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Supplementary Data from The Reversible Fourth-Generation EGFR Tyrosine Kinase Inhibitor OBX02–011 Overcomes C797S-Mediated Resistance in Lung Cancer

Osimertinib is an irreversible third-generation EGFR tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non–small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02–011, a reversible fourth-generation EGFR-TKI that overcomes the EGFR C797S mutation. Compared with approved EGFR-TKIs, OBX02–011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. In addition, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02–011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02–011 may be a promising new EGFR-TKI to overcome C797S-mediated resistance in NSCLC.Significance:OBX02–011 is designed to target EGFR C797S and can overcome EGFR double and triple mutations to effectively treat lung cancer.

Supplementary Table from The Reversible Fourth-Generation EGFR Tyrosine Kinase Inhibitor OBX02–011 Overcomes C797S-Mediated Resistance in Lung Cancer

Objective : Osteoporosis result from age-related decline in the number of osteoblast progenitors in the bone marrow. Probiotics have beneficial effects on the host, when administered in appropriate amounts. This study investigated the effects of probiotics expressing specific genes, especially the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing Lactobacillus plantarum CJNU 3003 (LP) on ovariectomized rats.Methods : Twenty-eight female Wistar rats (250–300 g, 12 weeks old) were divided into four groups : the sham (control), the ovariectomy (OVX)-induced osteoporosis group (OVX), the OVX and LP (OVX/LP), OVX and genetically modified BMP-2-expressing LP (OVX/LP with BMP) groups. The three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily. At 16 weeks post-OVX, blood was collected from the heart and the bilateral tibiae were extracted and were scanned by ex-vivo micro-computed tomography and stained with hematoxylin-and-eosin (H&E) and Masson’s trichrome stain for pathological assessment. The serum levels of osteocalcin (OC), rat C-telopeptide of type I collagen (CTX-I), BMP-2, and receptor activator of nuclear factor-ĸB ligand (RANKL) were measured.Results : The 3D-micro-computed tomography images showed that the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups. No significant differences were detected in trabecular thickness (Tb.Th) between control and OVX/LP with BMP groups (p>0.05). Furthermore, a tendency toward increased BMD, trabecular bone volume, Tb.Th, and trabecular number and decreased trabecular separation was found in rats in the OVX/LP with BMP groups when compared with the OVX and OVX/LP groups (p>0.05). The H&E and Masson’s trichrome stained sections showed a thicker trabecular bone in the OVX/LP with BMP group compared with the OVX and OVX/LP groups. There was no difference in serum levels of OC, CTX and RANKL control and OVX/LP with BMP groups (p>0.05). In contrast, significant differences were found in OC and CTX-1 levels between the OVX and OVX/LP with BMP groups (p

The purpose of this study was to determine the efficacy of highly concentrated tricalcium phosphate (TCP) biocomposite screws on local bone formation in a rabbit model of osteoporosis induced by bilateral ovariohysterectomy (OHE). Fourteen 24-week-old female New Zealand rabbits (weight, 3-3.5 kg) were divided into two groups: 1) OHE and biodegradable poly(lactic-co-glycolic acid) (PLGA) without s-TCP plate or screw insertion (OHE/BSR) group; and 2) OHE and biocomposite PLGA with highly concentrated s-TCP plate and screw insertion (OHE/HCCP). Both groups underwent bilateral OHE and had two different types of screws and plates inserted at the proximal tibia. Bilateral tibiae were extracted at 25 weeks post-OHE. The extracted tibiae were scanned with ex-vivo micro-computed tomography (micro-CT). Parameters including bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) were evaluated after staining the tibial samples with hematoxylin and eosin (H&E) and Masson's trichrome. We then performed pathological assessments. Micro-CT images revealed improved new bone formation near the implant in the OHE/HCCP group with higher values of BMD, BV/TV, and Tb.N but lower values of Tb.Th and Tb.Sp than the OHE/BSR group. Analyses of H&E and Masson's trichrome staining showed better new bone formation around the implant in the OHE/HCCP group than in the OHE/BSR group. The use of highly concentrated TCP biocomposite screw and plate might improve local bone formation and facilitate osteoconductivity in an osteoporotic rabbit model. This article is protected by copyright. All rights reserved.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFR

Osimertinib is an irreversible third-generation EGFR tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non–small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02–011, a reversible fourth-generation EGFR-TKI that overcomes the EGFR C797S mutation. Compared with approved EGFR-TKIs, OBX02–011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. In addition, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02–011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02–011 may be a promising new EGFR-TKI to overcome C797S-mediated resistance in NSCLC. Significance: OBX02–011 is designed to target EGFR C797S and can overcome EGFR double and triple mutations to effectively treat lung cancer.

