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 Table of Contents  
Year : 2019  |  Volume : 5  |  Issue : 1  |  Page : 10-16

Inhibitory effect of hyaluronidase-4 in a rat spinal cord hemisection model

1 Department of Orthopaedic, The Second Affiliated Hospital of Nantong University, Nantong 226001, China; Department of Advanced Medicine, Kanazawa Medical University Graduate School of Medical Science, Uchinada, Ishikawa 920-0293, Japan
2 Department of Advanced Medicine, Kanazawa Medical University Graduate School of Medical Science, Uchinada, Ishikawa 920-0293, Japan
3 Department of Orthopaedic Surgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei, China

Date of Submission06-Nov-2018
Date of Acceptance17-Dec-2018
Date of Web Publication28-Mar-2019

Correspondence Address:
Prof. Ping Liu
Department of Orthopaedic Surgery, Liyuan Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ctm.ctm_30_18

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Objective: In this study, the effects of anti-hyaluronidase-4 (Hyal-4) antibody on the histological changes of Hyal-4 and the corresponding chondroitin sulfate proteoglycan (CSPG) expression in the rat spinal cord hemisection model were examined.
Methods: After creating a rat spinal cord hemisection injury, experiments were conducted by administering anti-Hyal-4 antibody or control immunoglobulin G by intraspinal injection as a single dose, or intrathecal administration, using osmotic pumps, as multiple doses. Frozen sections of the injured spinal cord were made after a single-dose administration on days 1 and 4 and 1 week or at 1, 2, 3, and 4 weeks after the start of pump-aided injections. Immunofluorescence studies were then conducted using CS56 for CSPGs and anti-glial fibrillary acidic protein antibody for reactive astrocytes.
Results: No difference was observed between the test and control groups in the single-dose administration of the antibody. In pump-aided administration, CSPGs in the control group decreased at 4 weeks, but those in the anti-Hyal-4 antibody administered group did not.
Conclusion: Persistent suppression of Hyal-4 allowed CSPGs to remain and also increase in the rat spinal cord hemisection model, confirming Hyal-4 as an endogenous digestive enzyme of CSPGs.

Keywords: Chondroitin sulfate proteoglycans, hyaluronidase-4, inhibitory effect of hyaluronidase-4, rat spinal cord hemisection model, spinal cord injury

How to cite this article:
Wang X, Yokoyama M, Liu P. Inhibitory effect of hyaluronidase-4 in a rat spinal cord hemisection model. Cancer Transl Med 2019;5:10-6

How to cite this URL:
Wang X, Yokoyama M, Liu P. Inhibitory effect of hyaluronidase-4 in a rat spinal cord hemisection model. Cancer Transl Med [serial online] 2019 [cited 2020 May 28];5:10-6. Available from: http://www.cancertm.com/text.asp?2019/5/1/10/255123

  Introduction Top

After spinal cord injury, reactive astrocytes aggregate around damaged area to express chondroitin sulfate proteoglycans (CSPGs) and inhibit axonal regeneration by forming hollowed-out glial scar.[1],[2] CSPGs are ubiquitously present on the cell surface of animal cells and in the extracellular matrix of connective tissue.[3] In the central nervous system, CSPGs are expressed only at the developmental stage, as extracellular inhibitory molecules that regulate the direction of extension of axons,[4] but not in the normal adult spinal cord. These CSPGs can be digested by chondroitinase ABC (ChABC), an enzyme that degrades chondroitin sulfate (CS), purified from bacteria called Proteus vulgaris.[5] Postspinal cord injury, digestion of CSPGs by ChABC, has been shown to enhance regeneration of axons and promote functional recovery in rats.[6] Iseda et al. reported a difference in CSPG expression between rat spinal cord contusion injury and cut injury models; the CSPG expression peaked at about 2 weeks after contusion injury and persisted to inhibit axonal outgrowth thereafter, but the same was peaked at about 3 weeks after cut injury, which then decreased naturally.[7] From this, the presence of a CSPG-digesting enzyme in the spinal cord was inferred, the existence of which was elucidated by Kaneiwa et al. in 2010, posing human hyaluronidase-4 (Hyal-4) as a CS-specific hydrolytic enzyme.[8] Tachi et al. reported that it was also expressed in the rat spinal cord cut injury model.[9] Since the presence of Hyal-4 was confirmed expressing in the damaged part of the spinal cord and Hyal-4 was expressed around the periphery of CSPGs, we supposed that Hyal-4 may digest CSPGs. To confirm this phenomenon, we histologically studied the changes in CSPGs in the rat spinal cord hemisection model, treated with anti-Hyal-4 antibody.

