Protective Effects of PEP-1-GSTA2 Protein in Hippocampal Neuronal Cell Damage Induced by Oxidative Stress.

Glutathione S-transferase alpha 2 (GSTA2), a member of the glutathione S-transferase family, plays the role of cellular detoxification against oxidative stress. Although oxidative stress is related to ischemic injury, the role of GSTA2 against ischemia has not been elucidated. Thus, we studied whether GSTA2 prevents ischemic injury by using the PEP-1-GSTA2 protein which has a cell-permeable protein transduction domain. We revealed that cell-permeable PEP-1-GSTA2 transduced into HT-22 cells and markedly protected cell death via the inhibition of reactive oxygen species (ROS) production and DNA damage induced by oxidative stress. Additionally, transduced PEP-1-GSTA2 promoted mitogen-activated protein kinase (MAPK), and nuclear factor-kappaB (NF-κB) activation. Furthermore, PEP-1-GSTA2 regulated Bcl-2, Bax, cleaved Caspase-3 and -9 expression protein levels. An in vivo ischemic animal model, PEP-1-GSTA2, markedly prevented the loss of hippocampal neurons and reduced the activation of microglia and astrocytes. These findings indicate that PEP-1-GSTA2 suppresses hippocampal cell death by regulating the MAPK and apoptotic signaling pathways. Therefore, we suggest that PEP-1-GSTA2 will help to develop the therapies for oxidative-stress-induced ischemic injury.

Synaptic Cell Adhesion Molecule 3 (SynCAM3) Deletion Promotes Recovery from Spinal Cord Injury by Limiting Glial Scar Formation.

Synaptic cell adhesion molecules (SynCAMs) play an important role in the formation and maintenance of synapses and the regulation of synaptic plasticity. SynCAM3 is expressed in the synaptic cleft of the central nervous system (CNS) and is involved in the connection between axons and astrocytes. We hypothesized that SynCAM3 may be related to the astrocytic scar (glial scar, the most important factor of CNS injury treatment) through extracellular matrix (ECM) reconstitution. Thus, we investigated the influence of the selective removal of SynCAM3 on the outcomes of spinal cord injury (SCI). SynCAM3 knock-out (KO) mice were subjected to moderate compression injury of the lower thoracic spinal cord using wild-type (WT) (C57BL/6JJc1) mice as controls. Single-cell RNA sequencing analysis over time, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and immunohistochemistry (IHC) showed reduced scar formation in SynCAM3 KO mice compared to WT mice. SynCAM3 KO mice showed improved functional recovery from SCI by preventing the transformation of reactive astrocytes into scar-forming astrocytes, resulting in improved ECM reconstitution at four weeks after injury. Our findings suggest that SynCAM3 could be a novel therapeutic target for SCI.

Bazedoxifene, a Selective Estrogen Receptor Modulator, Promotes Functional Recovery in a Spinal Cord Injury Rat Model.

In research on various central nervous system injuries, bazedoxifene acetate (BZA) has shown two main effects: neuroprotection by suppressing the inflammatory response and remyelination by enhancing oligodendrocyte precursor cell differentiation and oligodendrocyte proliferation. We examined the effects of BZA in a rat spinal cord injury (SCI) model. Anti-inflammatory and anti-apoptotic effects were investigated in RAW 264.7 cells, and blood-spinal cord barrier (BSCB) permeability and angiogenesis were evaluated in a human brain endothelial cell line (hCMEC/D3). In vivo experiments were carried out on female Sprague Dawley rats subjected to moderate static compression SCI. The rats were intraperitoneally injected with either vehicle or BZA (1mg/kg pre-SCI and 3 mg/kg for 7 days post-SCI) daily. BZA decreased the lipopolysaccharide-induced production of proinflammatory cytokines and nitric oxide in RAW 264.7 cells and preserved BSCB disruption in hCMEC/D3 cells. In the rats, BZA reduced caspase-3 activity at 1 day post-injury (dpi) and suppressed phosphorylation of MAPK (p38 and ERK) at dpi 2, hence reducing the expression of IL-6, a proinflammatory cytokine. BZA also led to remyelination at dpi 20. BZA contributed to improvements in locomotor recovery after compressive SCI. This evidence suggests that BZA may have therapeutic potential to promote neuroprotection, remyelination, and functional outcomes following SCI.

