Comparative Analysis of the Expression of Chondroitin Sulfate Subtypes and Their Inhibitory Effect on Axonal Growth in the Embryonic, Adult, and Injured Rat Brains.

BACKGROUND: Chondroitin sulfate glycosaminoglycans (CS-GAGs) are the primary inhibitory GAGs for neuronal growth after central nervous system (CNS) injury. However, the inhibitory or permissive activity of CS-GAG subtypes is controversial and depends on the physiological needs of CNS tissues. In this study, we investigated the characteristics and effects of CS-GAGs on axonal growth, which was isolated from the brain cortices of normal rat embryo at E18, normal adult rat brain and injured adult rat brain. METHODS: Isolated CS-GAGs from embryo, normal adult, and injured adult rat brains were used for analyzing their effect on attachment and axonal growth using modified spot assay with dorsal root ganglion (DRG) explants and cerebellar granule neurons (CGNs). CS-GAGs were separated using high performance liquid chromatography (HPLC), and the subtypes of CS-GAGs were analyzed. RESULTS: CS-GAGs of all three groups inhibited CGN attachment and axonal growth of DRGs. However, CS-GAGs of normal adult rat brain exhibited higher inhibitory activity than those of the other groups in both assays. When subtypes of CS-GAGs were analyzed using HPLC, CS-A (4S) was the most abundant in all three groups and found in largest amount in normal adult rat brain. In contrast, unsulfated CS (CS0) and CS-C (6S) were more abundant by 3-4-folds in E18 group than in the two adult groups. CONCLUSION: When compared with the normal adult rat brain, injured rat brain showed relatively similar patterns to that of embryonic rat brain at E18 in the expression of CS subtypes and their inhibitory effect on axonal growth. This phenomenon could be due to differential expression of CS-GAGs subtypes causing decrease in the amount of CS-A and mature-type CS proteoglycan core proteins.

Activin A and BMP chimera (AB204) induced bone fusion in osteoporotic spine using an ovariectomized rat model.

BACKGROUND CONTEXT: Recombinant human bone morphogenic protein 2 (rhBMP2) has been used to induce bone fusion in patients with spinal fusion surgery. However, the effectiveness of rhBMP2 in the bone fusion process is limited in osteoporosis patients, and a high dose of rhBMP2 for enough bone fusion sometimes provokes side effects. Therefore, substitutes for rhBMP2 with a higher therapeutic potency are needed, and already several studies have published the effectiveness of Activin A/BMP2 chimera (AB204) in new bone formation process in vitro and in vivo. PURPOSE: In the present study, we provide evidence that bone fusion activity of AB204 is superior to that of rhBMP2 in osteoporotic rat models. STUDY DESIGN/SETTINGS: An in vivo animal study was carried out. METHODS: A total of 40 Sprague-Dawley rats underwent bilateral ovariectomy. At 6 weeks after ovariectomy, a lumbar spinal bone fusion model of bilateral intertransverse process was performed. All rats were randomly divided into four groups as follows: rats receiving 5 µg of rhBMP2 (Group I), rats receiving 10 µg of rhBMP2 (Group II), rats receiving 5 µg of AB204 (Group III), and rats receiving 10 µg of AB204 (Group IV). Simple radiographs were performed at 6 and 12 weeks after bone fusion, and direct palpation, micro-CT, and immunohistochemistry (hematoxylin-eosin stain and Masson's trichrome stain) were performed at 12 weeks after bone fusion. The qualitative degree of bone fusion was assessed as manual fusion score from direct palpation, and radio-histologic fusion score from simple radiographs, micro-CT, and immunohistochemistry. Also, the quantitative degree of bone fusion was assessed using fusion bone volume by micro-CT and serum osteocalcin level as bone turnover markers. RESULTS: The change of body weight was not different among the groups during follow-up. The qualitative degree of bone fusion assessed by direct palpation, simple radiographs, micro-CT, and histologic evaluation was significantly different among the four groups. Also, the quantitative degree of bone fusion including fusion bone volume and serum osteocalcin was significantly different among the groups. Especially, in manual fusion score, radio-histologic fusion score, and fusion bone volume, the AB204 group revealed superior results to the rhBMP2 group when using the same dose. Furthermore, even the low-dose AB204 group (Group III) showed superior results to the high-dose rhBMP2 group (Group II) in radio-histologic fusion score and fusion bone volume. CONCLUSION: The effect of bone fusion in osteoporotic rats was significantly higher in the AB204 group than in the rhBMP2 group. CLINICAL SIGNIFICANCE: If further organized animal studies and clinical trials are provided, AB204 may be a good substitute for rhBMP2 in osteoporotic spinal fusion surgery, as a superior osteogenesis inducer.

Activin A/BMP2 Chimera (AB204) Exhibits Better Spinal Bone Fusion Properties than rhBMP2.

OBJECTIVE: To compare the spinal bone fusion properties of activin A/BMP2 chimera (AB204) with recombinant human bone morphogenetic protein (rhBMP2) using a rat posterolateral spinal fusion model. METHODS: The study was designed to compare the effects and property at different dosages of AB204 and rhBMP2 on spinal bone fusion. Sixty-one male Sprague-Dawley rats underwent posterolateral lumbar spinal fusion using one of nine treatments during the study, that is, sham; osteon only; 3.0 µg, 6.0 µg, or 10.0 µg of rhBMP2 with osteon; and 1.0 µg, 3.0 µg, 6.0 µg, or 10.0 µg of AB204 with osteon. The effects and property on spinal bone fusion was calculated at 4 and 8 weeks after treatment using the scores of physical palpation, simple radiograph, micro-computed tomography, and immunohistochemistry. RESULTS: Bone fusion scores were significantly higher for 10.0 µg AB204 and 10.0 µg rhBMP2 than for osteon only or 1.0 µg AB204. AB204 exhibited more prolonged osteoblastic activity than rhBMP2. Bone fusion properties of AB204 were similar with the properties of rhBMP2 at doses of 6.0 and 10.0 µg, but, the properties of AB204 at doses of 3.0 µg exhibited better than the properties of rhBMP2 at doses of 3.0 µg. CONCLUSION: AB204 chimeras could to be more potent for treating spinal bone fusion than rhBMP2 substitutes with increased osteoblastic activity for over a longer period.