  Materials and Methods Top

The current animal study protocol was approved by and conducted following the guidelines of Kanazawa Medical University Ethics Committee.

Fabrication of rat spinal cord hemisection model

Forty-two female Sprague–Dawley (SD) rats (Sankyo Laboratory Service, Tokyo, Japan) were deeply anesthetized with peritoneal injection of 45 mg/kg of pentobarbital sodium (Somnopentyl, Kyoritsu Pharmaceutical, Tokyo, Japan). The lamina of the 9th and 10th thoracic vertebrae was excised to expose the 11th medulla. Postdural incision, the right side of the spinal cord was sectioned using a surgical blade. The cut was placed beyond the midline to ensure a full half cut.

Anti-hyaluronidase-4 antibody

The anti-Hyal-4 antibody was ordered from Invitrogen (Carlsbad, USA). Peptide synthesis was carried out from the amino acid sequence of rat Hyal-4, and rabbits were immunized to obtain serum. The serum was then purified on an affinity column to which the immunization peptide was bound, and the obtained antibody was used as a rabbit anti-Hyal-4 polyclonal antibody.[9]

Preparation of single-dose model

Immediately after sectioning the right half of the rat spinal cord, 1 mg/cm2 of anti-Hyal-4 antibody was injected into the left half of the spinal cord, 2 mm proximal to the center of the cut, using a 33G needle syringe (75 RN, HAMILTON, Reno, USA) mL at a rate of 1.0–1.2 μL/min. Control animals received 1 mg/mL of rabbit immunoglobulin G (IgG) (Sigma-Aldrich, St. Louis, USA) in a similar manner.

Preparation of continuous administration model

One hundred microliters of anti-Hyal-4 antibody or IgG was filled in an osmotic pump (0.11 μL/h/28 days, MODEL 1004, ALZET, DURECT, Cupertino, USA). A polyethylene tube with an outer diameter of 0.61 mm and an inner diameter of 0.28 mm (PE-10, Intramedic, Clay Adams, Parsippany, USA) was connected, immersed in 0.9% saline and loaded at 37°C for 48 h. After cutting the spinal cord of the rat, fenestrations between the 12th and 13th lumbar vertebra were made. The dura mater was exposed and incised with a micro pupil and a cusp blade, and then, the tube was inserted under the dura so as to cross the injured part. The osmotic pump was subcutaneously placed at the distal side of the intervertebral space.

Tissue processing and immunofluorescence methodology

Following perfusion fixation method, the animals were sacrificed at different time points: 1 day, 4 days, and 1 week postsurgery for the single administration model and 1 week, 2 weeks, 3 weeks, and 4 weeks postsurgery for the continuous administration model. Spinal cord samples, with a length of 20 mm excised along the craniocaudal direction around the damaged area, were obtained and put in 25% sucrose containing phosphate-buffered saline (PBS) solution overnight. The tissues were then embedded in OCT compound (Sakura Finetek Japan, Tokyo, Japan), and frozen sections, sliced 20 μm along the coronal plane, were made. After washing the compound with PBS solution, the section was blocked with PBS solution containing 10% normal goat serum (NGS) and 0.3% Triton X-100 for 2 h at room temperature. Postwash, the section was treated with the primary antibody and incubated overnight at 4°C. As the primary antibodies, mouse mAB CS56 (Sigma-Aldrich) at a dilution of 1:200 was used for CSPGs, and rabbit mAB glial fibrillary acidic protein (GFAP) (D1F4Q) (Cell Signaling Technology, Beverly, USA) at a dilution of 1:200 was used for reactive astrocyte. Sections were washed with PBS solution and then reacted with secondary antibody for 1 h at room temperature. The secondary antibodies were Alexa Fluor 488 goat anti-mouse IgG and Alexa Fluor 594 goat anti-rabbit IgG (Life Technologies), both of which were diluted in PBS containing 3% NGS and 0.3% Triton X-100 at a ratio of 1:200. After washing, the sections were stained with DAPI (Life Technologies) and cover glass mounted with ProLong Gold Antifade Reagent. As a negative control, a fluorescent antibody method was performed on normal spinal cords of two 10-week-old female SD rats using CS56, GFAP antibody in the same manner as described above. The images of the stained sections were then obtained using BZ-9000 (KEYENCE, Osaka, Japan) fluorescent microscope.

