Introduction
Traumatic spinal cord injury (SCI) is a frequent condition that significantly burdens societies (Ackery et al., 2004; Ahuja et al., 2017; Singh et al., 2014). SCI patients have numerous therapeutic challenges since neurologic recovery is limited despite rehabilitation.
There is a growing body of data and knowledge regarding SCI mechanisms and the resulting histopathologic effects. The secondary injury after SCI begins with the migration of inflammatory cells and marked inflammation at the injury site, resulting in cell toxicity and neuronal damage (Donnelly & Popovich, 2008; Hausmann, 2003). Myelin is one of the key inhibitory factors following SCI. Unlike Schwann cells in the peripheral nervous system, spinal cord oligodendrocytes do not clear injured axons or myelin debris following SCI. Also, oligodendrocytes do not recruit macrophages or microglia to assist in this process (Lemons et al., 1999).
In the subacute phase of secondary injury following SCI, astrocytes become reactive and produce intermediate filaments through a process called astrogliosis, leading to the formation of a glial or perilesional scar around the injury site. This perilesional scar has tremendous effects in restoring the blood-brain barrier, supporting wound contraction, minimizing leukocyte infiltration, and limiting neuronal damage and demyelination (Faulkner et al., 2004). However, it inhibits later neuronal plasticity, axonal regeneration, and sprouting (Fitch & Silver, 1997; Herrmann et al., 2008; Karimi-Abdolrezaee & Billakanti, 2012). Expression of chondroitin sulfate proteoglycans (CSPG) is one of the major parts of glial scar formation (Jones et al., 2002; Morgenstern et al., 2002). Thus, glial scar removal or clearance by targeting CSPGs in the perilesional scar may create an environment conducive to spinal cord regeneration. 
Chondroitinase ABC (ChABC), a bacterial endolyase, removes glycosaminoglycan chains from CSPG, which causes CSPG proteolysis after its formation (Fawcett, 2015; Wang et al., 2008). ChABC-targeted proteolysis is safer than enzyme therapy, which has been used previously (Guth et al., 1980). Enzyme therapies (including trypsin, hyaluronidase, elastase, elastase plus trypsin, or vehicle) can dissolve blood vessel walls, causing hemorrhage (Guth et al., 1980). Therefore, ChABC is a promising alternative therapy for promoting axonal plasticity in SCI, not only in the acute phase by preventing glial scar formation, but also in the chronic SCI phase through the digestion of the glial scar and promoting regeneration and functional recovery (Filous et al., 2010; Houle et al., 2006). Bradbury et al. (2002) showed, for the first time, the beneficial effects of ChABC on axonal regeneration and functional improvement after SCI. Despite the vast number of preclinical studies in this area (Filous et al., 2010; Houle et al., 2006; Massey et al., 2008), the exact mechanism of action is not known. However, much of the mechanism has been identified. Here, we systematically review the literature to summarize all potential histopathological effects of intrathecal administration of ChABC in spinal cord injured rats.
Materials and Methods
Our systematic review was conducted according to the PRISMA (preferred reporting items for systematic reviews and meta-analysis) 2020 checklist (Page et al., 2021).
Information sources and search strategy
We performed a comprehensive electronic search of studies published in PubMed, Scopus, Web of Science, Embase, and the Cochrane Library until November 22, 2021. We did not limit the search strategy by study type, language, or publication date. 

Eligibility criteria, selection process, and data extraction
Two independent reviewers performed the screening, and the third reviewer resolved any discrepancies. The inclusion criteria were experimental animal studies on SCI models in adult rats that investigated intrathecal ChABC treatment. To explore the precise histopathologic effects of ChABC on the SCI recovery process, we limited our inclusion criteria to intrathecal administration of ChABC. We included a study if it had at least 2 intervention groups: 1 intrathecal ChABC treatment group and 1 control group (i.e. sham surgery or no treatment), and the study reported histopathological outcomes. According to the group’s neurosurgeons’ opinions, we preliminarily defined 5 histopathologic outcome groups as the most clinically relevant outcomes of interest, including: 
1. Lesion breadth, also known as cavity volume, cyst size, myelin to cavity size ratio, or atrophic area.
2. Serotonergic (5HT) neurons’ plasticity, defined as the total number of 5HT fibers crossing the lesion or the 5HT fibers’ side branches in the lesion site.
3. Corticospinal tract (CST) plasticity, defined as the total number of CST fibers in the lesion site, CST fibers crossing the lesion site, or CST fibers’ side branches in the lesion site.
4. Sensory neurons’ plasticity, defined as the total number of sensory fibers in the lesion site, the number of sensory fibers crossing the lesion, or the sensory fibers’ side branches in the lesion site.
5. Electrophysiological outcomes, defined by postsynaptic cord dorsum potentials (CDPs), the amplitude of action potential volleys, and neuronal conduction latency. 
Studies included review articles, case reports, and case series. However, conference abstracts and posters without a published paper in a peer-reviewed journal were excluded. Also, we manually searched the references of the reviewed articles to identify any additional related articles.
Two independent reviewers performed the data extraction into the forms, and in the event of any discrepancy, a SCI specialist was consulted.