Objective : Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI.Methods : Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining.Results : Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p

Aryl hydrocarbon receptor (AhR) antagonism can mitigate cellular damage associated with cerebral ischaemia and reperfusion (I/R) injury. This study investigated the neuroprotective effects of AhR antagonist administration before reperfusion in a rat stroke model and influence of the timing of AhR antagonist administration on its neuroprotective effects. Magnetic resonance imaging (MRI) was performed at baseline, immediately after, and 3, 8, and 24 h after ischaemia in the sham, control (I/R injury), TMF10 (trimethoxyflavone [TMF] administered 10 min post-ischaemia), and TMF50 (TMF administered 50 min post-ischaemia) groups. The TMF treatment groups had significantly fewer infarcts than the control group. At 24 h, the relative apparent diffusion coefficient values of the ischaemic core and peri-infarct region were significantly higher and relative T2 values were significantly lower in the TMF10 groups than in the control group. The TMF treatment groups showed significantly fewer terminal deoxynucleotidyl transferase dUTP nick-end labelling positive (+) cells (%) in the peri-infarct region than the control group. This study demonstrated that TMF treatment 10 or 50 min after ischaemia alleviated brain damage. Furthermore, the timing of AhR antagonist administration affected the inhibition of cellular or vasogenic oedema formation caused by a transient ischaemic stroke.

Aryl hydrocarbon receptors (AhRs) have been reported to be important mediators of ischemic injury in the brain. Furthermore, the pharmacological inhibition of AhR activation after ischemia has been shown to attenuate cerebral ischemia-reperfusion (IR) injury. Here, we investigated whether AhR antagonist administration after ischemia was also effective in ameliorating hepatic IR injury. A 70% partial hepatic IR (45-minute ischemia and 24-hour reperfusion) injury was induced in rats. We administered 6,2',4'-trimethoxyflavone (TMF, 5 mg/kg) intraperitoneally 10 minutes after ischemia. Hepatic IR injury was observed using serum, magnetic resonance imaging-based liver function indices, and liver samples. TMF-treated rats showed significantly lower relative enhancement (RE) values and serum alanine aminotransferase (ALT) and aspartate aminotransferase levels than did untreated rats at three hours after reperfusion. After 24 hours of reperfusion, TMF-treated rats had significantly lower RE values, ΔT1 values, serum ALT levels, and necrotic area percentage than did untreated rats. The expression of the apoptosis-related proteins, Bax and cleaved caspase-3, was significantly lower in TMF-treated rats than in untreated rats. This study demonstrated that inhibition of AhR activation after ischemia was effective in ameliorating IR-induced liver injury in rats.

Objective The aim of this study was to evaluate the effect for biodegradable screws containing bone morphogenetic protein-2 (BMP-2) in an osteoporotic rat model. Methods Twenty-four female Wistar rat (250–300 g, 12 weeks of age) were randomized into four groups. Three groups underwent bilateral ovariectomy (OVX). Biodegradable screws with or without BMP-2 were inserted in the proximal tibia in two implantation groups. The extracted proximal metaphysis of the tibiae were scanned by exo-vivo micro-computed tomography. Evaluated parameters included bone mineral density (BMD), trabecular bone volume (BV/TV), trabecular number, trabecular thickness, and trabecular separation (Tb.Sp). The tibia samples were pathologically evaluated by staining with by Hematoxylin and Eosin, and trichrome. Results Trabecular formation near screw insertion site was evident only in rats receiving BMP-2 screws. BMD and BV/TV significantly differed between controls and the OVX and OVX with screw groups. However, there were no significant differences between control and OVX with screw BMP groups. Tb.Sp significantly differed between control and OVX and OVX with screw groups (p

Background: The classic animal model of Parkinson’s disease (PD) using neurotoxin can only simulate fixed stages of the disease by causing irreversible damage to the nigrostriatal system. Objectives: To develop an optogenetic PD model that can modulate the severity of disease by optical stimulation by introducing the halorhodopsin (NpHR) gene into the substantia nigra compacta. Methods: Fifteen rats received injections of engineered AAV with NpHR-YFP gene into the substantia nigra. They were then subjected to illumination of 590-nm light wavelengths with 3 optical stimulation conditions, i.e., frequency-width: 5 Hz-10 ms (n = 5), 5 Hz-100 ms (n = 5), and 50 Hz-10 ms (n = 5). Eleven rats received 6-hydroxydopamine injections to establish the conventional PD model. Results: The optogenetic models showed characteristic PD manifestations, similar to those of the conventional models; the severity of forelimb akinesia correlated with the total illumination value (frequency × width). The group with a low illumination value (5 Hz-10 ms) was comparable to the conventional partial model whereas the groups with high illumination values (5 Hz-100 ms and 50 Hz-10 ms) were similar to the conventional complete model. Conclusions: An optogenetic PD model has the advantage of more appropriately representing various PD stages by controlling illumination parameters.