Quantification of fluorescent stain

For each group at each time point, images from three rats were obtained, and for each rat, three slices in the vicinity of the center were selected. Then, using BZ-II Analyzer (KEYENCE), the area of positive stain was calculated within a range of 4000 μm × 4000 μm. The range of luminance was set at 10 (0–255), and nonspecific areas such as the meninges and nerve root tissues were excluded.

Statistical test

Descriptive statistics are expressed as mean ± standard deviation. For the data showing the immunostainability of each antibody at each time point (n = 9 in each group), obtained by quantification, analysis of variance was performed using Bonferroni Dunn's test. In addition, Mann–Whitney U-test was conducted for comparison between the two groups at the same point in each model. P < 0.05 was considered as statistically significant. We used StatView 5.0 (SAS Institute, Cary, USA) for all statistical tests.

  Results Top

Single dose

From 1 day after injection, pale dyeability of GFAP and CSPGs was observed in the damaged area in both groups [Figure 1]a, [Figure 1]d, [Figure 1]j, [Figure 1]m. At the 4th day, the scarred part stained with only CSPGs and its surroundings gave GFAP staining [Figure 1]b, [Figure 1]e, [Figure 1]i, [Figure 1]k, [Figure 1]n, [Figure 1]q, [Figure 1]r. After 1 week, the scar tissue stained with CSPGs increased in the inside of the cut surface, and the stainability of GFAP became stronger at the margin [Figure 1]c, [Figure 1]f, [Figure 1]l, [Figure 1]o, [Figure 1]p.
Figure 1: Day 1 (a, d, g, j, m, and p) and day 4 (b, c, and d) fluorescent staining of chondroitin sulfate proteoglycans and reactive astrocytes in a single administration of anti-hyaluronidase-4 (chondroitin sulfate proteoglycans) (green) of CS56 and immunostaining properties of glial fibrillary acidic protein (reactive astrocytes) at 1 week (c, f, i, l, o, and r) (red). The immunostainability of CS56 was observed from 1 day after administration in both groups (a and j), and the damaged part was confirmed until 1 week. It gradually increased to the center (a-c and j-l). The immunostainability of glial fibrillary acidic protein was observed from 1 day after administration (d and m) in both groups and gradually increased around the injured area until 1 week (d-f and m-o). In the double-stained image, a region showing immunostaining properties of glial fibrillary acidic protein around the region showing immunostaining of CS56 in the injured part at any point (g-i and p-r). In all images, the head side is the left side, and the scale bar is 500 μm