Efficacy for Whitlockite for Augmenting Spinal Fusion.

Whitlockite (WH) is the second most abundant inorganic component of human bone, accounting for approximately 25% of bone tissue. This study investigated the role of WH in bone remodeling and formation in a mouse spinal fusion model. Specifically, morphology and composition analysis, tests of porosity and surface area, thermogravimetric analysis, an ion-release test, and a cell viability test were conducted to analyze the properties of bone substitutes. The MagOss group received WH, Group A received 100% beta-tricalcium phosphate (ß-TCP), Group B received 100% hydroxyapatite (HAp), Group C received 30% HAp/70% ß-TCP, and Group D received 60% HAp/40% ß-TCP (n = 10 each). All mice were sacrificed 6 weeks after implantation, and micro-CT, hematoxylin and eosin (HE) staining, and Masson trichome (MT) staining and immunohistochemistry were performed. The MagOss group showed more homogeneous and smaller grains, and nanopores (<500 nm) were found in only the MagOss group. On micro-CT, the MagOss group showed larger fusion mass and better graft incorporation into the decorticate mouse spine than other groups. In the in vivo experiment with HE staining, the MagOss group showed the highest new bone area (mean: decortication group, 9.50%; A, 15.08%; B, 15.70%; C, 14.76%; D, 14.70%; MagOss, 22.69%; p < 0.0001). In MT staining, the MagOss group demonstrated the highest new bone area (mean: decortication group, 15.62%; A, 21.41%; B, 22.86%; C, 23.07%; D, 22.47%; MagOss, 26.29%; p < 0.0001). In an immunohistochemical analysis for osteocalcin, osteopontin, and CD31, the MagOss group showed a higher positive area than other groups. WH showed comparable bone conductivity to HAp and ß-TCP and increased new bone formation. WH is likely to be used as an improved bone substitute with better bone conductivity than HAp and ß-TCP.

Genetic Therapy for Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration can cause chronic lower back pain (LBP), leading to disability. Despite significant advances in the treatment of discogenic LBP, the limitations of current treatments have sparked interest in biological approaches, including growth factor and stem cell injection, as new treatment options for patients with chronic LBP due to IVD degeneration (IVDD). Gene therapy represents exciting new possibilities for IVDD treatment, but treatment is still in its infancy. Literature searches were conducted using PubMed and Google Scholar to provide an overview of the principles and current state of gene therapy for IVDD. Gene transfer to degenerated disc cells in vitro and in animal models is reviewed. In addition, this review describes the use of gene silencing by RNA interference (RNAi) and gene editing by the clustered regularly interspaced short palindromic repeats (CRISPR) system, as well as the mammalian target of rapamycin (mTOR) signaling in vitro and in animal models. Significant technological advances in recent years have opened the door to a new generation of intradiscal gene therapy for the treatment of chronic discogenic LBP.

An Injectable Hyaluronan-Methylcellulose (HAMC) Hydrogel Combined with Wharton's Jelly-Derived Mesenchymal Stromal Cells (WJ-MSCs) Promotes Degenerative Disc Repair.