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on glial scar formation after spinal cord injury in rats.

OBJECT: This study investigated the effects of granulocyte colony-stimulating factor (G-CSF) on glial scar formation after spinal cord injury (SCI) in rats and compared the therapeutic effects between G-CSF and granulocytemacrophage colony-stimulating factor (GM-CSF) to evaluate G-CSF as a potential substitute for GM-CSF in clinical application. METHODS: Rats were randomly assigned to 1 of 4 groups: a sham-operated group (Group 1), an SCI group without treatment (Group 2), an SCI group treated with G-CSF (Group 3), and an SCI group treated with GM-CSF (Group 4). G-CSF and GM-CSF were administered via intraperitoneal injection immediately after SCI. The effects of G-CSF and GM-CSF on functional recovery, glial scar formation, and axonal regeneration were evaluated and compared. RESULTS: The rats in Groups 3 and 4 showed better functional recovery and more decreased cavity sizes than those in Group 2 (p < 0.05). Both G-CSF and GM-CSF suppressed intensive expression of glial fibrillary acidic protein around the cavity at 4 weeks and reduced the expression of chondroitin sulfate proteoglycans (p < 0.05). Also, early administration of G-CSF and GM-CSF protected axon fibers from destructive injury and facilitated axonal regeneration. There were no significant differences in comparisons of functional recovery, glial scar formation, and axonal regeneration between G-CSF and GM-CSF. CONCLUSIONS: G-CSF suppressed glial scar formation after SCI in rats, possibly by restricting the expression of glial fibrillary acidic protein and chondroitin sulfate proteoglycans, which might facilitate functional recovery from SCI. GM-CSF and G-CSF had similar effects on glial scar formation and functional recovery after SCI, suggesting that G-CSF can potentially be substituted for GM-CSF in the treatment of SCI.

Improvement in sensory function via granulocyte-macrophage colony-stimulating factor in rat spinal cord injury models.

OBJECT: The aim in this study was to determine whether granulocyte-macrophage colony-stimulating factor (GM-CSF) leads to sensory improvement in rat spinal cord injury (SCI) models. METHODS: Thirty male Sprague-Dawley rats were included in this study: 10 in the sham group (laminectomy alone without SCI), 10 in the SCI group (SCI treated with phosphate-buffered saline), and 10 in the GM-CSF treatment group (SCI treated with GM-CSF). A locomotor function test and pain sensitivity test were conducted weekly for 9 weeks after SCI or sham injury. Spinal tissue samples from all rats were immunohistochemically examined for the expression of calcitonin gene-related peptide (CGRP) and abnormal sprouting at Week 9 post-SCI. RESULTS: Granulocyte-macrophage colony-stimulating factor treatment improves functional recovery after SCI. In the tactile withdrawal threshold and frequency of the hindlimb paw, the GM-CSF group always responded with a statistically significant lower threshold than the SCI group 9 weeks after SCI (p < 0.05). The response of the forelimb and hindlimb paws to cold in the GM-CSF group always reflected a statistically significant lower threshold than in the SCI group 9 weeks after injury (p < 0.05). Decreased CGRP expression, observed by density and distribution area, was noted in the GM-CSF group (optical density 113.5 ± 20.4) compared with the SCI group (optical density 143.1 ± 18.7; p < 0.05). CONCLUSIONS: Treatment with GM-CSF results in functional recovery, improving tactile and cold sense recovery in a rat SCI model. Granulocyte-macrophage colony-stimulating factor also minimizes abnormal sprouting of sensory nerves after SCI.

GM-CSF inhibits glial scar formation and shows long-term protective effect after spinal cord injury.

OBJECT: This study investigated the effects of granulocyte macrophage-colony stimulating factor (GM-CSF) on the scar formation and repair of spinal cord tissues in rat spinal cord injury (SCI) model. METHODS: Sprague-Dawley male rats (8 weeks old) were randomly divided into the sham-operated group, spinal cord injury group, and injury with GM-CSF treated group. A spinal cord injury was induced at T9/10 levels of rat spinal cord using a vascular clip. GM-CSF was administrated via intraperitoneal (IP) injection or on the dural surface using Gelfoam at the time of SCI. The morphological changes, tissue integrity, and scar formation were evaluated until 4 weeks after SCI using histological and immunohistochemical analyses. RESULTS: The administration of GM-CSF either via IP injection or local treatment significantly reduced the cavity size and glial scar formation at 3-4 weeks after SCI. GM-CSF also reduced the expression of core proteins of chondroitin sulfate proteoglycans (CSPGs) such as neurocan and NG2 but not phosphacan. In particular, an intensive expression of glial fibriallary acidic protein (GFAP) and neurocan found around the cavity at 4 weeks was obviously suppressed by GM-CSF. Immunostaining for neurofilament (NF) and Luxol fast blue (LFB) showed that GM-CSF preserved well the axonal arrangement and myelin structure after SCI. The expression of GAP-43, a marker of regenerating axons, also apparently increased in the rostral grey matter by GM-CSF. CONCLUSION: These results suggest that GM-CSF could enhance long-term recovery from SCI by suppressing the glial scar formation and enhancing the integrity of axonal structure.