Objective To evaluate the efficacy of biocomposite screws used in ovariectomy (OVX)-induced osteoporotic rats. Methods Twenty-four female Wistar rats (250–300 g, 12 weeks old) were divided into 4 groups: sham group (control), OVX-induced osteoporosis group (OVX), OVX and biodegradable poly(lactic- co -glycolic acid) (PLGA) without tricalcium phosphate (β-TCP) screw insertion group (OVX/BSR), and OVX and biocomposite (PLGA with β-TCP) screw insertion group (OVX/CSR). Three groups underwent bilateral OVX, and of these, 2 groups had 2 different types of screw inserted at the proximal tibia. At 25 weeks after OVX, the bilateral tibias were extracted. The extracted tibiae were scanned by ex vivo micro-computed tomography (CT) and stained with hematoxylin and eosin (H&E) and with Masson's trichrome stain for pathological assessment. Results Compared with the ovariectomized groups, the control group had the highest values for bone mineral density (BMD), bone volume (BV)/total volume (TV), and trabecular number (Tb.N) and the lowest values for trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). In the pairwise comparison among ovariectomized groups, the OVX/CSR group had significantly higher BMD, BV/TV, and Tb.N values than the other 2 groups (OVX and OVX/BSR) and significantly lower Tb.Sp. Micro-CT scans showed clear evidence of new trabecular formation near the screw insertion site in the OVX/CSR group only. Analyses of H&E- and Masson's trichrome–stained sections showed more and thicker trabecular bone around the implant in the OVX/CSR group compared with the OVX and OVX/BSR groups. Conclusions Biocomposite screws can improve local bone quality and facilitate osteoconductivity in an osteoporotic rat model.

OBJECTIVE Functional and neural tissue recovery has been reported in many animal studies conducted with stem cells. However, the combined effect of cytokines and stem cells has not yet been adequately researched. Here, we analyzed the additive effects of granulocyte colony-stimulating factor (GCSF) on adipose-derived stem cells (ADSCs) infusion in the treatment of acute spinal cord injury (SCI) in rats. METHODS Four days after intrathecal infusion tubes implantation in Sprague-Dawley rats, SCI was induced with an infinite horizon impactor. In the Sham group (n=5), phosphate-buffered saline was injected 3, 7, and 14 days after SCI. GCSF, ADSCs, and ADSCs with GCSF were injected at the same time in the GCSF (n=8), ADSC (n=8), and ADSC+GCSF groups (n=7), respectively. RESULTS The ADSC and ADSC+GCSF groups, but not the GCSF group, showed significantly higher Basso-Beattie-Bresnahan scores than the Sham group during 8 weeks (p

While pure mesenchymal stem cell (MSC) treatment for spinal cord injury (SCI) is known to be safe, its efficacy is insufficient. Therefore, gene-modified stem cells are being developed to enhance the effect of pure MSCs. We investigated the effect of stem cell therapy through the transfection of a Wnt3a-producing gene that stimulates axonal regeneration. MSCs obtained from the human umbilical cord blood (hMSCs) were multiplied, cultivated, and transfected with the pLenti-Wnt3a-GFP viral vector to produce Wnt3a-secreting hMSCs. A total of 50 rats were injured with an Infinite Horizon impactor at the level of the T7-8 vertebrae. Rats were divided into five groups according to the transplanted material: (1) phosphate-buffered saline injection group (sham group, n = 10); (Pertz et al. Proc Natl Acad Sci USA 105:1931–1936, 39) Wnt3a protein injection group (Wnt3a protein group, n = 10); (3) hMSC transplantation group (MSC group, n = 10); (4) hMSCs transfected with the pLenti vector transplantation group (pLenti-MSC group, n = 10); (5) hMSCs transfected with the pLenti+Wnt3a vector transplantation group (Wnt3a-MSC group, n = 10). Behavioral tests were performed daily for the first 3 days after injury and then weekly for 8 weeks. The injured spinal cords were extracted, and axonal regeneration markers including choline acetyltransferase (ChAT), growth-associated protein 43 (GAP43), and microtubule-associated protein 2 (MAP2) were investigated by immunofluorescence, RT-PCR, and western blotting. Seven weeks after the transplantation (8 weeks after SCI), rats in the Wnt3a-MSC group achieved significantly higher average scores in the motor behavior tests than those in the other groups (p