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The area of the region positive for CS56 immunostaining was found to be 19.5 ± 4.2 × 103 μm2 on day 1, 48.6 ± 7.8 × 103 μm2 on day 4, and 126.0 ± 20.3 × 103 μm2 at 1 week, after injection of anti-Hyal-4 antibody. Corresponding control samples showed 17.8 ± 3.5 × 103 μm2 on day 1, 55.1 ± 7.9 103 μm2 on day 4, and 112.0 ± 19.7 103 μm2 at 1 week [Figure 2]a, after injection of IgG antibody. Similarly, the area of the region positive for GFAP was found to be 29.1 ± 7.1 × 103 μm2 on day 1, 39.0 ± 7.8 × 103 μm2 on day 4, and 95.0 ± 14.3 × 103 μm2 at 1 week after injection of anti-Hyal-4 antibody. Results revealed a gradual increase in the CSPG and GFAP expression from day 1 to 1 week after injection [Figure 2]b. Statistical analysis revealed a significant increase in CS56 and GFAP expression from day 1 to 1 week after injection, and no significant difference was observed in CS56 and GFAP expression, between the test and control groups at the assessed time points [Figure 2]a and [Figure 2]b.
Figure 2: Chondroitin sulfate proteoglycans (a) in the damaged area after single administration of anti-hyaluronidase-4 antibody, control immunoglobulin G. Temporal transition of sexual astrocyte (b) distribution area. The data in the graph shown are average values of data of three slices per slice (nine slices total) at each time point and show standard deviation with error bars. Analysis of variance was performed from data at each time point, and the immunostainability of CS56 and glial fibrillary acidic protein showed a significant difference between each time point from 1 day to 1 week in both groups. The same comparison between the two groups at the time point showed no significant difference between CS56 and glial fibrillary acidic protein (* =P < 0.05, NS = not significant)

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Continuous administration

From 1 week of continuous infusion, animals showed positive staining of CS56 filling the damaged area and GFAP staining around the cut surface [Figure 3]a, [Figure 3]e, [Figure 3]m, and [Figure 3]q. Thereafter, in both groups, the stainability of CS56, GFAP became stronger, the stainability of CS56 attenuated at 4 weeks [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]f, [Figure 3]g, [Figure 3]h, [Figure 3]n, [Figure 3]o, [Figure 3]p, [Figure 3]r, [Figure 3]s, [Figure 3]t in the IgG administration group but strongly stained even after 4 weeks in the anti-Hyal-4 administration group BD, FH, NP, RT. The region showing immunostainability was found to be 119.0 ± 25.6 × 103 μm2 at 1 week, 181.2 ± 19.8 × 103 μm2 at 2 weeks, 270.9 ± 29.2 × 103 μm2 at 3 weeks, and 305.6 ± 39.0 × 103 μm2 at 4 weeks of continuous infusion of anti-Hyal-4 antibody. In the control group, the CS56-positive area was 115.8 ± 21.6 × 103 μm2 at 1 week, 176.1 ± 23.8 × 103 μm2 at 2 weeks, 250.3 ± 27.1 × 103 μm2 at 3 weeks, and 173.2 ± 26.2 × 103 μm2 at 4 weeks. Thus, a decrease in the CS56 expression was observed at 4 weeks in the control group, while the same was not observed in the anti-Hyal-4 antibody group [Figure 4]a. Similarly, the GFAP-positive area was found to be 97.2 ± 15.0 × 103 μm2 at 1 week, 187.7 ± 19.6 × 103 μm2 at 2 weeks, 254.6 ± 29.3 × 103 μm2 at 3 weeks, and 295.8 ± 41.5 × 103 μm2 at 4 weeks of administration of anti-Hyal-4 antibody. While in the control group, the GFAP-positive area was 94.2 ± 22.6 × 103 μm2 at 1 week, 191.9 ± 25.7 103 μm2 at 2 weeks, 295.6 ± 20.5 103 μm2 at 3 weeks, and 302.3 ± 32.7 103 μm2 at 4 weeks of IgG administration. Thus, GFAP showed an expression peak at 3–4 weeks in both the groups [Figure 4]b. In the statistical test, a significant increase in CS56 expression was observed up to 3 weeks in control group, followed by a decline at week 4. However, under the influence of anti-Hyal-4 antibody, the CS56 expression continued to increase even at week 4 [Figure 4]a.
Figure 3: Chondroitin sulfate proteoglycans and reactive astrocyte fireflies during continuous administration of anti-hyaluronidase-4 antibody, control immunoglobulin G, photograph stained image 3 weeks (c, g, k, o, s, and w), 2 weeks (b, f, j, n, r, and v), 1 week after administration (a, e, i, m, q, and u), immunostaining properties of CS56 at 4 weeks (d, h, l, p, t and x) (green), and glial fibrillary acidic protein immunostaining (red). The immunostainability of CS56 increases mainly in injured areas from 1 week to 3 weeks after administration in both groups (a-c and m-o). At 4 weeks, the anti-hyaluronidase-4 antibody-treated group increased (d) but decreased in the immunoglobulin G-administered group (p). The immunostainability of glial fibrillary acidic protein is 1–4 weeks (e-h and q-t). In the double-stained image, at any time point, the area of the immunostaining of CS56 in the damaged area was surrounded by glial fibrillary acidic protein. A region showing immunostainability was found (i-l and u-x). In all images, the head side is the left side and the scale bar is 500 μm

