Joongkee Min

Optogenetic inactivation of the subthalamic nucleus improves forelimb akinesia in a rat model of Parkinson disease.

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.

Adipose tissue-derived stem cells in the ovariectomy-induced postmenopausal osteoporosis rat model

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.

Are human dental papilla-derived stem cell and human brain-derived neural stem cell transplantations suitable for treatment of Parkinson's disease?

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 Parkinsons 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. [18F]-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.

Optogenetic Inhibition of the Subthalamic Nucleus Reduces Levodopa-Induced Dyskinesias in a Rat Model of Parkinson's Disease

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.

Analysis of equivalent parameters of two spinal cord injury devices: the New York University impactor versus the Infinite Horizon impactor.

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.

Wnt3a-producing fibroblasts in ovariectomy-induced osteoporosis in a rat model

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.

Implantation of adipose tissue-derived mesenchymal stem cells in degenerative intervertebral disc of rat: When is the most effective time during the degeneration period

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.

Is There Additive Therapeutic Effect When GCSF Combined with Adipose-Derived Stem Cell in a Rat Model of Acute Spinal Cord Injury?

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<0.01), but no significant difference between the ADSC and ADSC+GCSF groups. In the ladder rung test, all four groups were significantly different from each other, with the ADSC+GCSF group showing the best improvement (p<0.01). On immunofluorescent staining (GAP43, MAP2), western blotting (GAP43), and reverse transcription polymerase chain reaction (GAP43, nerve growth factor), the ADSC and ADSC+GCSF groups showed higher levels than the Sham and GCSF groups. Conclusion Our analyses suggest that the combination of GCSF and ADSCs infusions in acute SCI in the rat does not have a significant additive effect. Hence, when combination agents for SCI stem cell therapy are considered, molecules other than GCSF, or modifications to the methodology, should be investigated.

Changes of the Electrophysiological Study in Dogs with Acute Spinal Cord Injury

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.

Biodegradable Screws Containing Bone Morphogenetic Protein-2 in an Osteoporotic Rat Model

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<0.05), and between the OVX and OVX with screw BMP group (p<0.05), with no statistically significant difference between control and OVX with screw BMP groups. Over the 12 weeks after surgery, bone lamellae in direct contact with the screw developed more extensive and thicker trabecular bone around the implant in the OVX with screw BMP group compared to the OVX with screw group. Conclusion Biodegradable screws containing BMP-2 improve nearby bone conditions and enhance ostoeintegration between the implant and the osteoporotic bone.