Background Context The New York University (NYU) impactor and the Infinite Horizon (IH) impactor are used to create spinal cord injury (SCI) models. However, the parameters of these two devices that yield equivalent SCI severity remain unclear. Purpose To identify equivalent parameters, rats with SCIs induced by either device set at various parameters were subjected to behavioral and histologic analyses. Study Design This is an animal laboratory study. Methods Groups of eight rats acquired SCIs by dropping a 10 g rod from a height of 25 mm or 50 mm by using the NYU device or by delivering a force of 150 kdyn, 175 kdyn, 200 kdyn, or 250 kdyn by using the IH impactor. All injured rats were tested weekly for 8 weeks by using the Basso, Beattie, and Bresnahan (BBB) test and the ladder rung test. On the 10th week, the lesion volume of each group was measured by using a 9.4 Tesla magnetic resonance imaging (MRI), and the spinal cords were subjected to histologic analysis using anterograde biotinylated dextran amine (BDA) tracing and immunofluorescence staining with an anti-protein kinase C-gamma (PKC-γ) antibody. Results Basso, Beattie, and Bresnahan test scores between the 25 mm and the 200 kdyn groups as well as between the 50 mm and and 250 kdyn groups were very similar. Although it was not statistically significant, the mean scores of the ladder rung test in the 200 kdyn group were higher than the 25 mm group at all assessment time points. There was a significantly different cavity volume only between the 50 mm and the 200 kdyn groups. Midline sagittal images of the spinal cord on the MRI revealed that the 25 mm group predominantly had dorsal injuries, whereas the 200 kdyn group had deeper injuries. Anterograde tracing with BDA showed that in the 200 kdyn group, the dorsal corticospinal tract of the caudal area of the lesion was labeled. Similar labeling was not observed in the 25 mm group. Immunofluorescence staining of PKC-γ also revealed strong staining of the dorsal corticospinal tract in the 200 kdyn group but not in the 25 mm group. Conclusions The 25 mm injuries generated by the NYU impactor are generally equivalent to the 200 kdyn injuries generated by using the IH impactor. However, differences in the ladder rung test scores, MRI images, BDA traces, and PKC-γ staining demonstrate that the two devices exert qualitatively different impacts on the spinal cord.

Background: The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the amelioration of levodopa-induced dyskinesia. However, electrical deep brain stimulation cannot specifically activate or inactivate selected types of neurons. Objectives: We applied optogenetics as an alternative treatment to deep brain stimulation for levodopa-induced dyskinesia, and also to confirm that the mechanism of levodopa-induced dyskinesia amelioration by subthalamic nucleus deep brain stimulation is mediated through neuronal inhibition. Methods: 6-hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus (AAV) or hSynapsin1-YFP AAV. Two weeks after viral injections, all rats were treated with daily injections of levodopa. Then, the optic fiber was implanted into the ipsilateral subthalamic nucleus. We performed various behavioral tests to evaluate the changes in levodopa-induced dyskinesias after optogenetic expression and illumination in the subthalamic nucleus. Results: The behavioral tests revealed that optical inhibition of the subthalamic nucleus significantly ameliorated levodopa-induced dyskinesia by reducing the duration of the dyskinesias as well as the severity of axial dyskinesia. Conclusions: These findings will provide a useful foundation for the future development of optogenetic modulation systems that could be considered as an approach to dyskinesia therapy.

We performed optogenetic inactivation of rats' entopeduncular nucleus (EP, homologous to primates' globus pallidus interna (GPi)) and investigated the therapeutic effect in a rat model of PD. 6-Hydroxydopamine (6-OHDA)-induced hemiparkinsonian rats were injected with either a virus for halorhodopsin expression that is used to inactivate GABAergic neurons or a control virus injection and received optic fiber insertion. All the rats were illuminated by 590 nm of light. Each rat was then subjected to sequential sessions of stepping tests under controlled illumination patterns. The stepping test is a reliable evaluation method for forelimb akinesia. The number of adjusting steps was significantly higher in experimental (optogene with reporter gene expression) (5Hz - 10ms: 15.7 ± 1.9, 5Hz - 100ms: 16.0 ± 1.8, continuous: 21.6 ± 1.9) than control rats (reporter gene expression) (5Hz-10ms: 1.9 ± 1.1, 5Hz-100ms: 2.6 ± 1.0, continuous: 2.5 ± 1.2) (p0.001). Continuous EP illumination showed a significantly higher improvement of forelimb akinesia than other illumination patterns (p0.01). Optogene expression in the GABAergic neurons of the EP was confirmed by immunohistochemistry. Optogenetic inhibition of EP was effective to improve contralateral forelimb akinesia. However, further studies using prolonged illumination are needed to investigate the best illumination pattern for optogenetic stimulation.

