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Background Magnesium supplementation has potential for use in nerve regeneration. The expression of some magnesium transporter genes is reflective of the intracellular magnesium levels. Objective To assess the expression of various magnesium transporter genes as they relate to neurological alterations in a sciatic nerve injury model. Methods Sciatic nerve injury was induced in rats, which were then fed either basal or high magnesium diets. Magnesium concentrations and 5 magnesium transporter genes (SLC41A1, MAGT1, CNNM2, TRPM6, and TRPM7) were measured in the tissue samples. Results The high magnesium diet attenuated cytoskeletal loss in a dose-dependent manner in isolated nerve explants. The high magnesium diet augmented nerve regeneration and led to the restoration of nerve structure, increased S-100, and neurofilaments. This increased regeneration was consistent with the improvement of neurobehavioral and electrophysiological assessment. The denervated muscle morphology was restored with the high magnesium diet, and that was also highly correlated with the increased expression of desmin and acetylcholine receptors in denervated muscle. The plasma magnesium levels were significantly elevated after the animals consumed a high magnesium diet and were reciprocally related to the down-regulation of CNNM2, MagT1, and SCL41A1 in the blood monocytes, nerves, and muscle tissues of the nerve crush injury model. Conclusion The increased plasma magnesium levels after consuming a high magnesium diet were highly correlated with the down-regulation of magnesium transporter genes in monocytes, nerves, and muscle tissues after sciatic nerve crush injury. The study findings suggest that there are beneficial effects of administering magnesium after a nerve injury.

Purpose Parkinson's disease (PD) is a progressive degenerative central nervous system disorder that particularly impairs motor function. As PD advances, gait disorders become more pronounced and are often difficult to treat with current pharmacological therapies. Physical activity improves both mobility in and the daily living activities of patients with PD. Mitochondrial alterations and oxidative stress contribute to PD progression. Therefore, the association between mitochondria and exercise in PD and the implicated regulation of mitochondrial proteins was explored in this study. Methods In this study, we developed a unilateral 6-hydroxydopamine rat model of PD and executed 4 weeks of treadmill training. Motor behavior was evaluated through gait change analysis (the CatWalk method) and rotational testing. The viability of dopaminergic neurons, mitochondrial function, and oxidative stress in the substantia nigra and striatum were investigated through Western blot and immunohistochemical staining. Key findings Treadmill training improved the performance of gait parameters in terms of maximal area, swing speed, stride length, and stance phase; treadmill training also reduced methamphetamine-induced rotation. This training not only improved dopaminergic neuron viability but also recovered mitochondrial function and attenuated oxidative stress in PD rats. The mechanism may be associated with the facilitation of mitochondrial turnover, including facilitation of mitochondrial fusion, fission, and clearance accompanying increased quantities of mitochondria. Significance Treadmill exercise improved gait speed and balance, reduced oxidative stress, improved mitochondrial fusion and fission, increased mitochondrial amounts, and potentially attenuated dopaminergic neuron degeneration. Consequently, mitochondrial quality was improved in PD rats.

Transferring exogenous mitochondria has therapeutic effects on damaged heart, liver, and lung tissues. Whether this protective effect requires the symbiosis of exogenous mitochondria in host cells remains unknown. Here xenogenic mitochondria derived from a hamster cell line were applied to ischemic rat brains and rat primary cortical neurons. Isolated hamster mitochondria, either through local intracerebral or systemic intra-arterial injection, significantly restored the motor performance of brain-ischemic rats. The brain infarct area and neuronal cell death were both attenuated by the exogenous mitochondria. Although internalized mitochondria could be observed in neurons and astrocytes, the low efficacy of mitochondrial internalization could not completely account for the high rate of rescue of the treated neural cells. We further illustrated that disrupting electron transport or ATPase synthase in mitochondria significantly attenuated the protective effect, suggesting that intact respiratory activity is essential for the mitochondrial potency on neural protection. These results emphasize that nonsymbiotic extracellular mitochondria can provide an effective cell defense against acute injurious ischemic stress in the central nervous system.