Risk of bias assessment
The risk of bias of the included studies was evaluated using the quality assessment tool for SCI animal models (Hassannejad et al., 2016) to assess pathophysiological events following traumatic SCI experiments. This assessment tool evaluates the studies regarding 15 domains: animal species, applying suitable tests, severity of the SCI, level of SCI, age/weight, experimental group sizes, description of strain, description of control groups, statistical analysis details, regulation of ethical issues, bladder expression, the blindness of measurements, genetic background, method of random allocation to experimental groups, and details of animal exclusions during the study.
If each column had no risk of bias, it was scored as positive “+,” and if the presence of risk of bias was unclear due to insufficient descriptions in the article, it was scored as “–,” and high risk of bias in each column was scored as negative. Differences in assessment were discussed during a consensus meeting. A total score was computed by adding the number of positive scores, and high quality was defined as fulfilling 8 or more (more than 50%) of the 15 internal validity criteria. Finally, the risk of bias for each included article was assessed using the data extraction form. 
Results 
A total of 3857 records were retrieved during database and bibliography searches, of which 2144 were unique after duplicates were removed. After screening by title and abstract, 1957 articles were excluded, and 187 papers were selected for full-text assessment. Of the 187 articles, 156 were excluded due to lack of intervention criteria (n=63), lack of a control group (n=1), conference abstracts without a peer-reviewed paper (n=38), review articles (n=2), and lack of histopathological outcome measures of interest (n=52). Seventeen studies (Barritt et al., 2006; Caggiano et al., 2005; Führmann et al., 2018; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Massey et al., 2008; Mountney et al., 2013; Pan et al., 2018; Shields et al., 2008; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009) were included in the qualitative analysis based on the following PICO criteria: (Figure 1) P, adult rats; I, intrathecal ChABC administration; C, sham surgery or non-treated injured animals; O, as entered in the method section. All included studies had a low risk of bias except one, which had a moderate risk of bias (Table 1).
All studies used adult rats, of which 11 studies used the Sprague Dawley (Führmann et al., 2018; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Massey et al., 2008; Mountney et al., 2013; Shields et al., 2008; Shinozaki et al., 2016; Tom et al., 2009; Xia et al., 2015; Yang et al., 2009), 3 the Wistar (Barritt et al., 2006; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018), 2 the Lister hooded (García-Alías et al., 2008; Wang et al., 2011), and 1 the Long-Evans rats (Caggiano et al., 2005). Although all studies stated the size or number of the animals treated with ChABC and controls, only 7 mentioned the total number of animals sampled (a total of 440 rats) (Barritt et al., 2006; Führmann et al., 2018; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018; Shields et al., 2008; Shinozaki et al., 2016). The most common injury model in these articles was transection injury (García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Massey et al., 2008; Pan et al., 2018; Shields et al., 2008; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015). Compression injury (Caggiano et al., 2005; Führmann et al., 2018; Karimi-Abdolrezaee et al., 2010) and contusion injury (Barritt et al., 2006; Mountney et al., 2013; Shinozaki et al., 2016; Tom et al., 2009; Yang et al., 2009) were other injury methods. One study deployed both transection and contusion models (Tom et al., 2009). The size, level, and severity of the injury, as well as the injection time and whether unilateral or bilateral injury were used (Table 2).
The results, based on the phase of treatment, methodological details, and key findings of the included studies, are presented in Table 3. The treatment phase was defined as the time of ChABC administration (the first dose in multiple-dose regimens), and the studies were grouped as acute (within 24 hours after injury), subacute (5 or 7 days after injury), or chronic (4 or 6 weeks after injury). Four studies reported results of ChABC treatment on SCI lesion breadth (Caggiano et al., 2005; Führmann et al., 2018; Pan et al., 2018; Shinozaki et al., 2016). Eight studies evaluated the effects of ChABC treatment on serotonergic fiber plasticity and regeneration after SCI through anti-serotonin (5-hydroxytryptamine [5-HT]) immunostaining (Barritt et al., 2006; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Mountney et al., 2013; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011). CST remodeling after ChABC treatment was assessed in 12 studies (Barritt et al., 2006; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Massey et al., 2008; Shields et al., 2008; Shinozaki et al., 2016; Wang et al., 2014; Xia et al., 2015; Yang et al., 2009). Of the 17 studies, 8 used biotinylated dextran amine (BDA)-labeling for axonal tracing (Barritt et al., 2006; García-Alías et al., 2008; García-Alías et al., 2011; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009), one used WGA labeling (Shinozaki et al., 2016), and another used protein kinase C-ƴ (Ishikawa et al., 2015). Two high-quality studies used cholera toxin B-subunit (CTB) labeling in the acute SCI model to assess the potential effects of ChABC treatment on sensory neurons (Massey et al., 2008; Shields et al., 2008). The other study used calcitonin gene-related peptide (CGRP) immunohistochemistry (Barritt et al., 2006). Again, 2 other high-quality studies reported electrophysiological outcome measures of interest (García-Alías et al., 2011; Yang et al., 2009).

Acute phase 
Lesion breadth
One study used transection injury models (Pan et al., 2018), and two used compression (Caggiano et al., 2005; Führmann et al., 2018) to report the effects of acute ChABC administration on lesion breadth. In transection and contusion models, ChABC administration significantly decreased lesion length, cyst length at the injury site, hole size, and atrophic area volume (Pan et al., 2018; Shinozaki et al., 2016). However, acute administration of ChABC did not change the lesion length in the compression injury model. 

Plasticity of serotonergic neurons
Two studies used transection injury models (Ishikawa et al., 2015; Kim et al., 2006), two studies used contusion (Barritt et al., 2006; Mountney et al., 2013), and one study used both transection and contusion models (Tom et al., 2009) to assess the effects of acute ChABC administration on serotonergic neuron plasticity. In transection models, ChABC increased the number of serotonergic fibers at the injury site and rostral part of the injury, but it had a weak effect on reaching the caudal sites. In contusion injury models, treatment positively affected the ventral caudal horn in contrast to the caudal dorsal horn, where it did not change.

Plasticity of corticospinal neurons 
Four studies used transection injury models (García-Alías et al., 2008; Kim et al., 2006; Wang et al., 2014; Xia et al., 2015), and 2 used contusion (Barritt et al., 2006; Yang et al., 2009) to assess changes in CST neuron plasticity in response to acute ChABC administration. In transection models, treatment increased the number of CST fibers and their rostral length, but results were inconsistent at the injury site and in the caudal regions. Axonal sprouting was also increased at the injury site. One of two contusion studies reported increased CST fiber growth into and beyond the injury site, as well as increased terminal arborization (Barritt et al., 2006). In contrast, the other study (Yang et al., 2009) reported no difference in fiber length or number. The only compression study reported increased fiber growth and sprouting at the injury site and in the caudal regions (Führmann et al., 2018). To improve conduction, more studies on contusion and compression models are warranted.

Plasticity of sensory neurons
One study used transection injury models (Shields et al., 2008), and another used both compression and contusion models (Barritt et al., 2006). Both studies observed that acute administration of ChABC promoted afferent fiber plasticity and growth into and beyond the injury site. However, none of the included studies evaluated electrophysiological outcomes during the acute phase. 

Subacute phase
Lesion breadth
Two studies that used compressive injury models (Caggiano et al., 2005; Führmann et al., 2018) reported effects of ChABC subacute administration on lesion breadth, showing weak positive effects. Concerning the other outcome of interest, we found no studies reporting on the evidence for the effect of ChABC administration on serotonergic, sensory, and corticospinal neuronal plasticity. 

Chronic phase
Lesion breadth
Only 1 contusion injury study (Shinozaki et al., 2016) assessed lesion breadth changes in response to chronic administration of ChABC and found no influence of its treatment on lesion breadth changes. However, two studies reported increased tissue or axonal preservation with this treatment, either treadmill rehabilitation (Shinozaki et al., 2016) or neural progenitor cell transplantation (Karimi-Abdolrezaee et al., 2010). 

Serotonergic neurons’ plasticity
Chronic administration of Ch-ABC significantly increased the number and length of serotonergic fibers and sprouting at the rostral epicenter and caudal side of the lesion site (Karimi-Abdolrezaee et al., 2010; Shinozaki et al., 2016).

Corticospinal neuron plasticity

Chronic administration of ChABC significantly increased CST axon crossing and sprouting and rostral regrowth length (Karimi-Abdolrezaee & Billakanti, 2012; Karimi-Abdolrezaee et al., 2010; Shinozaki et al., 2016), but in caudal regions, there was no consensus on treatment effects. Two of the included studies (Karimi-Abdolrezaee & Billakanti, 2012; Shinozaki et al., 2016) reported no effects in the caudal regions. Overall, none of the included studies evaluated electrophysiological outcomes during the chronic phase. 