Intervertebral disc (IVD) degeneration is one of the predominant causes of chronic low back pain (LBP), which is a leading cause of disability worldwide. Despite substantial progress in cell therapy for the treatment of IVD degeneration, significant challenges remain for clinical application. Here, we investigated the effectiveness of hyaluronan-methylcellulose (HAMC) hydrogels loaded with Wharton's Jelly-derived mesenchymal stromal cell (WJ-MSCs) in vitro and in a rat coccygeal IVD degeneration model. Following induction of injury-induced IVD degeneration, female Sprague-Dawley rats were randomized into four groups to undergo a single intradiscal injection of the following: (1) phosphate buffered saline (PBS) vehicle, (2) HAMC, (3) WJ-MSCs (2 × 104 cells), and (4) WJ-MSCs-loaded HAMC (WJ-MSCs/HAMC) (n = 10/each group). Coccygeal discs were removed following sacrifice 6 weeks after implantation for radiologic and histologic analysis. We confirmed previous findings that encapsulation in HAMC increases the viability of WJ-MSCs for disc repair. The HAMC gel maintained significant cell viability in vitro. In addition, combined implantation of WJ-MSCs and HAMC significantly promoted degenerative disc repair compared to WJ-MSCs alone, presumably by improving nucleus pulposus cells viability and decreasing extracellular matrix degradation. Our results suggest that WJ-MSCs-loaded HAMC promotes IVD repair more effectively than cell injection alone and supports the potential clinical use of HAMC for cell delivery to arrest IVD degeneration or to promote IVD regeneration.

Multimodal therapy strategy based on a bioactive hydrogel for repair of spinal cord injury.

Traumatic spinal cord injury results in permanent and serious neurological impairment, but there is no effective treatment yet. Tissue engineering approaches offer great potential for the treatment of SCI, but spinal cord complexity poses great challenges. In this study, the composite scaffold consists of a hyaluronic acid-based hydrogel, decellularized brain matrix (DBM), and bioactive compounds such as polydeoxyribonucleotide (PDRN), tumor necrosis factor-α/interferon-γ primed mesenchymal stem cell-derived extracellular vesicles (TI-EVs), and human embryonic stem cell-derived neural progenitor cells (NPC). The composite scaffold showed significant effects on regenerative prosses including angiogenesis, anti-inflammation, anti-apoptosis, and neural differentiation. In addition, the composite scaffold (DBM/PDRN/TI-EV/NPC@Gel) induced an effective spinal cord regeneration in a rat spinal cord transection model. Therefore, this multimodal approach using an integrated bioactive scaffold coupled with biochemical cues from PDRN and TI-EVs could be used as an advanced tissue engineering platform for spinal cord regeneration.

Tat-Thioredoxin-like protein 1 attenuates ischemic brain injury by regulation of MAPKs and apoptosis signaling.

Thioredoxin-like protein 1 (TXNL1), one of the thioredoxin superfamily known as redox-regulator, plays an essential in maintaining cell survival via various antioxidant and anti-apoptotic mechanisms. It is well known that relationship between ischemia and oxidative stress, however, the role of TXNL1 protein in ischemic damage has not been fully investigated. In the present study, we aimed to determine the protective role of TXNL1 against on ischemic injury in vitro and in vivo using cell permeable Tat-TXNL1 fusion protein. Transduced Tat-TXNL1 inhibited ROS production and cell death in H2O2-exposed hippocampal neuronal (HT-22) cells and modulated MAPKs and Akt activation, and pro-apoptotic protein expression levels in the cells. In an ischemia animal model, Tat-TXNL1 markedly decreased hippocampal neuronal cell death and the activation of astrocytes and microglia. These findings indicate that cell permeable Tat-TXNL1 protects against oxidative stress in vitro and in vivo ischemic animal model. Therefore, we suggest Tat-TXNL1 can be a potential therapeutic protein for ischemic injury. [BMB Reports 2023; 56(4): 234-239].

Protective effects of cell permeable Tat-PIM2 protein on oxidative stress induced dopaminergic neuronal cell death.