We investigated the effect of in vitro cultured Wnt3a-producing fibroblasts on positive bone balance and regeneration of the osteopenic skeleton. Thirty-six female Wistar rats (250–300 g, aged 12 weeks) were randomized into three groups: a control group (sham-operated), ovariectomy (OVX) group, and OVX with Wnt-cell injection (OVX with Wnt) group. Wnt3a-producing fibroblasts were injected into the lateral tail vein at 2 and 4 weeks after OVX. Both tibias were removed at 5, 6, 7, and 8 weeks after OVX, and pathological and micro-CT evaluations were performed. We also evaluated β-catenin expression by immunohistochemical staining. For bone metabolism detection, we evaluated the expression of bone alkaline phosphatase (BALP), osteocalcin, and C-telopeptide of collagen type I. Bone mineral density, trabecular bone volume, trabecular number, trabecular thickness were higher, while trabecular seperation was lower, in the OVX with Wnt group than in the OVX group. BALP and osteocalcin levels were significantly higher in the OVX with Wnt group compared to the control and OVX groups. β-catenin expression was significantly lower in the OVX group than in the other two groups. Based on these results, we hypothesized that Wnt3a-producing fibroblasts may be effective for the induction of bone formation.

This study investigated the efficacy of adult adipose tissue-derived stem cells in restoring bone using an osteoporotic rat model. Thirty-six female Wistar rats (250–300 g, 12 weeks) were randomized into three equal groups: SHAM group (sham-operated), ovariectomy-induced (OVX) group and OVX with stem cell injection group (OVX with stem). Femur extraction and blood sampling were performed at 5, 6, 7 and 8 weeks. The proximal femoral metaphysis was scanned by micro-CT and evaluated for changes in various histomorphometric parameters. β-catenin expression was determined using H&E and immunohistochemistry. Bone metabolism was assessed using measurements of C-telopeptide of collagen type I (CTX), osteocalcin, and bone alkaline phosphatase (BALP). There was a trend for an increase in the bone mineral density (BMD), bone volume/trabecular bone volume (BV/TV) and trabecular number (Tb.N) in the OVX with stem group as compared with the OVX group, and decrease in the OVX with stem group as compared with SHAM group. The trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) of the OVX group were increased compared with those values in the SHAM and OVX with stem group. BALP levels were higher in the OVX group as compared with that in the other two groups and osteocalcin levels were highest in the SHAM group and slightly increased in the OVX with stem group as compared with the OVX group. This study may help us gain an understanding of the role of MSCs in the pathophysiology and treatment of osteoporosis.

This study aimed to investigate whether stem cell transplantation in acute stage is safe, and determined the relationship between the treatment effects and the degree of intervertebral disc degeneration. IVD rat model was created by needle aspiration. Thirty rats were divided into 5 groups as follow: normal (disc without injury), sham (only disc injury without injection), Day 3 (cell injection at 3 day after disc degeneration), Day 7 (cell injection at 7 day after disc degeneration), and Day 14 (cell injection at 14 day after disc degeneration). Magnetic resonance imaging (MRI) and histological evaluation were performed 4 times, 2 weeks after the degeneration was induced and 2, 4, and 6 weeks after cell transplantation at the level of 3 discs. We also evaluated the expression of the disc regeneration markers, such as aggrecan, collagen II, and SOX-9, by western blotting. Day 3 and Day 7 groups showed significantly lesser reduction of the disc height and better restoration of the MRI signal intensity of the intervertebral disc. In hematoxylin and eosin staining of annulus fibrosus, Day 3 and Day 7 groups showed a better grading than Day 14 group did, according to the Nishimura and Mochida grading system. The results of the western blotting revealed patterns similar to the radiologic findings. We revealed that transplanted ADMSCs promote the regeneration of the nucleus pulposus and extracellular matrix. Additionally, we demonstrated that regenerative stem cell therapy is more effective when applied at relatively early stages of degeneration.