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Figure 4: Chondroitin sulfate proteoglycans (a) in the damaged area after continuous administration of anti-hyaluronidase-4 antibody, control immunoglobulin G (a) and reaction around the injured area. Temporal transition of sexual astrocyte (b) distribution area. The data in the graph shown are average values of data of three slices per slice (nine slices total) at each time point and show standard deviation with error bars. Analysis of variance was performed from data at each time point, and the immunostainability of CS56 was significantly different between each time point until 4 weeks in both groups, and the immunostainability of glial fibrillary acidic protein was significantly higher. Significant difference was observed up to 3 weeks in both groups, but no significant difference was observed in anti-hyaluronidase-4 administered group at 4 weeks (b). In the comparison between CS56 at the same time point, 4-week immunoglobulin G administration group was significantly decreased (a). In the comparison between the two groups at the same time of glial fibrillary acidic protein, a significant difference was observed at 3 weeks but at other time points a significant difference (b) (* =P < 0.05, NS = not significant)

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Relationship between chondroitin sulfate proteoglycans and reactive astrocytes and hyaluronidase-4

The relationship between CSPGs and the distribution of reactive astrocytes was assessed using a double-stained image. In continuous administration group, both the anti-Hyal-4 antibody and the IgG administered rats showed the dyeability of CS56 mainly in the damaged area from 1 week of administration, and the stainability of GFAP surrounded the CS56 region [Figure 3]i and [Figure 3]u. In the anti-Hyal-4 antibody-treated group, the stainability of CS56 and GFAP gradually increased at 2–4 weeks [Figure 3]j, [Figure 3]k, [Figure 3]l. In the IgG administration group, the stainability of the GFAP region gradually increased at 2–4 weeks, while the staining of CS56 region gradually increased at 2–3 weeks but decreased at 4 weeks [Figure 3]v, [Figure 3]w, [Figure 3]x. The GFAP area did not penetrate the center of the damaged area, and CS56 filled the damaged area over time [Figure 3]i, [Figure 3]j, [Figure 3]k, [Figure 3]l and [Figure 3]u, [Figure 3]v, [Figure 3]w, [Figure 3]x. In the strong enlargement, the CS56 region was observed toward the damaged part in a partially overlapping manner from the GFAP region of the damaged part border [Figure 5].
Figure 5: A CS56 region was present toward the injured part in a partially overlapping manner with the glial fibrillary acidic protein region existing at the edge of the double staining enhanced image damaged part of CS56 and glial fibrillary acidic protein. Head side is left and the keel bar is 50 μm

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The Hyal-4 region[9] reported by Tachi et al. is present at the periphery of the damaged part and is similar to the GFAP region shown in this experiment, and both Hyal-4 and reactive astrocytes surround the CSPGs It was shown to exist as.

Staining of normal spinal cord

Normal spinal cord, serving as a negative control, demonstrated no strong staining of CS56 and GFAP [Figure 6].
Figure 6: Fluorescent staining images of chondroitin sulfate proteoglycans and reactive astrocytes in normal spinal cord. Immunostaining properties of CS56 (chondroitin sulfate proteoglycans) (a, green) and immunostaining properties of glial fibrillary acidic protein (reactive astrocytes) (b, red) were hardly observed in normal spinal cord tissues. Scale bar is 1000 μm