Background: Oxidative stress is considered as one of the main causes of Parkinson's disease (PD), however the exact etiology of PD is still unknown. Although it is known that Proviral Integration Moloney-2 (PIM2) promotes cell survival by its ability to inhibit formation of reactive oxygen species (ROS) in the brain, the precise functional role of PIM2 in PD has not been fully studied yet. Objective: We investigated the protective effect of PIM2 against apoptosis of dopaminergic neuronal cells caused by oxidative stress-induced ROS damage by using the cell permeable Tat-PIM2 fusion protein in vitro and in vivo. Methods: Transduction of Tat-PIM2 into SH-SY5Y cells and apoptotic signaling pathways were determined by Western blot analysis. Intracellular ROS production and DNA damage was confirmed by DCF-DA and TUNEL staining. Cell viability was determined by MTT assay. PD animal model was induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and protective effects were examined using immunohistochemistry. Results: Transduced Tat-PIM2 inhibited the apoptotic caspase signaling and reduced the production of ROS induced by 1-methyl-4-phenylpyridinium (MPP+) in SH-SY5Y cells. Furthermore, we confirmed that Tat-PIM2 transduced into the substantia nigra (SN) region through the blood-brain barrier and this protein protected the Tyrosine hydroxylase-positive cells by observation of immunohistostaining. Tat-PIM2 also regulated antioxidant biomolecules such as SOD1, catalase, 4-HNE, and 8-OHdG which reduce the formation of ROS in the MPTP-induced PD mouse model. Conclusion: These results indicated that Tat-PIM2 markedly inhibited the loss of dopaminergic neurons by reducing ROS damage, suggesting that Tat-PIM2 might be a suitable therapeutic agent for PD.

Tat-RAN attenuates brain ischemic injury in hippocampal HT-22 cells and ischemia animal model.

Oxidative stress plays a key role in the pathogenesis of neuronal injury, including ischemia. Ras-related nuclear protein (RAN), a member of the Ras superfamily, involves in a variety of biological roles, such as cell division, proliferation, and signal transduction. Although RAN reveals antioxidant effect, its precise neuroprotective mechanisms are still unclear. Therefore, we investigated the effects of RAN on HT-22 cell which were exposed to H2O2-induced oxidative stress and ischemia animal model by using the cell permeable Tat-RAN fusion protein. We showed that Tat-RAN transduced into HT-22 cells, and markedly inhibited cell death, DNA fragmentation, and reactive oxygen species (ROS) generation under oxidative stress. This fusion protein also controlled cellular signaling pathways, including mitogen-activated protein kinases (MAPKs), NF-κB, and apoptosis (Caspase-3, p53, Bax and Bcl-2). In the cerebral forebrain ischemia animal model, Tat-RAN significantly inhibited both neuronal cell death, and astrocyte and microglia activation. These results indicate that RAN significantly protects against hippocampal neuronal cell death, suggesting Tat-RAN will help to develop the therapies for neuronal brain diseases including ischemic injury.

Tat-GSTpi Inhibits Dopaminergic Cells against MPP+-Induced Cellular Damage via the Reduction of Oxidative Stress and MAPK Activation.

Glutathione S-transferase pi (GSTpi) is a member of the GST family and plays many critical roles in cellular processes, including anti-oxidative and signal transduction. However, the role of anti-oxidant enzyme GSTpi against dopaminergic neuronal cell death has not been fully investigated. In the present study, we investigated the roles of cell permeable Tat-GSTpi fusion protein in a SH-SY5Y cell and a Parkinson's disease (PD) mouse model. In the 1-methyl-4-phenylpyridinium (MPP+)-exposed cells, Tat-GSTpi protein decreased DNA damage and reactive oxygen species (ROS) generation. Furthermore, this fusion protein increased cell viability by regulating MAPKs, Bcl-2, and Bax signaling. In addition, Tat-GSTpi protein delivered into the substantia nigra (SN) of mice brains protected dopaminergic neuronal cell death in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD animal model. Our results indicate that the Tat-GSTpi protein inhibited cell death from MPP+- and MPTP-induced damage, suggesting that it plays a protective role during the loss of dopaminergic neurons in PD and that it could help to identify the mechanism responsible for neurodegenerative diseases, including PD.

Protective effect of GK2 fused BLVRA protein against oxidative stress-induced dopaminergic neuronal cell damage.

