A multi-channel collagen conduit with aligned Schwann cells and endothelial cells for enhanced neuronal regeneration in spinal cord injury.

Traumatic spinal cord injury (SCI) leads to Wallerian degeneration and the accompanying disruption of vasculature leads to ischemia, which damages motor and sensory function. Therefore, understanding the biological environment during regeneration is essential to promote neuronal regeneration and overcome this phenomenon. The band of Büngner is a structure of an aligned Schwann cell (SC) band that guides axon elongation providing a natural recovery environment. During axon elongation, SCs promote axon elongation while migrating along neovessels (endothelial cells [ECs]). To model this, we used extrusion 3D bioprinting to develop a multi-channel conduit (MCC) using collagen for the matrix region and sacrificial alginate to make the channel. The MCC was fabricated with a structure in which SCs and ECs were longitudinally aligned to mimic the sophisticated recovering SCI conditions. Also, we produced an MCC with different numbers of channels. The aligned SCs and ECs in the 9-channel conduit (9MCC-SE) were more biocompatible and led to more proliferation than the 5-channel conduit (5MCC-SE) in vitro. Also, the 9MCC-SE resulted in a greater healing effect than the 5MCC-SE with respect to neuronal regeneration, remyelination, inflammation, and angiogenesis in vivo. The above tissue recovery results led to motor function repair. Our results show that our 9MCC-SE model represents a new therapeutic strategy for SCI.

Multi-modulation of immune-inflammatory response using bioactive molecule-integrated PLGA composite for spinal fusion.

Despite current developments in bone substitute technology for spinal fusion, there is a lack of adequate materials for bone regeneration in clinical applications. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commercially available, but a severe inflammatory response is a known side effect. Bone graft substitutes that enhance osteogenesis without adverse effects are needed. We developed a bioactive molecule-laden PLGA composite with multi-modulation for bone fusion. This bioresorbable composite scaffold was considered for bone tissue engineering. Among the main components, magnesium hydroxide (MH) aids in reduction of acute inflammation affecting disruption of new bone formation. Decellularized bone extracellular matrix (bECM) and demineralized bone matrix (DBM) composites were used for osteoconductive and osteoinductive activities. A bioactive molecule, polydeoxyribonucleotide (PDRN, PN), derived from trout was used for angiogenesis during bone regeneration. A nano-emulsion method that included Span 80 was used to fabricate bioactive PLGA-MH-bECM/DBM-PDRN (PME2/PN) composite to obtain a highly effective and safe scaffold. The synergistic effect provided by PME2/PN improved not only osteogenic and angiogenic gene expression for bone fusion but also improved immunosuppression and polarization of macrophages that were important for bone tissue repair, using a rat model of posterolateral spinal fusion (PLF). It thus had sufficient biocompatibility and bioactivity for spinal fusion.

Concentration-Dependent Efficacy of Recombinant Human Bone Morphogenetic Protein-2 Using a HA/ß-TCP Hydrogel Carrier in a Mini-Pig Vertebral Oblique Lateral Interbody Fusion Model.

Bone morphogenetic protein-2 (BMP-2) is used in the treatment of degenerative spinal disease and vertebral fractures, spine fusion, dental surgery, and facial surgery. However, high doses are associated with side effects such as inflammation and osteophytes. In this study, we performed spinal fusion surgery on mini-pigs using BMP-2 and a HA/ß-TCP hydrogel carrier, and evaluated the degree of fusion and osteophyte growth according to time and dosage. Increasing the dose of BMP-2 led to a significantly higher fusion rate than was observed in the control group, and there was no significant difference between the 8-week and 16-week samples. We also found that the HA + ß-TCP hydrogel combination helped maintain the rate of BMP-2 release. In conclusion, the BMP-2-loaded HA/ß-TCP hydrogel carrier used in this study overcame the drawback of potentially causing side effects when used at high concentrations by enabling the sustained release of BMP-2. This method is also highly efficient, since it provides mineral matter to accelerate the fusion rate of the spine and improve bone quality.

Mediating Effects of Anger Expression in the Relationship of Work Stress with Burnout among Hospital Nurses Depending on Career Experience.