OBJECTIVE This study describes a method for inducing spinal cord injuries in dogs by using balloon catheters via laminectomy and the subsequent changes in the electrophysiological response. METHODS Female Beagle (Orient Bio, Seongnam, Korea) dogs weighing 10 kg at the time of injury were used. Under inhalation anesthesia, a posterior midline approach laminectomy was performed. A silicone balloon catheter (size 6 Fr; Sewoon Medical, Cheonan, Korea) was then inserted into the vertebral canal at the center of T10. The balloon was inflated to the maximum volume for 1, 2, or 3 days. Open field testing was performed for evaluating motor functions of the hindlimbs. Motor evoked potentials (MEPs) induced by electrical and magnetic stimulation were recorded before and after spinal cord injury. RESULTS Open field testing yielded locomotor scores of 0 or 1 for dogs subjected to compression for 3 days. These dogs showed no obvious improvement throughout the observation period, and the tonus of their hindlimbs was flaccid. In contrast, motor functions of dogs that had experienced compression for 1 or 2 days were variable, and all dogs showed spastic tonus in their hindlimbs. In dogs subjected to after compression for 3 days, electrically stimulated MEPs for the hindlimbs showed a significant amplitude reduction. Further, hindlimb movements were not evoked by magnetic stimulation of the cervical spine and vertex area. CONCLUSION Compression for 3 days with a balloon catheter is a safe, reproducible, and reliable method for evaluating electrophysiological changes in a dog model of complete spinal cord injury.

A double toxin-double lesion strategy is appropriate for mimicking of striatonigral degeneration. Because knowledge of human pathology is limited, animal models must be well characterized prior to testing of therapeutic approaches to treat multiple system atrophy. In double-toxin animal models, however, reduced contralateral rotation after apomorphine injection is restored within a few weeks via an unknown mechanism; the animals thus revert to PD status. We assessed this phenomenon using multitracer microPET and tissue staining.Five adult male Wistar rats received injections of 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle (MFB), followed 3 weeks later by injections of quinolinic acid (QA) into the ipsilateral striatum. Apomorphine-induced rotation tests were performed 1 week after each injection, and 6 and 10 weeks after QA injection. Rotarod tests were performed weekly after 6-OHDA injection. MSA-p status was characterized by microPET 5 and 10 weeks after QA injection using the tracers 2-deoxy-2-[(18)F]-fluoro-D-glucose ([(18)F]-FDG) and [(18)F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([(18)F]-FP-CIT). Histological changes were evaluated by tyrosine hydroxylase (TH) and cresyl violet staining.The numbers of apomorphine-induced rotations increased contralaterally after 6-OHDA lesions were created, but decreased significantly after QA administration (p = 0.007). Five weeks after QA injection, however, contralateral rotation again increased and persisted for 1 month. Rotarod rotation differed significantly between the intact and PD states (p0.05), but not between the PD and MSA-p states. MicroPET revealed glucose hypometabolism and dopamine transporter (DAT) impairment on the lesioned side of the striatum 1 and 2 months after QA lesion surgery. Loss of nigral cells was confirmed by TH immunostaining, and striatal atrophy was observed upon cresyl violet staining.Pathological changes consistent with MSA-p can be generated by the double toxin-double lesion method and persist during follow-up. Behavioral tests, such as drug-induced rotation and rotarod tests, are not appropriate for long-term follow-up in the MSA-p model, suggesting the need for development of more appropriate behavioral tests.

Axonal regeneration is a prerequisite for recovery from spinal cord injury. Here, we investigated whether Wnt3a-secreting fibroblasts exert a favorable effect on spinal cord regeneration in spinal cord-injured rats. Spinal cord injury (SCI) was induced in rats (n = 21) using an NYU impactor. One week after SCI, rats were assigned to a Wnt3a-secreting fibroblast transplantation group (Wnt group, n = 7), a L929 fibroblast transplantation group (vehicle group, n = 7), and contusion only group (sham group, n = 7). Motor function was tested weekly for 6 weeks. Manganese-enhanced magnetic resonance imaging (ME-MRI) was performed twice, once before cell transplantation and again 5 weeks after cell transplantation. After ME-MRI, expression of the axonal regeneration marker GAP-43 was assessed by immunohistochemistry (IHC). In the Wnt group, the mean Basso–Beattie–Bresnahan score was higher than that of the vehicle and sham groups throughout the observation period. The Wnt group also exhibited stronger signal intensity on ME-MRI, and IHC revealed that GAP-43 was highly expressed in the injured spinal cord in the Wnt group. These results strongly suggest that transplanted Wnt3a secreting fibroblasts promote axonal regeneration and functional improvement after SCI. Although further investigation will be necessary to clarify the intracellular mechanism by which Wnt signaling promotes axonal regeneration and functional improvement, this approach could be a highly promising therapeutic strategy for SCI.