Functional outcome
Most studies used the Basso, Beattie, Bresnahan (BBB) scale (for hindlimb), ladder walk test/staircase reaching test, grid-walking/strength test, hindlimb contact placing response/vertical exploration (rearing) and sticker removal tests (for forelimb function), single pellet reaching task, fine touch and mechanical hyperalgesia assessment, spinal cord evoked potential and motor evoked potential assessment to evaluate motor and sensory functional outcomes (Caggiano et al., 2005; Führmann et al., 2018; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Mountney et al., 2013; Pan et al., 2018; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009). Also, one study used residual urine volumes to test for autonomic functions (Caggiano et al., 2005). Overall, the functional outcome were mixed, with 3 studies showing no improvement in functional outcomes after ChABC administrations (Führmann et al., 2018; Mountney et al., 2013; Tom et al., 2009) and 9 showing that ChABC administration alone (García-Alías et al., 2008) or combined with neurotrophin NT-3 secretion and NR2D expression (García-Alías et al., 2011) or NPCs (Karimi-Abdolrezaee et al., 2010), or antisense vimentin cDNA (Xia et al., 2015) or poly(glycerol sebacate) (Pan et al., 2018) or insulin+ methylprednisolone (Yang et al., 2009) or transplant mediated axonal remodeling or rehabilitation (Ishikawa et al., 2015; Shinozaki et al., 2016; Wang et al., 2011) improved functional outcomes, particularly motor functions (Table 3). 
Discussion
SCI is a devastating clinical condition that results in rapid-onset and long-term disability related to the central nervous system. Several underlying mechanisms have been identified as factors responsible for primary and secondary damage following SCI. Primary mechanisms include neural death and axonal injury, which subsequently lead to sensorimotor disruption (de Almeida et al., 2023). After the primary complications, secondary mechanisms are initiated, including inflammation, vascular changes, ion disproportion, glutamate excitotoxicity, and radical formation, which result in additional complications, including progressive neural death, edema, hyperpyrexia, and paralysis (de Almeida et al., 2023; Korovessis, 2019). Inflammation and granulocyte colony-stimulating factor (G-CSF) production are two major components of glial scar formation that hamper recovery after SCI (Shechter et al., 2011). A correlation between inflammation and G-CSF expression via inflammatory cytokines has been reported in the literature (Shechter et al., 2011). The evidence suggests that inflammatory cytokines enhance GSK3β expression, which, in turn, promotes demyelination and neuronal degeneration (Nagai et al., 2016; Renault-Mihara et al., 2011). Furthermore, GSK3β may prevent post-SCI neuronal regeneration by modulating G-SCF discharge (Nagai et al., 2016). Therefore, therapeutic agents are designed to disrupt the molecular mechanisms underlying complications related to SCI. 
Although several therapeutic approaches, including pharmacological, stem cell-based, and enzyme-based approaches, have been developed for SCI, the management of patients with SCI remains challenging in developing or even developed countries. The difficulty in treating SCI patients is related to environmental factors in the injured area (de Almeida et al., 2023). However, advances in understanding SCI pathology have led to de novo research into new treatment approaches. Recent studies have focused on the secondary mechanisms involved in SCI to prevent further damage and facilitate neural regeneration. Kwon et al. (2011a) systematically reviewed the pre-clinical studies of neuroprotective agents already prescribed in humans. This study documented the potential effect of non-invasive medications for acute SCI, including erythropoietin, progesterone, estrogen, riluzole, polyethylene glycol, atorvastatin, magnesium, minocycline, inosine, NSAID, anti-CD11, and pioglitazone. In 2010, Cadotte and Fehlings, highlighted the effects of riluzole, anti-Rho antibody, and surgical decompression on SCI, thereby establishing evidence for translating preclinical research into clinical research (Cadotte & Fehlings, 2011). 
In 2011, another systematic review by Kwon et al. (2011b) aimed to investigate in vivo studies on the efficacy of intraspinal ChABC, anti-Nogo antibody, and anti-Rho antibody strategies, providing evidence for translating preclinical findings into human studies and clinical trials. They found that the Nogo receptor on neural cells prevents neural growth, suggesting that an anti-Nogo antibody is a promising therapeutic approach to promote neural recovery following SCI. Also, clinical trials have been conducted to investigate the effects of anti-Nogo in patients with acute and chronic SCI. Additionally, although riluzole is a sodium glutamate antagonist used in patients with amyotrophic lateral sclerosis (ALS), it is a potential drug for SCI patients due to its neuroprotective effect. A recent systematic review of the efficacy of riluzole in SCI, including animal and human studies, showed that riluzole is also a promising pharmacological agent that improves behavioral outcomes, promotes histological sparing, and exhibits electrophysiological effects following SCI, while diminishing the sequelae of this condition (Srinivas et al., 2019). 
Several key components, such as inflammation, oxidative stress, and edema, have been considered in the pathophysiology of SCI and can inform the development of effective therapeutic modalities. For instance, oxidative stress is putatively involved in the secondary deterioration of SCI. Therefore, depletion of reactive oxidative stress could be an effective approach to prevent SCI-associated secondary deterioration in patients. Medications, including the glucocorticoid steroid and the non-glucocorticoid 21-aminosteroid tirilazad, also have antioxidant activity that significantly enhances recovery after SCI (Jia et al., 2012). Likewise, edema is another essential component of the pathophysiology of SCI, which is initiated rapidly within minutes following injury (Rowland et al., 2008). Leonard et al. (2015) observed that deterioration of edema-induced injury was associated with more severe complications. Therefore, a systematic review of treatments targeting edema in SCI identified 3 main approaches to eliminate edema: inhibition of aquaporin 4 (AQP4), immunosuppression, and surgery. Furthermore, trifluoperazine, which prevents AQP4 localization, was proposed to be the most effective treatment, significantly reducing edema within a week after injection (Masterman & Ahmed, 2021). 
Lemons et al.’s (1999) investigation is the first to report an increase in CSPGs at the injury site and in adjacent areas after SCI. Moreover, they showed that astrocytes are a source of CSPG, leading to a lack of neural regeneration. Besides, they suggested that exogenous ChABC administration could degrade CSPG, which indicates the role of CSPGs as inhibitory outgrowth factors. These findings also unveil a potential therapeutic effect of ChABC. Further studies have documented that local injection of ChABC is associated with protein-based neuronal regeneration (Barritt et al., 2006; Ishikawa et al., 2015). Moreover, Lee et al. (2010) found that ChABC administration affected CSPG and inflammation, reducing CSPG and GSK3β levels and accelerating neural growth (Yılmaz & Kaptanoğlu, 2015). 
 A systematic review and meta-analysis conducted by Yousefifard et al. (2022) demonstrated that ChABC has a moderate effect on locomotor function in animal models of SCI, with no differences in injury severity. They also revealed that the induction model of SCI and the number of ChABC injections did not influence the efficacy of ChABC on locomotor function in SCI. In mouse models, Carter et al. (2008) showed that ChABC was neuroprotective for cortical layer V projection neurons after ICV infusion. ChABC also prevented cell atrophy after localized delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. 
Additional studies designed based on the combination of other treatment options like stem cells (Jevans et al., 2021), tissue engineering approaches (Raspa et al., 2021), sucrose (Raspa et al., 2019), photobiomodulation therapy (Janzadeh et al., 2020) antisense vimentin cDNA (Xia et al., 2015), poly(glycerol sebacate) (Pan et al., 2018), insulin + methylprednisolone (Yang et al., 2009) and rehabilitation (Ishikawa et al., 2015; Shinozaki et al., 2016; Wang et al., 2011) with ChABC showed improvement of the efficiency of Ch-ABC in SCI and improved functional outcomes. Additionally, a combination of ChABC and delayed injection of adeno-associated virus encoding the L1 cell adhesion molecule or pluripotent stem cell-derived NSCs in mouse models showed enhanced locomotor recovery after treatment (Lee et al., 2012; Suzuki et al., 2017). Furthermore, multiple injections of ChABC in Rhesus monkeys were associated with increased corticospinal axon growth, increased synapse formation by corticospinal terminals in the gray matter caudal to the lesion, and improved hand function (Rosenzweig et al., 2019). In this review, the functional outcomes assessed included motor, sensory, and autonomic functions, including forelimb and hindlimb function, gait, pellet-reaching task, fine-touch and mechanical hyperalgesia assessment, and residual urine volume assessment. Although the results were heterogeneous, most studies showed that ChABC administration improved functional outcomes in animal models.
This systematic review entails the histological aspects of the efficacy of intrathecal ChABC in SCI rats. Our comprehensive search identified 17 eligible studies assessing histopathological outcomes of intrathecal ChABC administration following SCI in rats. When analyzing treatment results by treatment phase, our results showed that ChABC treatment in the acute phase reduced necrosis and atrophic areas, increased tissue preservation, and increased sensory neuron plasticity and growth into and beyond the injury site. Moreover, ChABC increased the number of serotonergic fibers (5-HT), reduced neuronal apoptosis, increased the digestion of CSPG, promoted the differentiation of stem cells into neurons, and increased GAP-43, NG-2, chondroitin 4 sulfate, and BDA levels. 
Regardless of the treatment phase, ChABC promoted the survival of serotonergic fibers, plasticity, and regrowth beyond the injury site, as well as rostral global plasticity and the survival of CST fibers. To the best of our knowledge, this is the first systematic review to evaluate the histopathological effects of intrathecal ChABC treatment. To make the results of the ChABC treatment more tangible, we compared them with studies using combined ChABC treatment. In a systematic review by Kwon et al. (2011b), the putative mechanisms underlying intraspinal ChABC injection for acute SCI were assessed. In this regard, axonal germination/growth of fibers, specifically serotonergic fibers, and the neuroprotective effect of intra-spinal ChABC, preventing neural atrophy, were the most promising mechanisms. Our results indicated that CST and serotonergic fibers sprouting, terminal arborization, and crossing were significantly increased after ChABC treatment, similar to the results of ChABC treatment in combination with rehabilitation (Marsh et al., 2011), Nogo-A inhibitors (Zhao et al., 2013), and increased neurotrophin-3 levels (Massey et al., 2008). We also found that ascending fiber regeneration was significantly promoted after intrathecal ChABC administration. This finding was reproduced in 3 combinational treatment studies using neurotrophin-3, cell plant (Massey et al., 2008), and conditioning agents such as zymosan (Harel et al., 2012; Steinmetz et al., 2005). Grimpe et al. (2005) and Vavrek et al. (2007) reported that a combination of cell transplant and ChABC increased CST axon regeneration rostrally and promoted their elongation through the injury site and to the caudal sites. The articles we included in this area did not yield consistent results. While another study (García-Alías et al., 2008) reported increased regrowth length of CST axons into and beyond the injury site, the results of four other studies (Kim et al., 2006; Shinozaki et al., 2016; Wang et al., 2014; Yang et al., 2009) showed no significant effects on regrowth to caudal sites for ChABC treatment. It seems that when a substantial atrophic area is present after injury, a graft is necessary to provide a tissue scaffold for regenerating axons to traverse the injury site. Although it is well known that ChABC improves axonal sprouting and neural function by degrading CSPGs, the exact molecular mechanism underlying this process remains unclear. However, Hu et al. (2021) explored the key role of CSPGs in axon regeneration and neural apoptosis. They showed that the caspase activity is significantly increased within 2 to 11 weeks after injury. Importantly, ChABC reduces the total amount of functional caspase-3, validating the anti-apoptotic activity reported by Kwon et al. (2011b). Protein tyrosine phosphatase sigma is an example of a CSPG receptor involved in SCI-induced retrograde neural apoptosis. Correspondingly, the expression of PTPs, which was associated with neural apoptosis, decreased following ChABC treatment, demonstrating ChABC’s anti-apoptotic activity in SCI. Regarding functional outcomes, we found that ChABC ­­ssociated with better motor functions in animal models with SCI (García-Alías et al., 2011; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018; Xia et al., 2015; Yang et al., 2009).