OBJECTIVES: This study examined the mediating effects of anger expression in the relationship between work stress and burnout among nurses with more than or less than 3 years of career experience. METHODS: A cross-sectional study was conducted with 454 nurses working at three university hospitals in South Korea. Nurses completed a survey consisting of demographic questions, the Maslach Burnout Inventory, the Work Stress Scale, and the Korean version of the Anger Expression Inventory. A multiple-group mediation analysis was performed using structural equation modeling. RESULTS: In total (N = 454), work stress directly affected burnout, and all anger expressions indirectly affected the relationship of work stress with burnout. Different mediating effects of anger expression style according to career experience were shown; anger-out and anger-in in nurses with less experience (n = 184) and only anger-in in those with more experience (n = 270) partially mediated the relationship of work stress with burnout. CONCLUSIONS AND CLINICAL RELEVANCE: The indirect effect of anger-in style was higher than other anger expression styles in all nurses; suppression of anger caused by work stress can increase burnout. Anger-out played a functional role in the relationship of work stress with burnout among nurses who had less career experience. Therefore, to reduce burnout, creating a work environment with collaborative culture including authentic leadership and providing anger management programs will help to manage nurses' anger properly. For new nurses, promoting work and life balance, and creating a culturally empowering work environment to help them use anger-out expression constructively, are important to reduce burnout.

Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model.

Generation of autologous human motor neurons holds great promise for cell replacement therapy to treat spinal cord injury (SCI). Direct conversion allows generation of target cells from somatic cells, however, current protocols are not practicable for therapeutic purposes since converted cells are post-mitotic that are not scalable. Therefore, therapeutic effects of directly converted neurons have not been elucidated yet. Here, we show that human fibroblasts can be converted into induced motor neurons (iMNs) by sequentially inducing POU5F1(OCT4) and LHX3. Our strategy enables scalable production of pure iMNs because of the transient acquisition of proliferative iMN-intermediate cell stage which is distinct from neural progenitors. iMNs exhibited hallmarks of spinal motor neurons including transcriptional profiles, electrophysiological property, synaptic activity, and neuromuscular junction formation. Remarkably, transplantation of iMNs showed therapeutic effects, promoting locomotor functional recovery in rodent SCI model. Together, our advanced strategy will provide tools to acquire sufficient human iMNs that may represent a promising cell source for personalized cell therapy.

Acute and 13-week subchronic toxicity studies of hot-water extract of Cynanchi wilfordii Radix in Sprague-Dawley rats.

Cynanchi wilfordii Radix (CWR) is a herbal medicinal plant that is well-known and used in Asian countries as a health food. In this study, acute and 13-week subchronic oral toxicity studies of hot-water extract of CWR (CWR-WE) were performed in Sprague-Dawley rats. For the acute toxicity study, CWR-WE was administered once orally to five male and five female rats at doses of 800, 2000, and 5000 mg/kg. Mortality, clinical signs, and body weight changes were monitored over 14 days. There were no treatment-related changes in these parameters and the approximate lethal dose of CWR-WE in male and female rats was determined to be > 5000 mg/kg. For the subchronic toxicity study, CWR-WE was administered orally once daily to male and female rats for 13 consecutive weeks at doses of 0 (vehicle control), 500, 1000, and 2000 mg/kg/day (n = 10 rats/sex/group). There were no toxicologically significant changes with regard to clinical signs, body weight, food consumption, ophthalmology, urinalysis, hematology, clinical chemistry, organ weights, necropsy findings, and histopathological findings. These results suggest that the oral no observed adverse-effect level of CWR-WE is > 2000 mg/kg/day for both sexes, although target organs were not identified.

Effect of the type of electrical stimulation on spinal fusion in a rat posterolateral spinal fusion model.

BACKGROUND CONTEXT: Posterolateral fusion (PLF) with autogenous iliac bone graft is one of the most common surgical procedures for lumbar spinal disease. However, its limited success demands new biologically competent graft enhancers or substitutes. Although the use of direct current (DC) electrical stimulation has been shown to increase rate of successful spinal fusions, little is known about the effect of the type of current in DC stimulation. PURPOSE: To evaluate the effects of various DC stimulators on the strength and success rate of posterolateral fusion facilitated by using a nitinol mesh container, in rats. STUDY DESIGN: This was an experimental animal study. METHODS: A conductive, tubular nitinol mesh container was used to carry small pieces of bone grafts. The nitinol mesh container received electrical stimulation via a lead that connected the container to different types of DC stimulators. Sixty male Sprague-Dawley rats were divided into three groups (N=20 in each): a control group that underwent PLF with a nitinol container filled with autograft, a constant DC group that received a nitinol container and constant DC (100 µA), and a pulsed DC group that received a nitinol container and pulsed DC (100 µA, 100 Hz, 200 µs). The rats underwent PLF between L4 and L5, and transverse processes were grafted with bilateral iliac grafts. A stimulator was implanted subcutaneously. The rats were sacrificed 8 weeks postsurgery, and lumbar spines were removed. Spinal fusion was evaluated by microcomputed tomography, manual testing, biomechanical testing, histologic examination, and molecular analysis. RESULTS: All animals in the DC stimulation groups displayed solid fusion, whereas only 70% of control animals showed solid fusion. Radiographic images, biomechanical testing, histologic examination, and molecular analysis revealed improved fusion in the order control group

