parnigoni A, et al. Revisiting the hallmarks of cancer: the role of hyaluronan. Semin Cancer Biol 2020;62:9–19;
124. Irvine SF, Gigout S, Štepánková K, et al. 4-methylumbelliferone enhances neuroplasticity in the central nervous system: potential oral treatment for SCI. bioRxiv 2023.
125. Štepánková K, Chudíčková M, Šimková Z, et al. Oral administration of 4-methylumbelliferone reduces glial scar and promotes anatomical plasticity. bioRxiv 2023.
126. Even-Ram S, Yamada KM. Cell migration in 3D matrix. Curr Opin Cell Biol 2005;17(5):524–532;
127. Zuo J, Ferguson TA, Hernandez YJ, et al. Neuronal matrix metalloproteinase-2 degrades and inactivates a neurite-inhibiting chondroitin sulfate proteoglycan. J Neurosci 1998;18(14):5203–5211;
128. Krystosek A, Seeds NW. Peripheral neurons and Schwann cells secrete plasminogen activator. J Cell Biol 1984;98(2):773–776;
129. Turk V, Stoka V, Vasiljeva O, et al. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta Proteins Proteom 2012;1824(1):68–88;
130. Ellis RC, O'Steen WA, Hayes RL, et al. Cellular localization and enzymatic activity of cathepsin B after spinal cord injury in the rat. Exp Neurol 2005;193(1):19–28;
131. Jacobs BL, Fornal CA. Activity of serotonergic neurons in behaving animals. Neuropsychopharmacology 1999;21(2):9S-15S;
132. Wei K, Glaser JI, Deng L, et al. Serotonin affects movement gain control in the spinal cord. J Neurosci 2014;34(38):12690–12700;
133. Sakai M, Matsunaga M, Kubota A, et al. Reduction in excessive muscle tone by selective depletion of serotonin in intercollicularly decerebrated rats. Brain Res 2000;860(1–2):104–111;
134. Weidner N, Ner A, Salimi N, et al. Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury. Proc Natl Acad Sci 2001;98(6):3513–3518;
135. Aoki M, Fujito Y, Satomi H, et al. The possible role of collateral sprouting in the functional restitution of corticospinal connections after spinal hemisection. Neurosci Res 1986;3(6):617–627;
136. Li WWY, Yew DTW, Chuah MI, et al. Axonal sprouting in the hemisected adult rat spinal cord. Neuroscience 1994;61(1):133–139;
137. Curcio M, Bradke F. Axon regeneration in the central nervous system: facing the challenges from the inside. Annu Rev Cell Dev Biol 2018;34(1):495–521;
138. Bareyre FM, Kerschensteiner M, Raineteau O, et al. The injured spinal cord spontaneously forms a new intraspinal circuit in adult rats. Nat Neurosci 2004;7(3):269–277;
139. Dyck S, Kataria H, Alizadeh A, et al. Perturbing chondroitin sulfate proteoglycan signaling through LAR and PTPσ receptors promotes a beneficial inflammatory response following spinal cord injury. J Neuroinflammation 2018;15(1):90;
Information & Authors
Information
Published In
Journal of Neurotrauma
Volume 40 • Issue Number 23-24 • December 2023
Pages: 2500 - 2521
PubMed: 37606910
Copyright
© Adrianna J. Milton et al. 2023; Published by Mary Ann Liebert, Inc.
Open Access
This Open Access article is distributed under the terms of the Creative Commons License (CC-BY) (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.
History
Published in print: December 2023
Published online: 30 November 2023
Published ahead of print: 11 October 2023
Published ahead of production: 22 August 2023
Authors
Authors' Contributions
Adrianna J. Milton was responsible for conceptualization, methodology, data curation, visualization, formal analysis, writing – original draft preparation, and review and editing. Jessica C.F. Kwok was responsible for conceptualization, methodology, writing – review and editing, supervision, resources, and funding acquisition. Jacob McClellan was responsible for data curation, software, visualization, and formal analysis. Sabre G. Randall was responsible for data curation, visualization, investigation, validation, and writing – reviewing and editing. Justin D. Lathia was responsible for visualization, investigation, writing – reviewing and editing, supervision, and project administration. Philippa M. Warren was responsible for conceptualization, methodology, resources, funding acquisition, writing – review and editing, supervision, and project administration. Daniel J. Silver was responsible for methodology, investigation, resources, funding acquisition, writing – original draft preparation, review and editing, visualization, and project administration. Jerry Silver was responsible for conceptualization, methodology, resources, writing – original draft preparation, review and editing, visualization, supervision, project administration, and funding acquisition.
Author Disclosure Statement
No competing financial interests exist.
Funding Information
Financial support was provided by Wings for Life, the Ohio Department of Higher Education Third Frontier Program, The Brumagin-Nelson Fund, The Griffin Family, The Timothy Brodigan Trust, The Kaneko Family Fund, and the NIH National Institute of Neurological Disorders and Stroke (NINDS) (grants 1R01NS101105 and 1R011NS113831). We gratefully acknowledge the support of the Government of Canada's New Frontiers in Research Fund (Mend the Gap), from grant NFRFT-2020-00238, the Czech Science Agency (grant 19-10365S to J.C.F.K.) and the Medical Research Council UK (project grant MR/S011110/1 to J.C.F.K. and P.M.W.).
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