Conclusion
In summary, our systematic review of the available evidence for finding the histopathological effects of intrathecal ChABC administration in spinal cord injured rats suggests that this treatment can reduce necrosis and atrophic area and increase tissue preservation, increase sensory neuron plasticity and growth into and beyond the injury site, reduce the apoptosis of neurons, promotes the survival of serotonergic fibers, plasticity and regrowth to and beyond the injury site, the global plasticity and survival rostrally of CST fibers, increase digestion of the CSPG, differentiation of stem cells to neurons and the GAP-43, NG-2, chondroitin 4 sulfate, BDA level. ChABC treatment was associated with improved functional outcome in rats, mice, and primates. Although these findings are promising, further studies can provide additional evidence to support a final assessment of the risks and benefits of ChABC in SCI.
Strengths and limitations
As this is the first systematic review on the histopathological effects of ChABC treatment in SCI, results can be used to better understand the mechanisms underlying positive functional outcomes, understand the possible adverse effects to look for, and discover the pitfalls in studies that require more attention in future work. The most important limitation of this study is the small sample size in animal studies, which may both increase the risk of selection bias and render most effects non-significant. We grouped studies by intervention phase and injury model, and in some groups, there was only one study available, leaving very little evidence to deduct points. Furthermore, because studies using different enzyme administration regimens are limited, we did not summarize results by regimen. This limitation can affect the results and may be a source of the non-convergence.
However, clinical studies have higher priority and can more directly benefit individuals with SCI, whereas preclinical studies provide the fundamental basis for clinically approved treatments. Our study emphasizes the indispensable role of animal studies in advancing treatments for SCI and provides insight for bridging preclinical studies to patient care. This study also provided a comprehensive review of the histopathological effects of ChABC on rat SCI models. To gain a clearer understanding of ChABC’s roles in SCI, we classified the reviewed studies by SCI phase, which is of high importance in this context.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran (Code: IR.TUMS.MEDICINE.REC.1398.886).

Data availability
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Declaration of generative AI and AI-assisted technologies in the writing process
No AI tool influenced the scientific content, data analysis, or conclusions of this work.

Funding
This study was extracted from the general medical doctorate thesis of Ali Anjomsho, approved by the Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. This work was funded by Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran (Grant No.: 97-02-38-40904). 

Authors' contributions
All authors contributed equally to the conception and design of the study, data collection and analysis, interception of the results and drafting of the manuscript. Each author approved the final version of the manuscript for submission.