Subchronic toxicity of Acorus gramineus rhizoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Acorus gramineus rhizoma (AGR) is the dry rhizome of Acorus gramineus Solander from the family Araceae that has been used as sedative, analgesic, diuretic, digestive and antifungal agent. AIM OF THE STUDY: To evaluate the no-observed-adverse-effect level (NOAEL) and the toxicity of AGR, following repeated oral administration to rats for 13 weeks. MATERIALS AND METHODS: AGR was administered by oral gavage to groups of rats (10 per group, each sex) at doses of 0 (control), 25, 74, 222, 667, or 2,000mg/kg/day, 5 times per week for 13 weeks. Mortality, clinical signs, body weights, food consumption, hematology, serum chemistry, urinalysis, vaginal cytology, sperm motility, sperm morphology, organ weights, gross and histopathological findings were compared between control and AGR groups. RESULTS: No mortality or remarkable clinical signs were observed during this 13-week study. No adverse effects on body weight, food consumption, urinalysis, hematology, serum chemistry, organ weights, gross lesion, histopathology, vaginal cytology, sperm motility or deformity were observed in any of the male or female rats treated with AGR. CONCLUSIONS: On the basis of these results, the NOAEL of AGR is determined to be 2,000mg/kg/day for male and female rats.

Hypoxia-specific, VEGF-expressing neural stem cell therapy for safe and effective treatment of neuropathic pain.

Vascular endothelial growth factor (VEGF) is an angiogenic cytokine that stimulates the differentiation and function of vascular endothelial cells. VEGF has been implicated in improving nervous system function after injury. However, uncontrolled overexpression of VEGF increases the risk of tumor formation at the site of gene delivery. For this reason, VEGF expression needs to be strictly controlled. The goal of the present study was to understand the effects of hypoxia-induced gene expression system to control VEGF gene expression in neural stem cells (NSCs) on the regeneration of neural tissue after sciatic nerve injury. In this study, we used the erythropoietin (Epo) enhancer-SV40 promoter system (EpoSV-VEGF-NSCs) for hypoxia-specific VEGF expression. We used three types of NSCs: DsRed-NSCs as controls, SV-VEGF-NSCs as uncontrolled VEGF overexpressing NSCs, and EpoSV-VEGF-NSCs. For comparison of VEGF expression at normoxia and hypoxia, we measured the amount of VEGF secreted. VEGF expression decreased at normoxia and increased at hypoxia for EpoSV-VEGF-NSCs; thus, EpoSV-VEGF-NSCs controlled VEGF expression, dependent upon oxygenation condition. To demonstrate the therapeutic effect of EpoSV-VEGF-NSCs, we transplanted each cell line in a neuropathic pain sciatic nerve injury rat model. The transplanted EpoSV-VEGF-NSCs improved sciatic nerve functional index (SFI), mechanical allodynia, and re-myelination similar to the SV-VEGF-NSCs. Additionally, the number of blood vessels increased to a level similar to that of the SV-VEGF-NSCs. However, we did not observe tumor generation in the EpoSV-VEGF-NSC animals that were unlikely to have tumor formation in the SV-VEGF-NSCs. From our results, we determined that EpoSV-VEGF-NSCs safely regulate VEGF gene expression which is dependent upon oxygenation status. In addition, we found that they are therapeutically appropriate for treating sciatic nerve injury.

Experimental Evaluation of Percutaneous Lumbar Laser Disc Decompression Using a 1414 nm Nd:YAG Laser.