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  Discussion Top

The role of glial scarring, formed from reactive astrocytes during spinal cord injury,[1],[2] was defined by Okada et al. as to minimize spinal cord injury, by preventing the lesion from expanding further.[10] It was achieved by observing the status of the spinal cord with contusion injury in mice knocked out of signal transducer and activator of transcription 3, a signal that causes reactive astrocytes to migrate to the site of injury. On the other hand, Nogo-A,[11] myelin-associated glycoprotein (MAG),[12] oligodendrocyte-myelin glycoprotein (OMgp),[13] Rho kinase,[14] which are myelin-related proteins derived from oligodendrocytes around the glial scar, and CSPGs[15],[16],[17] in glial scar tissue, semaphorin-3A[18],[19] expressed from fibroblasts, etc., are found to be widely distributed in injured areas and are known inhibitors of axonal regeneration. The glial scar was thought to have duality that inhibits spinal cord regeneration while minimizing the extent of spinal cord injury and thus it is thought that the preventing/removal of glial scar promotes spinal cord regeneration.

It is known that by forming a concentration gradient of CSPGs in the damaged part, a round structure called dystrophic endball is formed at the stump of damaged axon to suppress axon elongation.[20] In contrast, many cases have reported that the functional regeneration of axons occurs by ChABC-mediated digestion of CSPGs within the glial scar.[6],[7],[20],[21]

The myelin-related proteins Nogo-A, MAG, and OMgp bind ligands to the common receptor Nogo receptor,[12],[13],[22] Activates Rho kinase by activating Rho kinase by transmitting signals intracellularly through a low-affinity receptor (P75NTR) of a neurotrophic factor that binds to the Nogo receptor and forms a receptor complex and inhibits axon extension.[14],[23] However, administration of a Rho kinase inhibitor after spinal cord injury results in functional recovery and axonal regeneration is allowed.[24] Semaphorin-3A acts on the microtubule actin skeleton to destroy the neural growth cone, inhibition of which promotes axonal regeneration, regeneration of blood vessels, suppression of apoptosis, etc. A study also reports a significant motor function recovery, postsemaphorin-3A inhibition.[19] Since excluding axonal outgrowth by excluding the action of each of these inhibitory factors, it is considered that each of them independently has an axon elongation inhibiting action.

ChABC is an enzyme that degrades CS, but Hyal-4 is an endo-type hydrolase specific to CS present in vivo, as indicated by Kaneiwa et al.[8] It was thought that this Hyal-4 could digest CSPGs. However, the expression site of Hyal-4 is confined to the placenta, skeletal muscle, and testis in humans, and in mice, it is expressed only in testes and embryos on the 17th day of embryo, and it is not expressed in specific organs. It is highly likely that it is an enzyme with limited function.[25] Tachi et al. showed the expression of Hyal-4 in the injured spinal cord in the rat spinal cord injury models.[9] Hyal-4 acting in the damaged area was suppressed by administering anti-Hyal-4 antibody, which confirmed an increase in CSPGs, and thus, Hyal-4 was projected as an intrinsic digestive enzyme of CSPGs.

In our study, in anti-Hyal-4 antibody group, no change was observed at 1 week after administration. Owing to the in vivo half-life of anti-Hyal-4 antibody, which was less than a week, additional administration of antibodies was required to suppress the action of Hyal-4. The effect of anti-Hyal-4 antibody does not last until 3 weeks,[9] which is the peak of Hyal-4 expression, as indicated by Tachi et al. To suppress the action of Hyal-4, additional administration of antibody is required. Therefore, using an osmotic pump, continuous administration of anti-Hyal-4 antibody was carried out. As a result, the CSPG expression continued to increase at 4 weeks, while the same was found decreased in control group, after peaking at 3 weeks postinjury. The expression of CSPGs in the continuous administration of anti-Hyal-4 antibody group was almost the same as IgG administered group for up to 3 weeks, as the effect of endogenous Hyal-4 is known to appear around 3 weeks. Then, the CSPGs are digested endogenously in the IgG administration group, but in the anti-Hyal-4 antibody administration group, CSPGs were not digested and thus continued to increase. From this, it is confirmed that the endogenous Hyal-4 is expressed in cut-injured spinal cord, which digests the locally.