OBJECTIVE Previously, we presented the fabrication and characterization of a flexible penetrating microelectrode array (FPMA) as a neural interface device. In the present study, we aim to prove the feasibility of the developed FPMA as a chronic intrafascicular recording tool for peripheral applications. APPROACH For recording from the peripheral nerves of medium-sized animals, the FPMA was integrated with an interconnection cable and other parts that were designed to fit canine sciatic nerves. The uniformity of tip exposure and in vitro electrochemical properties of the electrodes were characterized. The capability of the device to acquire in vivo electrophysiological signals was evaluated by implanting the FPMA assembly in canine sciatic nerves acutely as well as chronically for 4 weeks. We also examined the histology of implanted tissues to evaluate the damage caused by the device. MAIN RESULTS Throughout recording sessions, we observed successful multi-channel recordings (up to 73% of viable electrode channels) of evoked afferent and spontaneous nerve unit spikes with high signal quality (SNR > 4.9). Also, minor influences of the device implantation on the morphology of nerve tissues were found. SIGNIFICANCE The presented results demonstrate the viability of the developed FPMA device in the peripheral nerves of medium-sized animals, thereby bringing us a step closer to human applications. Furthermore, the obtained data provide a driving force toward a further study for device improvements to be used as a bidirectional neural interface in humans.

BACKGROUND The inhibition of neuronal activity by electrical deep brain stimulation is one of the mechanisms explaining the therapeutic effects in patients with Parkinson disease (PD) but cannot specifically activate or inactivate different types of neurons. Recently, a new technology based on optogenetics has been developed to modulate the activity of specific neurons. However, the therapeutic effects of optical inactivation in the subthalamic nucleus (STN) have not been fully investigated. OBJECTIVE To perform various behavioral tests to evaluate changes in motor functions in a PD rat model after optogene expression and, unlike previous studies, to assess the therapeutic effects of direct optogenetic inactivation in the STN. METHODS 6-Hydroxydopamine-induced hemiparkinsonian rats received injections of hSynapsin1-NpHR-YFP adeno-associated virus or an equivalent volume of phosphate-buffered saline. Three weeks after injection of adeno-associated virus or phosphate-buffered saline, the optic fiber was implanted into the ipsilateral STN. A stepping test, a cylinder test, and an apomorphine-induced rotation test were performed in 3 sequential steps: during light-off state, during light stimulation, and again during light-off state. RESULTS Stepping tests revealed that optical inhibition of the STN significantly improved 6-hydroxydopamine-induced forelimb akinesia. PD motor signs, as assessed by cylinder and apomorphine tests, were not affected by optical inhibition. Immunofluorescence revealed that halorhodopsin was highly expressed and colocalized with vesicular glutamate transporter 2 in the STN. CONCLUSION Optogenetic inhibition in the STN may be effective in improving contralateral forelimb akinesia but not in changing forelimb preference or reducing dopaminergic receptor supersensitivity. These findings are useful as a basis for future studies on optogenetics in PD.

Transplantation of neural stem cells has been reported as a possible approach for replacing impaired dopaminergic neurons. In this study, we tested the efficacy of early-stage human dental papilla-derived stem cells and human brain-derived neural stem cells in rat models of 6-hydroxydopamine-induced Parkinson's disease. Rats received a unilateral injection of 6-hydroxydopamine into right medial forebrain bundle, followed 3 weeks later by injections of PBS, early-stage human dental papilla-derived stem cells, or human brain-derived neural stem cells into the ipsilateral striatum. All of the rats in the human dental papilla-derived stem cell group died from tumor formation at around 2 weeks following cell transplantation. Postmortem examinations revealed homogeneous malignant tumors in the striatum of the human dental papilla-derived stem cell group. Stepping tests revealed that human brain-derived neural stem cell transplantation did not improve motor dysfunction. In apomorphine-induced rotation tests, neither the human brain-derived neural stem cell group nor the control groups (PBS injection) demonstrated significant changes. Glucose metabolism in the lesioned side of striatum was reduced by human brain-derived neural stem cell transplantation. [(18)F]-FP-CIT PET scans in the striatum did not demonstrate a significant increase in the human brain-derived neural stem cell group. Tyrosine hydroxylase (dopaminergic neuronal marker) staining and G protein-activated inward rectifier potassium channel 2 (A9 dopaminergic neuronal marker) were positive in the lesioned side of striatum in the human brain-derived neural stem cell group. The use of early-stage human dental papilla-derived stem cells confirmed its tendency to form tumors. Human brain-derived neural stem cells could be partially differentiated into dopaminergic neurons, but they did not secrete dopamine.