BACKGROUND: Laser ablation under an epiduroscopic view allows for the vaporization of a small amount of the nucleus pulposus, causing a reduction in intradiscal pressure and relief of radicular pain. Currently, Ho:YAG and Nd:YAG lasers are commonly used for spinal diseases. However, the use of the Nd:YAG laser for intra-spinal procedures can be limited because of thermal injury and low efficacy. OBJECTIVE: To investigate the efficacy and safety of epiduroscopic laser ablation using a 1414 nm Nd:YAG laser, we examined that laser ablation was able to penetrate nucleus pulposus without heating surrounding tissues and without mechanical damage to surrounding tissue. STUDY DESIGN: Our experiment involved live and cadaveric animal studies and a human cadaveric study. SETTING: University in Korea. METHODS: Two live pigs, 3 porcine cadavers, and 2 human cadavers were used. For the in vitro study, intradiscal and epidural pressure and temperature were compared in vertebral columns obtained from 3 porcine cadavers before and after laser ablation. For the in vivo study, 2 pigs were used to simulate percutaneous epiduroscopic laser ablation. They were observed for behavioral changes and neurological deficits for one month after the laser ablation procedure. Two human cadavers were used for placing the laser fiber and epiduroscope in the correct target site through the sacral hiatus. Histological analysis was also performed to observe any damage around the ablated lesion. RESULTS: Both intradiscal and epidural pressure were markedly reduced immediately after laser ablation as compared with the pre-ablative state. The amount of the pressure decrease in the intradiscal space was significantly greater than that in the epidural space (45.8 ± 15.0 psi vs. 30.0 ± 9.6 psi, P = 0.000). The temperature beneath the ipsilateral spinal nerve, which was the nearest site to the laser probe, never exceeded 40° C. Histology revealed no evidence of thermal damage to surrounding structures, including the spinal nerves, end plates, and vertebrae, after laser ablation. All live pigs showed normal behavior without any sign of pain. In the human cadaveric study, there was no case of targeting failure or dural laceration. The mean time to reach the target region was less than 5 minutes. LIMITATIONS: The pressure measurements were performed on cadavers and not in vivo. Cadaver models cannot account for intradiscal pressure changes that occur during live muscle contraction and different positions, which may affect results. Moreover, although we controlled temperatures with heat baths, vascular and cerebrospinal fluid circulations were not simulated. Those circulations may change the temperature results in vivo. CONCLUSIONS: The 1414 nm Nd:YAG laser can be used effectively and safely under the guidance of a spinal epiduroscope in an in vivo porcine model and in a human cadaveric model. STUDY APPROVAL: Approval for the current study was granted by the Institutional Review Board of our institute (approval number: 1-2014-0049).

Antiapoptotic Effect of Highly Secreted GMCSF From Neuronal Cell-specific GMCSF Overexpressing Neural Stem Cells in Spinal Cord Injury Model.

STUDY DESIGN: Neuronal cell-specific gene expression system and neural stem cells (NSCs) were combined for treatment of spinal cord injury (SCI). OBJECTIVE: To verify the reproducibility of the neuronal cell-specific therapeutic gene overexpression system, we develop a neuronal cell-specific granulocyte-macrophage colony-stimulating factor expression system (NSE-GMCSF), and then examine the characteristics of GMCSF overexpression and protective effect on neural cells in vitro and vivo. SUMMARY OF BACKGROUND DATA: The stem cell transplantation is considered a promising therapy for SCI. However, stem cell monotherapy strategy is insufficient for complete recovery after SCI. Therefore, combined treatment method based on stem cells with other therapeutic system may be effective for improving the therapeutic efficacy. In this study, we established the gene and stem cell therapy platform based on NSCs and neuronal cell-specific gene expression system. METHODS: To examine the GMCSF expression pattern, we compared the amount of secreted GMCSF from the neuronal cell-specific GMCSF expressing NSCs with control GMCSF-expressing NSCs (respectively, NSE-GMCSF-NSCs vs. SV-GMCSF-NSCs) by ELISA in vitro and in vivo, and then verified the neuronal protective effect of these cells in vitro and vivo. RESULTS: The results showed that NSE-GMCSF-NSCs secreted more GMCSF compared with SV-GMCSF-NSCs in normoxia, hypoxia and cytotoxic conditions. The cell viability of NSE-GMCSF-NSCs was increased depending on the amount of secreted GMCSF in cytotoxic condition. In addition, the amount of secreted GMCSF by NSE-GMCSF-NSCs transplanted into injured spinal cord was significantly higher than SV-GMCSF-NSCs. Higher amount of secreted GMCSF decreased the expression of proapoptotic protein, Bax. CONCLUSION: In this study, we demonstrated that the neuronal cell-specific gene expression system induced overexpression of GMCSF in NSCs. These combined NSCs & gene therapy treatment protocol would be an effective therapeutic system for SCI. LEVEL OF EVIDENCE: N/A.