It is well known that oxidative stress is highly associated with Parkinson's disease (PD), and biliverdin reductase A (BLVRA) is known to have antioxidant properties against oxidative stress. In this study, we developed a novel N-acetylgalactosamine kinase (GK2) protein transduction domain (PTD) derived from adenosine A2A and fused with BLVRA to determine whether the GK2-BLVRA fusion protein could protect dopaminergic neuronal cells (SH-SY5Y) from oxidative stress in vitro and in vivo using a PD animal model. GK2-BLVRA was transduced into various cells, including SH-SY5Y cells, without cytotoxic effects, and this fusion protein protected SH-SY5Y cells and reduced reactive oxygen species production and DNA damage after 1-methyl-4-phenylpyridinium (MPP+ ) exposure. GK2-BLVRA suppressed mitogen-activated protein kinase (MAPK) activation and modulated apoptosis-related protein (Bcl-2, Bax, cleaved Caspase-3 and -9) expression levels. In the PD animal model, GK2-BLVRA transduced into the substantia nigra crossed the blood-brain barrier and markedly reduced dopaminergic neuronal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced animals. These results indicate that our novel PTD GK-2 is useful for the transduction of protein, and GK2-BLVRA exhibits a beneficial effect against dopaminergic neuronal cell death in vitro and in vivo, suggesting that BLVRA can be used as a therapeutic agent for PD.

Highly porous multiple-cell-laden collagen/hydroxyapatite scaffolds for bone tissue engineering.

Efficient vascularization within a scaffold is an essential criterion for evaluating the success of volumetric bone formation. Various strategies using angiogenic growth factors and cell-based approaches to induce effective osteogenic and angiogenic activities have been investigated. In this study, we propose a new highly porous multiple-cell-laden collagen/hydroxyapatite scaffold fabricated using a whipped bioink. After in vitro culturing of cells in the porous scaffolds for an extended culture period, osteogenic and angiogenic activities were significantly enhanced owing to the well-developed microporous cell-supporting matrix inducing efficient crosstalk between the adipose stem cells and endothelial cells compared to those of the normally bioprinted cell-constructs. Furthermore, the in vitro results were thoroughly evaluated by in vivo experiments using a posterolateral lumbar spinal fusion model of an ovariectomized mouse. Based on these results, the porous multiple-cell-laden scaffolds enhanced spine fusion in the event of osteoporosis.

Preclinical Study of Human Bone Marrow-Derived Mesenchymal Stem Cells Using a 3-Dimensional Manufacturing Setting for Enhancing Spinal Fusion.

Spinal fusion surgery is a surgical technique that connects one or more vertebrae at the same time to prevent movement between the vertebrae. Although synthetic bone substitutes or osteogenesis-inducing recombinant proteins were introduced to promote bone union, the rate of revision surgery is still high due to pseudarthrosis. To promote successful fusion after surgery, stem cells with or without biomaterials were introduced; however, conventional 2D-culture environments have resulted in a considerable loss of the innate therapeutic properties of stem cells. Therefore, we conducted a preclinical study applying 3D-spheroids of human bone marrow-dewrived mesenchymal stem cells (MSCs) to a mouse spinal fusion model. First, we built a large-scale manufacturing platform for MSC spheroids, which is applicable to good manufacturing practice (GMP). Comprehensive biomolecular examinations, which include liquid chromatography-mass spectrometry and bioinformatics could suggest a framework of quality control (QC) standards for the MSC spheroid product regarding the identity, purity, viability, and potency. In our animal study, the mass-produced and quality-controlled MSC spheroids, either undifferentiated or osteogenically differentiated were well-integrated into decorticated bone of the lumbar spine, and efficiently improved angiogenesis, bone regeneration, and mechanical stability with statistical significance compared to 2D-cultured MSCs. This study proposes a GMP-applicable bioprocessing platform and QC directions of MSC spheroids aiming for their clinical application in spinal fusion surgery as a new bone graft substitute.

Resolvin D3 Promotes Inflammatory Resolution, Neuroprotection, and Functional Recovery After Spinal Cord Injury.