In addition, expression of CSPGs was observed so as to fill the damaged area, with a small overlap over the margin showing GFAP stainability. The suppression of Hyal-4 showed no effect on the GFAP expression between the test and the control groups, despite the observed increase in CSPG. Tachi et al. reported the possibility that Hyal-4 expression from reactive astrocytes,[9] but the expression of Hyal-4 was similar to the expression of GFAP in this experiment. Astrocyte has the dual function of axon regeneration promotion and axonal regeneration suppression.[26] In a complete spinal cord cut-injury model of young rats, juvenile astrocytes were found mobilized into the damaged area supporting the axons cross over into the injured area, thus promoting regeneration.[27] In this way, since there are dichotomies in the properties of astrocytes, in the cleavage model, reactive astrocytes expressing CSPGs are influenced by the injury-related factors and Hyal-4, suggesting the possibility of regulating the synthesis and digestion of CSPGs.

CSPGs naturally decrease in the rat spinal cord cut-injury model, used in this experiment, but they do not decrease in a spinal cord crush-injury model.[7] Among the cytokines expressed during spinal cord injury, it was reported that IL-6 strongly induces neural stem cells to differentiate into astrocytes.[28] In addition, neural stem cells are present in the adult central nervous system, and it is reported that the endogenous neural stem cells proliferate when injured but mostly differentiate into astrocytes and form glial scars.[29] When the damage is severe, like that in spinal cord crush model, the expression of CSPGs increases in order to increase the astrocyte response to wall off the damage, so even if Hyal-4 is expressed, the expression of CSPGs might still be maintained.

It was believed that the injured spinal cord did not recover. However, in recent years, research has proven it wrong and has developed various promising approaches to resolve the issue, some of which are under clinical trials. In one such trial, after local transplantation of autologous olfactory mucosa-coated glial cells, half of the subjects demonstrated scar removal with improvement in paralysis.[30] In another study, several cases showed clinical improvements with subarachnoid administration of autologous marrow stromal cells, by lumbar puncture.[31] Apart from cell transplantation, administration of riluzole, an N-methyl-D-aspartic acid-type glutamate receptor inhibitor, has shown neuroprotection against secondary damage while also promoting axonal outgrowth.[32] Similarly, another clinical trial was conducted with Rho inhibitor Cethlin, a known neuroprotector and an axon extension inhibitor, where some patients showed improved motor function.[33] The neuroprotective effect of granulocyte colony-stimulating factor is effective in treating spinal cord injury and has obtained good results in clinical trials.[34] Administration of hepatocyte growth factor has also been shown to be effective in basic research,[35] and it is expected to be a substitute for massive steroid therapy in the future. Among these studies, there are many evidence that digesting CSPGs is effective for axon regeneration, and in recent years, combination of neural stem cell transplantation and ChABC administration,[19] or combined use of anti-Nogo-A antibody and ChABC,[36] rehabilitation combined with ChABC[37] and various other strategies of ChABC combination are explored, with some promising results. Because ChABC is a bacterial enzyme,[5] it can only be administered by intraspinal injection[7] or intrathecal injection.[6],[38] Since Hyal-4 is an endogenous enzyme, naturally expressed in the human spinal cord, it could be possible to administer the same under minimally invasive methods. Further, when the mechanism of the mechanism of secretion and production of Hyal-4 in astrocytes is elucidated, there is a possibility of amplifying it in vivo, aiding in intrinsic digestion of CSPGs in the damaged area.

In this study, differences in expression levels of CSPGs were observed by administering neutralizing antibodies of Hyal-4 in a rat spinal cord cut-injury model. In the future, axonal outgrowth affected by increased or decreased expression of Hyal-4 needs to be studied. Further, it is necessary to consider the effect of Hyal-4 and CSPGs in the regeneration and functional recovery and spinal cord in crush-injury model.


We thank Lee Seaman of Seaman Medical, Inc. (Bellingham, WA, USA) for providing professional English language editing of this article.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]


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