Conflict of interest
The authors declared no conflict of interest.Introduction
raumatic spinal cord injury (SCI) is a frequent condition that significantly burdens societies (Ackery et al., 2004; Ahuja et al., 2017; Singh et al., 2014). SCI patients have numerous therapeutic challenges since neurologic recovery is limited despite rehabilitation.
There is a growing body of data and knowledge regarding SCI mechanisms and the resulting histopathologic effects. The secondary injury after SCI begins with the migration of inflammatory cells and marked inflammation at the injury site, resulting in cell toxicity and neuronal damage (Donnelly & Popovich, 2008; Hausmann, 2003). Myelin is one of the key inhibitory factors following SCI. Unlike Schwann cells in the peripheral nervous system, spinal cord oligodendrocytes do not clear injured axons or myelin debris following SCI. Also, oligodendrocytes do not recruit macrophages or microglia to assist in this process (Lemons et al., 1999).
In the subacute phase of secondary injury following SCI, astrocytes become reactive and produce intermediate filaments through a process called astrogliosis, leading to the formation of a glial or perilesional scar around the injury site. This perilesional scar has tremendous effects in restoring the blood-brain barrier, supporting wound contraction, minimizing leukocyte infiltration, and limiting neuronal damage and demyelination (Faulkner et al., 2004). However, it inhibits later neuronal plasticity, axonal regeneration, and sprouting (Fitch & Silver, 1997; Herrmann et al., 2008; Karimi-Abdolrezaee & Billakanti, 2012). Expression of chondroitin sulfate proteoglycans (CSPG) is one of the major parts of glial scar formation (Jones et al., 2002; Morgenstern et al., 2002). Thus, glial scar removal or clearance by targeting CSPGs in the perilesional scar may create an environment conducive to spinal cord regeneration. 
Chondroitinase ABC (ChABC), a bacterial endolyase, removes glycosaminoglycan chains from CSPG, which causes CSPG proteolysis after its formation (Fawcett, 2015; Wang et al., 2008). ChABC-targeted proteolysis is safer than enzyme therapy, which has been used previously (Guth et al., 1980). Enzyme therapies (including trypsin, hyaluronidase, elastase, elastase plus trypsin, or vehicle) can dissolve blood vessel walls, causing hemorrhage (Guth et al., 1980). Therefore, ChABC is a promising alternative therapy for promoting axonal plasticity in SCI, not only in the acute phase by preventing glial scar formation, but also in the chronic SCI phase through the digestion of the glial scar and promoting regeneration and functional recovery (Filous et al., 2010; Houle et al., 2006). Bradbury et al. (2002) showed, for the first time, the beneficial effects of ChABC on axonal regeneration and functional improvement after SCI. Despite the vast number of preclinical studies in this area (Filous et al., 2010; Houle et al., 2006; Massey et al., 2008), the exact mechanism of action is not known. However, much of the mechanism has been identified. Here, we systematically review the literature to summarize all potential histopathological effects of intrathecal administration of ChABC in spinal cord injured rats.

Materials and Methods
Our systematic review was conducted according to the PRISMA (preferred reporting items for systematic reviews and meta-analysis) 2020 checklist (Page et al., 2021).

Information sources and search strategy
We performed a comprehensive electronic search of studies published in PubMed, Scopus, Web of Science, Embase, and the Cochrane Library until November 22, 2021. We did not limit the search strategy by study type, language, or publication date. 

Eligibility criteria, selection process, and data extraction
Two independent reviewers performed the screening, and the third reviewer resolved any discrepancies. The inclusion criteria were experimental animal studies on SCI models in adult rats that investigated intrathecal ChABC treatment. To explore the precise histopathologic effects of ChABC on the SCI recovery process, we limited our inclusion criteria to intrathecal administration of ChABC. We included a study if it had at least 2 intervention groups: 1 intrathecal ChABC treatment group and 1 control group (i.e. sham surgery or no treatment), and the study reported histopathological outcomes. According to the group’s neurosurgeons’ opinions, we preliminarily defined 5 histopathologic outcome groups as the most clinically relevant outcomes of interest, including: 
1. Lesion breadth, also known as cavity volume, cyst size, myelin to cavity size ratio, or atrophic area.
2. Serotonergic (5HT) neurons’ plasticity, defined as the total number of 5HT fibers crossing the lesion or the 5HT fibers’ side branches in the lesion site.
3. Corticospinal tract (CST) plasticity, defined as the total number of CST fibers in the lesion site, CST fibers crossing the lesion site, or CST fibers’ side branches in the lesion site.
4. Sensory neurons’ plasticity, defined as the total number of sensory fibers in the lesion site, the number of sensory fibers crossing the lesion, or the sensory fibers’ side branches in the lesion site.
5. Electrophysiological outcomes, defined by postsynaptic cord dorsum potentials (CDPs), the amplitude of action potential volleys, and neuronal conduction latency. 
Studies included review articles, case reports, and case series. However, conference abstracts and posters without a published paper in a peer-reviewed journal were excluded. Also, we manually searched the references of the reviewed articles to identify any additional related articles.
Two independent reviewers performed the data extraction into the forms, and in the event of any discrepancy, a SCI specialist was consulted.


Risk of bias assessment
The risk of bias of the included studies was evaluated using the quality assessment tool for SCI animal models (Hassannejad et al., 2016) to assess pathophysiological events following traumatic SCI experiments. This assessment tool evaluates the studies regarding 15 domains: animal species, applying suitable tests, severity of the SCI, level of SCI, age/weight, experimental group sizes, description of strain, description of control groups, statistical analysis details, regulation of ethical issues, bladder expression, the blindness of measurements, genetic background, method of random allocation to experimental groups, and details of animal exclusions during the study.
If each column had no risk of bias, it was scored as positive “+,” and if the presence of risk of bias was unclear due to insufficient descriptions in the article, it was scored as “–,” and high risk of bias in each column was scored as negative. Differences in assessment were discussed during a consensus meeting. A total score was computed by adding the number of positive scores, and high quality was defined as fulfilling 8 or more (more than 50%) of the 15 internal validity criteria. Finally, the risk of bias for each included article was assessed using the data extraction form. 