Enhanced Neurite Outgrowth by Intracellular Stimulation.

Electrical stimulation through direct electrical activation has been widely used to recover the function of neurons, primarily through the extracellular application of thin film electrodes. However, studies using extracellular methods show limited ability to reveal correlations between the cells and the electrical stimulation due to interference from external sources such as membrane capacitance and culture medium. Here, we demonstrate long-term intracellular electrical stimulation of undamaged pheochromocytoma (PC-12) cells by utilizing a vertical nanowire electrode array (VNEA). The VNEA was prepared by synthesizing silicon nanowires on a Si substrate through a vapor-liquid-solid (VLS) mechanism and then fabricating them into electrodes with semiconductor nanodevice processing. PC-12 cells were cultured on the VNEA for 4 days with intracellular electrical stimulation and then a 2-day stabilization period. Periodic scanning via two-photon microscopy confirmed that the electrodes pierced the cells without inducing damage. Electrical stimulation through the VNEA enhances cellular differentiation and neurite outgrowth by about 50% relative to extracellular stimulation under the same conditions. VNEA-mediated stimulation also revealed that cellular differentiation and growth in the cultures were dependent on the potential used to stimulate them. Intracellular stimulation using nanowires could pave the way for controlled cellular differentiation and outgrowth studies in living cells.

Multifunctional nanoparticles for gene delivery and spinal cord injury.

Methylprednisolone (MP) is a glucocorticoid that is used as an anti-inflammatory agent to the treat spinal cord injury (SCI). A low molecular weight chitosan was used to synthesize chitosan-MP conjugate, which was used to evaluate the gene therapy, anti-inflammatory and anti-apoptotic effects of MP. The cytotoxicity of chitosan-MP nanoparticles and the transfection efficiency of plasmid DNA were evaluated by MTT and luciferase assays. A chitosan-MP/pDNA complexes was injected into injured spinal cord to evaluate the anti-inflammatory and anti-apoptotic effects of these complexes using terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) and ED1 staining, respectively. In addition, to evaluate the distribution of chitosan-MP/pDNA complexes, pß-gal encapsulated chitosan-MP was injected into the injected site. Cell survival was similar in cells treated with chitosan-MP conjugate and untreated cells. Luciferase expression was higher in cells treated with the chitosan-MP/pDNA than cells treated with the chitosan/pDNA. The chitosan-MP/pDNA complexes also reduced apoptosis and inflammation at the injury site. These results suggest that chitosan-MP conjugation is an effective gene delivery system to treat SCI.

Human Adipose Stem Cells Improve Mechanical Allodynia and Enhance Functional Recovery in a Rat Model of Neuropathic Pain.

Stem cells are a promising source of tissue engineering due to their differentiation potential. Today, direct transplantation of stem cells for cell therapy is commonly performed. However, in cases of nerve injury, direct transplantation of cells could lead to secondary nerve damage. Male Sprague-Dawley rats were randomized into four groups: the phosphate-buffered saline epineural transplantation (PBS-ENT) group, the PBS intraneural transplantation (PBS-INT) group, the human adipose-derived stem cells epineural transplantation (hASCs-ENT) group, and human adipose-derived stem cells intraneural transplantation (hASCs-INT) group. Transplantation was conducted 1 week later after inflicting a crush injury with subsequent observation for 5 weeks. To evaluate pain, each group was examined with regard to paw withdrawal latency and evoked potentials. The sciatic functional index (SFI) was calculated to estimate functional recovery. The sciatic nerve was also examined histologically. The hASCs-ENT group showed a more rapid paw withdrawal threshold and SFI recovery than the other groups (p<0.05). The hASCs-ENT group also showed shorter initial latencies in both somatosensory evoked potential (SSEP) and motor evoked potential (MEP) than the PBS-INT group (p<0.05). In addition, the N1 latency of the MEP and the N1 and P1 latencies of the SSEP were significantly shorter than those of the PBS-INT group (p<0.05). Histological examination revealed that the transplanted groups showed better neural recovery and remyelination than the groups injected with PBS. These results show that the transplantation of hASCs into the injured sciatic nerve improved mechanical allodynia and functional recovery as determined by the paw withdrawal test, SFI analysis, and electrophysiological studies. ENT is superior to INT in terms of invasiveness and better outcomes.