Resolvins, a new family from the endogenous specialized pro-resolving mediators (SPMs), promote the resolution of the inflammatory response. Resolvin D3 (RvD3), a docosahexaenoic acid (DHA) product, has been known to suppress the inflammatory response. However, the anti-inflammatory and neuroprotective effects of RvD3 are not known in a model of spinal cord injury (SCI). Here, we investigated the anti-inflammatory and neuroprotective effect of RvD3 in a mouse model of SCI. Processes associated with anti-inflammation and angiogenesis were studied in RAW 264.7 cells and the human brain endothelial cell line hCMEC/D3, respectively. Additionally, female C57BL/6 mice were subjected to moderate compression SCI (20-g weight compression for 1 min) followed by intrathecal injection of vehicle or RvD3 (1 µg/20 µL) at 1 h post-SCI. RvD3 decreased the lipopolysaccharide (LPS)-induced production of inflammatory mediators and nitric oxide (NO) in RAW 264.7 cells and promoted an angiogenic effect in the hCMEC/D3 cell line. Treatment with RvD3 improved locomotor recovery and reduced thermal hyperalgesia in SCI mice compared with vehicle treatment at 14 days post-SCI. Remarkably, RvD3-treated mice exhibited reduced expression of inflammatory cytokines (TNF-α, IL6, IL1ß) and chemokines (CCL2, CCL3). Also, RvD3-treated mice exhibited increased expression of tight junction proteins such as zonula occludens (ZO)-1 and occludin. Furthermore, immunohistochemistry showed a decreased level of gliosis (GFAP, Iba-1) and neuroinflammation (CD68, TGF-ß) and enhanced neuroprotection. These data provide evidence that intrathecal injection of RvD3 represents a promising therapeutic strategy to promote inflammatory resolution, neuroprotection, and neurological functional recovery following SCI.

Monitoring of Functioning Status in Subjects With Chronic Stroke in South Korea Using WHODAS II.

OBJECTIVE: To follow up the long-term functioning in a community through assessing personal background and status based on the International Classification of Functioning, Disability and Health (ICF) after a stroke, by using a Korean version of World Health Organization Disability Assessment Scale II (K-WHODAS II). METHODS: We surveyed 146 patients diagnosed at the first-onset of acute stroke and discharged after Inha University Hospital, and 101 patients answered the K-WHODAS II survey. We analyzed the relationship of six functioning domains of K-WHODAS II with K-MMSE (Korean version of Mini-Mental State Examination) and K-MBI (Korean version of Modified Barthel Index) at admission and discharge, and personal background. All subjects were divided into five groups, according to the disease durations, to assess the functional changes and the differences of K-MMSE and K-MBI at the admission and discharge. RESULTS: K-MBI and K-MMSE at admission and discharge showed no significant differences in all five groups, respectively (p>0.05), reflecting no baseline disparity for long-term follow-up. All subjects showed positive gains of K-MBI and K-MMSE at discharge (p<0.05). The six functioning domains and total scores of K-WHODAS II had decreasing trends until 3 years after the stroke onset, but rose thereafter. Higher scores of K-MBI and K-MMSE, younger age, women, working status, higher educational level, and living with a partner were correlated with lower scores of K-WHODAS II (p<0.05). CONCLUSION: The long-term functioning after stroke was affected not only by cognitive and motor status in hospital, but also by certain kinds of personal background. K-WHODAS II may be used to monitor functioning status in a community and to assess personal backgrounds in subjects with chronic stroke.

Successful conservative treatment: multiple atypical fractures in osteoporotic patients after bisphosphate medication: a unique case report.

Bisphosphonates have been commonly used for the treatment of osteoporosis. However, there have been recent case reports of atypical fractures citing their long-term use, which inhibits the turnover of bone components. A 64-year-old woman visited the outpatient clinic with pain in her right thigh and ambulation difficulty. We found fractures at both pedicles of L4 vertebra. subtrochanteric region of right femur, and left femoral shaft upon a radiologic examination. She had taken intravenous ibandronic sodium for osteoporosis over 3 years. We changed the bishophonates to a parathyroid hormone because it was suspected that the multiple fractures were caused by the medication. Further, rehabilitation, including progressive weight bearing, was started. After 3 months of the conservative treatment, she was able to walk independently. In conclusion, it is necessary to evaluate the possibility of atypical fractures in osteoporotic patients when they complain of lower extremity pain and to consider alternative treatments instead of bisphosphonates.