Results 
A total of 3857 records were retrieved during database and bibliography searches, of which 2144 were unique after duplicates were removed. After screening by title and abstract, 1957 articles were excluded, and 187 papers were selected for full-text assessment. Of the 187 articles, 156 were excluded due to lack of intervention criteria (n=63), lack of a control group (n=1), conference abstracts without a peer-reviewed paper (n=38), review articles (n=2), and lack of histopathological outcome measures of interest (n=52). Seventeen studies (Barritt et al., 2006; Caggiano et al., 2005; Führmann et al., 2018; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Massey et al., 2008; Mountney et al., 2013; Pan et al., 2018; Shields et al., 2008; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009) were included in the qualitative analysis based on the following PICO criteria: (Figure 1) P, adult rats; I, intrathecal ChABC administration; C, sham surgery or non-treated injured animals; O, as entered in the method section. All included studies had a low risk of bias except one, which had a moderate risk of bias (Table 1).
All studies used adult rats, of which 11 studies used the Sprague Dawley (Führmann et al., 2018; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Massey et al., 2008; Mountney et al., 2013; Shields et al., 2008; Shinozaki et al., 2016; Tom et al., 2009; Xia et al., 2015; Yang et al., 2009), 3 the Wistar (Barritt et al., 2006; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018), 2 the Lister hooded (García-Alías et al., 2008; Wang et al., 2011), and 1 the Long-Evans rats (Caggiano et al., 2005). Although all studies stated the size or number of the animals treated with ChABC and controls, only 7 mentioned the total number of animals sampled (a total of 440 rats) (Barritt et al., 2006; Führmann et al., 2018; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018; Shields et al., 2008; Shinozaki et al., 2016). The most common injury model in these articles was transection injury (García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Massey et al., 2008; Pan et al., 2018; Shields et al., 2008; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015). Compression injury (Caggiano et al., 2005; Führmann et al., 2018; Karimi-Abdolrezaee et al., 2010) and contusion injury (Barritt et al., 2006; Mountney et al., 2013; Shinozaki et al., 2016; Tom et al., 2009; Yang et al., 2009) were other injury methods. One study deployed both transection and contusion models (Tom et al., 2009). The size, level, and severity of the injury, as well as the injection time and whether unilateral or bilateral injury were used (Table 2).
The results, based on the phase of treatment, methodological details, and key findings of the included studies, are presented in Table 3. The treatment phase was defined as the time of ChABC administration (the first dose in multiple-dose regimens), and the studies were grouped as acute (within 24 hours after injury), subacute (5 or 7 days after injury), or chronic (4 or 6 weeks after injury). Four studies reported results of ChABC treatment on SCI lesion breadth (Caggiano et al., 2005; Führmann et al., 2018; Pan et al., 2018; Shinozaki et al., 2016). Eight studies evaluated the effects of ChABC treatment on serotonergic fiber plasticity and regeneration after SCI through anti-serotonin (5-hydroxytryptamine [5-HT]) immunostaining (Barritt et al., 2006; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Mountney et al., 2013; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011). CST remodeling after ChABC treatment was assessed in 12 studies (Barritt et al., 2006; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Massey et al., 2008; Shields et al., 2008; Shinozaki et al., 2016; Wang et al., 2014; Xia et al., 2015; Yang et al., 2009). Of the 17 studies, 8 used biotinylated dextran amine (BDA)-labeling for axonal tracing (Barritt et al., 2006; García-Alías et al., 2008; García-Alías et al., 2011; Karimi-Abdolrezaee et al., 2010; Kim et al., 2006; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009), one used WGA labeling (Shinozaki et al., 2016), and another used protein kinase C-ƴ (Ishikawa et al., 2015). Two high-quality studies used cholera toxin B-subunit (CTB) labeling in the acute SCI model to assess the potential effects of ChABC treatment on sensory neurons (Massey et al., 2008; Shields et al., 2008). The other study used calcitonin gene-related peptide (CGRP) immunohistochemistry (Barritt et al., 2006). Again, 2 other high-quality studies reported electrophysiological outcome measures of interest (García-Alías et al., 2011; Yang et al., 2009).

Acute phase 
Lesion breadth
One study used transection injury models (Pan et al., 2018), and two used compression (Caggiano et al., 2005; Führmann et al., 2018) to report the effects of acute ChABC administration on lesion breadth. In transection and contusion models, ChABC administration significantly decreased lesion length, cyst length at the injury site, hole size, and atrophic area volume (Pan et al., 2018; Shinozaki et al., 2016). However, acute administration of ChABC did not change the lesion length in the compression injury model. 

Plasticity of serotonergic neurons
Two studies used transection injury models (Ishikawa et al., 2015; Kim et al., 2006), two studies used contusion (Barritt et al., 2006; Mountney et al., 2013), and one study used both transection and contusion models (Tom et al., 2009) to assess the effects of acute ChABC administration on serotonergic neuron plasticity. In transection models, ChABC increased the number of serotonergic fibers at the injury site and rostral part of the injury, but it had a weak effect on reaching the caudal sites. In contusion injury models, treatment positively affected the ventral caudal horn in contrast to the caudal dorsal horn, where it did not change.

Plasticity of corticospinal neurons 
Four studies used transection injury models (García-Alías et al., 2008; Kim et al., 2006; Wang et al., 2014; Xia et al., 2015), and 2 used contusion (Barritt et al., 2006; Yang et al., 2009) to assess changes in CST neuron plasticity in response to acute ChABC administration. In transection models, treatment increased the number of CST fibers and their rostral length, but results were inconsistent at the injury site and in the caudal regions. Axonal sprouting was also increased at the injury site. One of two contusion studies reported increased CST fiber growth into and beyond the injury site, as well as increased terminal arborization (Barritt et al., 2006). In contrast, the other study (Yang et al., 2009) reported no difference in fiber length or number. The only compression study reported increased fiber growth and sprouting at the injury site and in the caudal regions (Führmann et al., 2018). To improve conduction, more studies on contusion and compression models are warranted.

Plasticity of sensory neurons
One study used transection injury models (Shields et al., 2008), and another used both compression and contusion models (Barritt et al., 2006). Both studies observed that acute administration of ChABC promoted afferent fiber plasticity and growth into and beyond the injury site. However, none of the included studies evaluated electrophysiological outcomes during the acute phase. 


Subacute phase
Lesion breadth
Two studies that used compressive injury models (Caggiano et al., 2005; Führmann et al., 2018) reported effects of ChABC subacute administration on lesion breadth, showing weak positive effects. Concerning the other outcome of interest, we found no studies reporting on the evidence for the effect of ChABC administration on serotonergic, sensory, and corticospinal neuronal plasticity. 

Chronic phase
Lesion breadth
Only 1 contusion injury study (Shinozaki et al., 2016) assessed lesion breadth changes in response to chronic administration of ChABC and found no influence of its treatment on lesion breadth changes. However, two studies reported increased tissue or axonal preservation with this treatment, either treadmill rehabilitation (Shinozaki et al., 2016) or neural progenitor cell transplantation (Karimi-Abdolrezaee et al., 2010). 

Serotonergic neurons’ plasticity
Chronic administration of Ch-ABC significantly increased the number and length of serotonergic fibers and sprouting at the rostral epicenter and caudal side of the lesion site (Karimi-Abdolrezaee et al., 2010; Shinozaki et al., 2016).

Corticospinal neuron plasticity
Chronic administration of ChABC significantly increased CST axon crossing and sprouting and rostral regrowth length (Karimi-Abdolrezaee & Billakanti, 2012; Karimi-Abdolrezaee et al., 2010; Shinozaki et al., 2016), but in caudal regions, there was no consensus on treatment effects. Two of the included studies (Karimi-Abdolrezaee & Billakanti, 2012; Shinozaki et al., 2016) reported no effects in the caudal regions. Overall, none of the included studies evaluated electrophysiological outcomes during the chronic phase. 