Vascular endothelial growth factor-expressing neural stem cell for the treatment of neuropathic pain.

Previously, we determined that vascular endothelial growth factor (VEGF) improves the survival of neural stem cells (NSCs) transplanted into an ischemic environment and effectively enhances angiogenesis. Here, we applied NSCs expressing VEGF (SV-VEGF-NSCs) to treat neuropathic pain. In this study, our goal was to verify the therapeutic effect of SV-VEGF-NSCs by transplanting the cells in a sciatic nerve injury model. We compared the amount of VEGF secreted from DsRed-NSCs (control) or SV-VEGF-NSCs and observed that SV-VEGF-NSCs have a much higher expression level of VEGF. We next investigated whether transplantation with SV-VEGF-NSCs aids functional recovery and pain reduction. We confirmed that transplantation with SV-VEGF-NSCs enhances functional recovery, pain reduction, and remyelination as well as the number of blood vessels compared with the control groups. Our results show that VEGF aids functional recovery and pain reduction in a sciatic nerve injury model.

Acute and 28-Day Subacute Toxicity Studies of Hexane Extracts of the Roots of Lithospermum erythrorhizon in Sprague-Dawley Rats.

Lithospermum erythrorhizon has long been used as a traditional oriental medicine. In this study, the acute and 28-day subacute oral dose toxicity studies of hexane extracts of the roots of L. erythrorhizon (LEH) were performed in Sprague-Dawley rats. In the acute toxicity study, LEH was administered once orally to 5 male and 5 female rats at dose levels of 500, 1,000, and 2,000 mg/kg. Mortality, clinical signs, and body weight changes were monitored for 14 days. Salivation, soft stool, soiled perineal region, compound-colored stool, chromaturia and a decrease in body weight were observed in the extract-treated groups, and no deaths occurred during the study. Therefore, the approximate lethal dose (ALD) of LEH in male and female rats was higher than 2,000 mg/kg. In the subacute toxicity study, LEH was administered orally to male and female rats for 28 days at dose levels of 25, 100, and 400 mg/kg/day. There was no LEH-related toxic effect in the body weight, food consumption, ophthalmology, hematology, clinical chemistry and organ weights. Compound-colored (black) stool, chromaturia and increased protein, ketone bodies, bilirubin and occult blood in urine were observed in the male and female rats treated with the test substance. In addition, the necropsy revealed dark red discoloration of the kidneys, and the histopathological examination showed presence of red brown pigment or increased hyaline droplets in the renal tubules of the renal cortex. However, there were no test substance-related toxic effects in the hematology and clinical chemistry, and no morphological changes were observed in the histopathological examination of the kidneys. Therefore, it was determined that there was no significant toxicity because the changes observed were caused by the intrinsic color of the test substance. These results suggest that the no-observed-adverse-effect Level (NOAEL) of LEH is greater than 400 mg/kg/day in both sexes.

Subchronic toxicity study of Coptidis rhizoma in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Coptidis Rhizoma (CR) is a medical herb from the family Ranunculacease that has been used to treat gastroenteritis, dysentery, diabetes mellitus, and severe skin diseases. AIM OF THE STUDY: To evaluate the no-observed-adverse-effect level (NOAEL) and the toxicity of CR, following repeat oral administration to rats for 13 weeks. MATERIALS AND METHODS: CR was administered by oral gavage to groups of rats (n=10/group, each sex) at dose levels of 0 (control), 25, 74, 222, 667 or 2000 mg/kg/day 5 times per week for 13 weeks. Mortality, clinical signs, body weights, food consumption, hematology, serum chemistry, urinalysis, vaginal cytology and sperm morphology, organ weights, gross and histopathological findings were compared between control and CR groups. RESULTS: Urinalysis showed a significant increase in N-acety1-ß-glucosaminidase in males in the 2000 mg/kg/day group (P<0.01). However, no mortality or remarkable clinical signs were observed during this 13-week study. No adverse effects on body weight, food consumption, hematology, serum chemistry, organ weights, gross lesion, histopathology, vaginal cytology, sperm motility, or deformity were observed in the males or female rats treated with CR. CONCLUSIONS: On the basis of these results, the NOAEL of CR is determined to be 667 mg/kg/day for males and 2000 mg/kg/day for females.