Objective Recent studies have shown encouraging progress toward the use of autogenic and allogenic mesenchymal stem cells (MSCs) to arrest, or even lead to partial regeneration in, intervertebral disc (IVD) degeneration. However, this technology is still in its infancy, and further development is required. The aim of this study was to analyze whether rat adipose-derived mesenchymal stem cells (ADMSC) can differentiate towards IVD-like cells after treatment with transforming growth factor β3 (TGF-β3) in vitro. We also performed quantitative analysis of gene expression for ADMSC only, ADMSCs treated with TGF-β3, and co-cultured ADMSCs treated with TGF-β3. Methods ADMSCs were sub-cultured to homogeneity and used in fluorocytometry assays for CD11, CD45, and CD90/Thy1. ADMSCs were differentiated in spheroid culture towards the chondrogenic lineage by the presence of TGF-β3, dexamethasone, and ascorbate. We also co-cultured pure ADMSCs and nucleus pulposus cells in 24-well plates, and performed immunohistochemical staining, western blotting, and RT-PCR for quantitativeanalysis of gene expression. Results Results of fluorocytometry were positive for CD90/Thy1 and negative for CD11 and CD45. TGF-β3-mediated induction of ADMSCs led to the expression of the differentiation markers of intervertebral disc-like cells, such as aggrecan, collagen II, and sox-9. Co-cultured ADMSCs treated with TGF-β3 showed higher expression of differentiation markers and greater extracellular matrix production compared with ADMSCs treated with TGF-β3 alone. Conclusion ADMSC treated with TGF-β3 may be an attractive source for regeneration therapy in degenerative IVD. These findings may also help elucidate the pathologic mechanism of MSC therapy in the degeneration of IVD in vivo.

Wnt proteins are bifunctional axon guidance molecules, several of which appear to mediate guidance of corticospinal tract axons along the spinal cord. Here, we studied increasing effect on regeneration by Wnt-containing alginate scaffolds on spinal cord injury (SCI). A total of 32 rats were injured at the T7–8 level with an NYU impactor. According to transplantation materials, rats were classified into four groups: a Wnt3a-secreting fibroblast transplantation group (Wnt group, n = 8), a Wnt3a-secreting fibroblast with alginate transplantation group (Wnt + alginate group, n = 8), an alginate transplantation group (alginate group, n = 8), and a contusion-only group (sham group, n = 8). Behavioral tests were performed on the first, second, and third days after injury, and then weekly for 8 weeks. Five of the eight rats from each group were selected for manganese-enhanced magnetic resonance imaging (ME-MRI). Two rats from each group were examined for GAP43 and MAP2 expression using monoclonal and polyclonal primary antibodies, respectively. Seven weeks after transplantation (8 weeks after SCI), Wnt + alginate group rats achieved an average Basso–Beattie–Bresnahan locomotor score of 19.0, which was significantly higher than that of other groups. ME-MRI at 8 weeks after SCI revealed significantly higher relative signal intensities in the Wnt + alginate group. Gap43 and Map2 immunostaining, showed strong positive in the Wnt + alginate group. The Wnt + alginate complex exerted significantly enhanced recovery in a rat SCI model compared to alginate or Wnt3a alone. These results suggest that alginate scaffolds facilitate the regeneration of axon working with Wnt3a protein that promotes regeneration of the injured spinal cord.

Objective: Promoting axonal regeneration is one of essential goals to be achieved for effective repair from spinal cord injury (SCI). The authors performed an experiment on the effect of Wnt-3a producing fibroblast injection on SCI. Methods: SCI is performed by New York University (NYU) impactor in 21 rats, and 1 week after injury, the rats were grouped into Wnt-3a producing fibroblast transplantation group (Wnt group, n=7), fibroblast transplantation group (vehicle group, n=7), and no treatment group (sham group, n=7). For the behavioral tests, the Basso, Beattie and Bresnahan (BBB) locomotor rating scale were evaluated. Also, to study radiological evidence of axon regeneration, the rats underwent manganese enhanced MRI (ME-MRI) twice, before treatment and 5 weeks after treatment. Results: On average, the sham group and vehicle group rats attained BBB score of 10.4 and 12.1, respectively, whereas Wnt group rats achieved a significantly higher BBB score of 14.0 at 5 weeks after treatment. At 5 weeks after treatment, relative signal intensities of ME-MRI was significantly higher in the Wnt group than in the vehicle group and sham group at 5 weeks after injury. Conclusion: In this study, the authors observed that neurological recovery after SCI in rats were improved by Wnt-3a producing fibroblasts which were injected intramedullary at 1 week after SCI. (J Kor Neurotraumatol Soc 2010;6:1-5)