Functional outcome
Most studies used the Basso, Beattie, Bresnahan (BBB) scale (for hindlimb), ladder walk test/staircase reaching test, grid-walking/strength test, hindlimb contact placing response/vertical exploration (rearing) and sticker removal tests (for forelimb function), single pellet reaching task, fine touch and mechanical hyperalgesia assessment, spinal cord evoked potential and motor evoked potential assessment to evaluate motor and sensory functional outcomes (Caggiano et al., 2005; Führmann et al., 2018; García-Alías et al., 2008; García-Alías et al., 2011; Ishikawa et al., 2015; Kim et al., 2006; Mountney et al., 2013; Pan et al., 2018; Shinozaki et al., 2016; Tom et al., 2009; Wang et al., 2011; Xia et al., 2015; Yang et al., 2009). Also, one study used residual urine volumes to test for autonomic functions (Caggiano et al., 2005). Overall, the functional outcome were mixed, with 3 studies showing no improvement in functional outcomes after ChABC administrations (Führmann et al., 2018; Mountney et al., 2013; Tom et al., 2009) and 9 showing that ChABC administration alone (García-Alías et al., 2008) or combined with neurotrophin NT-3 secretion and NR2D expression (García-Alías et al., 2011) or NPCs (Karimi-Abdolrezaee et al., 2010), or antisense vimentin cDNA (Xia et al., 2015) or poly(glycerol sebacate) (Pan et al., 2018) or insulin+ methylprednisolone (Yang et al., 2009) or transplant mediated axonal remodeling or rehabilitation (Ishikawa et al., 2015; Shinozaki et al., 2016; Wang et al., 2011) improved functional outcomes, particularly motor functions (Table 3).
 
Discussion
SCI is a devastating clinical condition that results in rapid-onset and long-term disability related to the central nervous system. Several underlying mechanisms have been identified as factors responsible for primary and secondary damage following SCI. Primary mechanisms include neural death and axonal injury, which subsequently lead to sensorimotor disruption (de Almeida et al., 2023). After the primary complications, secondary mechanisms are initiated, including inflammation, vascular changes, ion disproportion, glutamate excitotoxicity, and radical formation, which result in additional complications, including progressive neural death, edema, hyperpyrexia, and paralysis (de Almeida et al., 2023; Korovessis, 2019). Inflammation and granulocyte colony-stimulating factor (G-CSF) production are two major components of glial scar formation that hamper recovery after SCI (Shechter et al., 2011). A correlation between inflammation and G-CSF expression via inflammatory cytokines has been reported in the literature (Shechter et al., 2011). The evidence suggests that inflammatory cytokines enhance GSK3β expression, which, in turn, promotes demyelination and neuronal degeneration (Nagai et al., 2016; Renault-Mihara et al., 2011). Furthermore, GSK3β may prevent post-SCI neuronal regeneration by modulating G-SCF discharge (Nagai et al., 2016). Therefore, therapeutic agents are designed to disrupt the molecular mechanisms underlying complications related to SCI. 
Although several therapeutic approaches, including pharmacological, stem cell-based, and enzyme-based approaches, have been developed for SCI, the management of patients with SCI remains challenging in developing or even developed countries. The difficulty in treating SCI patients is related to environmental factors in the injured area (de Almeida et al., 2023). However, advances in understanding SCI pathology have led to de novo research into new treatment approaches. Recent studies have focused on the secondary mechanisms involved in SCI to prevent further damage and facilitate neural regeneration. Kwon et al. (2011a) systematically reviewed the pre-clinical studies of neuroprotective agents already prescribed in humans. This study documented the potential effect of non-invasive medications for acute SCI, including erythropoietin, progesterone, estrogen, riluzole, polyethylene glycol, atorvastatin, magnesium, minocycline, inosine, NSAID, anti-CD11, and pioglitazone. In 2010, Cadotte and Fehlings, highlighted the effects of riluzole, anti-Rho antibody, and surgical decompression on SCI, thereby establishing evidence for translating preclinical research into clinical research (Cadotte & Fehlings, 2011). 
In 2011, another systematic review by Kwon et al. (2011b) aimed to investigate in vivo studies on the efficacy of intraspinal ChABC, anti-Nogo antibody, and anti-Rho antibody strategies, providing evidence for translating preclinical findings into human studies and clinical trials. They found that the Nogo receptor on neural cells prevents neural growth, suggesting that an anti-Nogo antibody is a promising therapeutic approach to promote neural recovery following SCI. Also, clinical trials have been conducted to investigate the effects of anti-Nogo in patients with acute and chronic SCI. Additionally, although riluzole is a sodium glutamate antagonist used in patients with amyotrophic lateral sclerosis (ALS), it is a potential drug for SCI patients due to its neuroprotective effect. A recent systematic review of the efficacy of riluzole in SCI, including animal and human studies, showed that riluzole is also a promising pharmacological agent that improves behavioral outcomes, promotes histological sparing, and exhibits electrophysiological effects following SCI, while diminishing the sequelae of this condition (Srinivas et al., 2019). 
Several key components, such as inflammation, oxidative stress, and edema, have been considered in the pathophysiology of SCI and can inform the development of effective therapeutic modalities. For instance, oxidative stress is putatively involved in the secondary deterioration of SCI. Therefore, depletion of reactive oxidative stress could be an effective approach to prevent SCI-associated secondary deterioration in patients. Medications, including the glucocorticoid steroid and the non-glucocorticoid 21-aminosteroid tirilazad, also have antioxidant activity that significantly enhances recovery after SCI (Jia et al., 2012). Likewise, edema is another essential component of the pathophysiology of SCI, which is initiated rapidly within minutes following injury (Rowland et al., 2008). Leonard et al. (2015) observed that deterioration of edema-induced injury was associated with more severe complications. Therefore, a systematic review of treatments targeting edema in SCI identified 3 main approaches to eliminate edema: inhibition of aquaporin 4 (AQP4), immunosuppression, and surgery. Furthermore, trifluoperazine, which prevents AQP4 localization, was proposed to be the most effective treatment, significantly reducing edema within a week after injection (Masterman & Ahmed, 2021). 
Lemons et al.’s (1999) investigation is the first to report an increase in CSPGs at the injury site and in adjacent areas after SCI. Moreover, they showed that astrocytes are a source of CSPG, leading to a lack of neural regeneration. Besides, they suggested that exogenous ChABC administration could degrade CSPG, which indicates the role of CSPGs as inhibitory outgrowth factors. These findings also unveil a potential therapeutic effect of ChABC. Further studies have documented that local injection of ChABC is associated with protein-based neuronal regeneration (Barritt et al., 2006; Ishikawa et al., 2015). Moreover, Lee et al. (2010) found that ChABC administration affected CSPG and inflammation, reducing CSPG and GSK3β levels and accelerating neural growth (Yılmaz & Kaptanoğlu, 2015). 
 A systematic review and meta-analysis conducted by Yousefifard et al. (2022) demonstrated that ChABC has a moderate effect on locomotor function in animal models of SCI, with no differences in injury severity. They also revealed that the induction model of SCI and the number of ChABC injections did not influence the efficacy of ChABC on locomotor function in SCI. In mouse models, Carter et al. (2008) showed that ChABC was neuroprotective for cortical layer V projection neurons after ICV infusion. ChABC also prevented cell atrophy after localized delivery to the spinal cord, suggesting a possible retrograde neuroprotective effect mediated at the injury site. 
Additional studies designed based on the combination of other treatment options like stem cells (Jevans et al., 2021), tissue engineering approaches (Raspa et al., 2021), sucrose (Raspa et al., 2019), photobiomodulation therapy (Janzadeh et al., 2020) antisense vimentin cDNA (Xia et al., 2015), poly(glycerol sebacate) (Pan et al., 2018), insulin + methylprednisolone (Yang et al., 2009) and rehabilitation (Ishikawa et al., 2015; Shinozaki et al., 2016; Wang et al., 2011) with ChABC showed improvement of the efficiency of Ch-ABC in SCI and improved functional outcomes. Additionally, a combination of ChABC and delayed injection of adeno-associated virus encoding the L1 cell adhesion molecule or pluripotent stem cell-derived NSCs in mouse models showed enhanced locomotor recovery after treatment (Lee et al., 2012; Suzuki et al., 2017). Furthermore, multiple injections of ChABC in Rhesus monkeys were associated with increased corticospinal axon growth, increased synapse formation by corticospinal terminals in the gray matter caudal to the lesion, and improved hand function (Rosenzweig et al., 2019). In this review, the functional outcomes assessed included motor, sensory, and autonomic functions, including forelimb and hindlimb function, gait, pellet-reaching task, fine-touch and mechanical hyperalgesia assessment, and residual urine volume assessment. Although the results were heterogeneous, most studies showed that ChABC administration improved functional outcomes in animal models.
This systematic review entails the histological aspects of the efficacy of intrathecal ChABC in SCI rats. Our comprehensive search identified 17 eligible studies assessing histopathological outcomes of intrathecal ChABC administration following SCI in rats. When analyzing treatment results by treatment phase, our results showed that ChABC treatment in the acute phase reduced necrosis and atrophic areas, increased tissue preservation, and increased sensory neuron plasticity and growth into and beyond the injury site. Moreover, ChABC increased the number of serotonergic fibers (5-HT), reduced neuronal apoptosis, increased the digestion of CSPG, promoted the differentiation of stem cells into neurons, and increased GAP-43, NG-2, chondroitin 4 sulfate, and BDA levels. 
Regardless of the treatment phase, ChABC promoted the survival of serotonergic fibers, plasticity, and regrowth beyond the injury site, as well as rostral global plasticity and the survival of CST fibers. To the best of our knowledge, this is the first systematic review to evaluate the histopathological effects of intrathecal ChABC treatment. To make the results of the ChABC treatment more tangible, we compared them with studies using combined ChABC treatment. In a systematic review by Kwon et al. (2011b), the putative mechanisms underlying intraspinal ChABC injection for acute SCI were assessed. In this regard, axonal germination/growth of fibers, specifically serotonergic fibers, and the neuroprotective effect of intra-spinal ChABC, preventing neural atrophy, were the most promising mechanisms. Our results indicated that CST and serotonergic fibers sprouting, terminal arborization, and crossing were significantly increased after ChABC treatment, similar to the results of ChABC treatment in combination with rehabilitation (Marsh et al., 2011), Nogo-A inhibitors (Zhao et al., 2013), and increased neurotrophin-3 levels (Massey et al., 2008). We also found that ascending fiber regeneration was significantly promoted after intrathecal ChABC administration. This finding was reproduced in 3 combinational treatment studies using neurotrophin-3, cell plant (Massey et al., 2008), and conditioning agents such as zymosan (Harel et al., 2012; Steinmetz et al., 2005). Grimpe et al. (2005) and Vavrek et al. (2007) reported that a combination of cell transplant and ChABC increased CST axon regeneration rostrally and promoted their elongation through the injury site and to the caudal sites. The articles we included in this area did not yield consistent results. While another study (García-Alías et al., 2008) reported increased regrowth length of CST axons into and beyond the injury site, the results of four other studies (Kim et al., 2006; Shinozaki et al., 2016; Wang et al., 2014; Yang et al., 2009) showed no significant effects on regrowth to caudal sites for ChABC treatment. It seems that when a substantial atrophic area is present after injury, a graft is necessary to provide a tissue scaffold for regenerating axons to traverse the injury site. Although it is well known that ChABC improves axonal sprouting and neural function by degrading CSPGs, the exact molecular mechanism underlying this process remains unclear. However, Hu et al. (2021) explored the key role of CSPGs in axon regeneration and neural apoptosis. They showed that the caspase activity is significantly increased within 2 to 11 weeks after injury. Importantly, ChABC reduces the total amount of functional caspase-3, validating the anti-apoptotic activity reported by Kwon et al. (2011b). Protein tyrosine phosphatase sigma is an example of a CSPG receptor involved in SCI-induced retrograde neural apoptosis. Correspondingly, the expression of PTPs, which was associated with neural apoptosis, decreased following ChABC treatment, demonstrating ChABC’s anti-apoptotic activity in SCI. Regarding functional outcomes, we found that ChABC ­­ssociated with better motor functions in animal models with SCI (García-Alías et al., 2011; Karimi-Abdolrezaee et al., 2010; Pan et al., 2018; Xia et al., 2015; Yang et al., 2009).