Subchronic oral toxicity of evodia fruit powder in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Evodia, a fruit from Evodia rutaecarpa, has been used in oriental medicine, and since its various pharmaceutical actions, including anti-cancer activity, have become known, evodia has been widely used as a dietary supplement. However, information regarding its toxicity is limited. MATERIALS AND METHODS: Evodia fruit from Evodia rutaecarpa (Juss.) Benth. var. officinalis (Dode) Huang (0, 25, 74, 222, 667, and 2000 mg/kg) was administered orally five times per week for 13 weeks. Clinical signs, body weight, food consumption, hematology, serum chemistry, urinalysis, vaginal cytology, sperm morphology, organ weight, and gross and histopathological findings were evaluated. RESULTS: Urinary ketone body excretion was detected in males at 667 and 2000 mg/kg and in females at 2000 mg/kg. An increase in absolute/relative liver weight was observed in both sexes at 2000 mg/kg. Although levels of serum alanine aminotransferase, glucose, total cholesterol, and triglycerides were significantly reduced in males and/or females at 200 and/or 667 and 2000 mg/kg, all values were within normal ranges and were considered non-adverse. In addition, no treatment-related differences in body weight, food consumption, hematology, vaginal cytology, sperm morphology, or gross and histopathological examination were detected. CONCLUSIONS: The subchronic no-observable-adverse-effect level for evodia fruit powder following oral administration in rats is greater than 2000 mg/kg.

Sub-acute toxicity study in female ICR mice following repetitive intramuscular injection of cervical cancer vaccines.

OBJECTIVES: The sub-acute toxic effects following repetitive intramuscular injection of two cervical cancer vaccines newly developed against human papillomaviruse (HPV)16/58/18 and HPV16 were investigated in female ICR (CrljOri: CD1) mice, and the no-observedadverse- effect-level (NOAEL) of the cervical cancer vaccines was estimated. METHODS: Female ICR mice (n=15 in each group) were exposed to a 1:1 mixture of two cervical cancer vaccines by repetitive intramuscular injection (once a week, 5 times) for 5 weeks. Mortality, body weight, organ weight, hematological/biochemical parameters, and histopathological effects were examined at different concentrations (0, 1×10(8), 5×10(8), and 2.5×10(9) copies/animal) of the cervical cancer vaccines. RESULTS: The cervical cancer vaccines did not show toxic responses for body weight, absolute/ relative organ weight, hematological/biochemical parameters, or histopathological parameters. CONCLUSIONS: Female ICR mice exposed to vaccines for cervical cancer did not show any toxic response. We suggest that a NOAEL of the vaccine following repetitive intramuscular injection for 5 weeks is >2.5×10(9) copies/animal.

Interaction of CD99 with its paralog CD99L2 positively regulates CD99L2 trafficking to cell surfaces.

Mouse CD99 and its paralog CD99-like 2 (CD99L2) are surface proteins implicated in cellular adhesion and migration. Although their distributions overlap in a wide variety of cells, their physical/functional relationship is currently unknown. In this study, we show the interaction between the two molecules and its consequence for membrane trafficking of mouse (m)CD99L2. The interaction was analyzed by bimolecular fluorescence complementation, immunoprecipitation, and fluorescence resonance energy transfer assays. When coexpressed, mCD99 formed heterodimers with mCD99L2, as well as homodimers, and the heterodimers were localized more efficiently at the plasma membrane than were the homodimers. Their interaction was cytoplasmic domain-dependent and enhanced mCD99L2 trafficking to the plasma membrane regardless of whether it was transiently overexpressed or endogenously expressed. Surface levels of endogenous mCD99L2 were markedly low on thymocytes, splenic leukocytes, and CTL lines derived from CD99-deficient mice. Importantly, the surface levels of mCD99L2 on mCD99-deficient cells recovered significantly when wild-type mCD99 was exogenously introduced, but they remained low when a cytoplasmic domain mutant of mCD99 was introduced. Our results demonstrate a novel role for mCD99 in membrane trafficking of mCD99L2, providing useful insights into controlling transendothelial migration of leukocytes.