Conclusion
In summary, our systematic review of the available evidence for finding the histopathological effects of intrathecal ChABC administration in spinal cord injured rats suggests that this treatment can reduce necrosis and atrophic area and increase tissue preservation, increase sensory neuron plasticity and growth into and beyond the injury site, reduce the apoptosis of neurons, promotes the survival of serotonergic fibers, plasticity and regrowth to and beyond the injury site, the global plasticity and survival rostrally of CST fibers, increase digestion of the CSPG, differentiation of stem cells to neurons and the GAP-43, NG-2, chondroitin 4 sulfate, BDA level. ChABC treatment was associated with improved functional outcome in rats, mice, and primates. Although these findings are promising, further studies can provide additional evidence to support a final assessment of the risks and benefits of ChABC in SCI.

Strengths and limitations
As this is the first systematic review on the histopathological effects of ChABC treatment in SCI, results can be used to better understand the mechanisms underlying positive functional outcomes, understand the possible adverse effects to look for, and discover the pitfalls in studies that require more attention in future work. The most important limitation of this study is the small sample size in animal studies, which may both increase the risk of selection bias and render most effects non-significant. We grouped studies by intervention phase and injury model, and in some groups, there was only one study available, leaving very little evidence to deduct points. Furthermore, because studies using different enzyme administration regimens are limited, we did not summarize results by regimen. This limitation can affect the results and may be a source of the non-convergence.
However, clinical studies have higher priority and can more directly benefit individuals with SCI, whereas preclinical studies provide the fundamental basis for clinically approved treatments. Our study emphasizes the indispensable role of animal studies in advancing treatments for SCI and provides insight for bridging preclinical studies to patient care. This study also provided a comprehensive review of the histopathological effects of ChABC on rat SCI models. To gain a clearer understanding of ChABC’s roles in SCI, we classified the reviewed studies by SCI phase, which is of high importance in this context.

Ethical Considerations
Compliance with ethical guidelines
This study was approved by the Ethics Committee of Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran (Code: IR.TUMS.MEDICINE.REC.1398.886).

Data availability
The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.
Declaration of generative AI and AI-assisted technologies in the writing process
No AI tool influenced the scientific content, data analysis, or conclusions of this work.

Funding
This study was extracted from the general medical doctorate thesis of Ali Anjomsho, approved by the Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. This work was funded by Sina Trauma and Surgery Research Center, Tehran University of Medical Sciences, Tehran, Iran (Grant No.: 97-02-38-40904). 

Authors' contributions
All authors contributed equally to the conception and design of the study, data collection and analysis, interception of the results and drafting of the manuscript. Each author approved the final version of the manuscript for submission.

Conflict of interest
The authors declared no conflict of interest.


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