Background Intermittent parathyroid hormone (PTH) can be used to treat osteoporosis of the spine and hip. However, whether it can be used to treat osteoporosis of the mandible is unclear. The purpose of this study was to explore the influence of applying intermittent PTH to ovariectomized rats on the trabecular bone microarchitecture of the mandible and femoral head. Methods Eighteen female rats were divided into three groups: the healthy group, ovariectomized (OVX) group, and OVX + PTH group. The OVX group and OVX + PTH group had an OVX at 8 weeks of age. The OVX + PTH group received intermittent PTH therapy for 12 weeks. The mandibles and femurs of all rats were removed at 20 weeks and were then scanned using microcomputed tomography (micro-CT). Results From the micro-CT analysis, the trabecular bone microarchitecture of the mandible and femoral head are offered as follows: (1) The bone volume fraction and trabecular thickness in the OVX group were lower than those in the healthy group. (2) The bone volume fraction and trabecular thickness in the OVX + PTH group approximated those in the healthy group. Conclusion The conclusions of this study regarding the trabecular bone microarchitecture of the mandible and femoral head are offered as follows: (1) The BV/TV and TbTh in the OVX group were lower than those in the healthy group. (2) The BV/TV and TbTh in the OVX + PTH group approximated those in the healthy group, therefore, intermittent PTH displayed high efficacy for treating femoral or mandibular deterioration of bone microstructure resulting from loss of ovarian function. Osteoporosis of the femur or mandible in the rats was ameliorated by intermittent PTH therapy.

Background Axon degeneration leads to cytoskeletal disassembly, metabolism imbalance, and mitochondrial dysfunction during neurodegeneration or nerve injury. Objective In this study, we assess the possibility of mitigating axon degeneration by local injection of mitochondria in a crushed sciatic nerve. Methods Sciatic nerve explants cocultured with mitochondria were assessed for the optimal dosage in local injection and nerve regeneration potential. The left sciatic nerve was crushed in Sprague-Dawley rats and then local injection of mitochondria into the distal end of the injured nerve was conducted for further assessment. Results Mitochondrial coculture attenuated cytoskeletal loss and oxidative stress in isolated nerve explants. In Vivo analyses also showed that mitochondrial transplantation improved animal neurobehaviors, electrophysiology of nerve conduction, and muscle activities. Mitochondria injection significantly attenuated the oxidative stress and increased the expression of neurotrophic factors both in injured nerves and denervated muscles, as well as restored muscular integrity, and increased the pool of muscular progenitor cells and total muscle weight. Conclusion Mitochondria injection can protect injured nerves from axonal degeneration both in Vitro and in Vivo. This improvement was accompanied with the expression of neurotrophic factors as well as the reduction of oxidative stress, which may account for the functional recovery of both injured nerves and denervated muscles.

Traumatic brain injury (TBI) results in neurological dysfunction and death through primary or secondary mechanisms. Here, we evaluated the effect of osmotic pump delivery of granulocyte colony-stimulating factor (G-CSF) on the histopathology and motor function recovery of rats after experimental TBI.Sprague-Dawley rats were used as experimental model by fluid percussion device to cause brain injury on the motor cortex area. The rats were simultaneously subjected to TBI and were implanted of min-osmotic pump containing recombinant human G-CSF (300 μg/700 μl) via intraperitoneal injection. Motor function was assessed by rotarod test. 5-bromo-2'-deoxyuridine (BrdU) was used to label the proliferating cells and their differentiation was evaluated by histology and immunohistochemistry.The G-CSF group showed significantly better motor function recovery than the control group, and the effect lasted up to 14 days after TBI. Moreover, the G-CSF group exhibited a greater increase in the number of BrdU-positive cells compared with the control group. The G-CSF group also had a significantly higher number of DCX-positive cells in the ipsilateral subventricular zone (SVZ) than the control group.These data suggest that the beneficial effect of delivering G-CSF via an osmotic pump may improve the motor function and enhance neurogenesis in the SVZ of the injured brain.

BACKGROUND Statins have therapeutic benefits for the management of several disorders. A short-term course of a high-dose statin pretreatment has demonstrated neuroprotective effects against neurological diseases. However, the molecular basis underlying the neuroprotective action of statins remains unclear. OBJECTIVE We investigated whether a short-term course of high-dose atorvastatin pretreatment has beneficial effects in protecting sciatic nerve from crush injury. METHODS Atorvastatin (5 mg/kg) or saline was given orally to Sprague-Dawley rats for 7 days before injury. The rats were subjected to crush injury in the left sciatic nerve with a vessel clamp. Biochemical, functional, electrophysiological, and morphological alterations occurring during injury-induced degeneration/regeneration were examined. RESULTS Atorvastatin improved injury-induced neurobehavioral/electrophysiological changes and axonal loss. Damage-associated alterations, including structural disruption, oxidative stress, inflammation, and apoptosis, were attenuated by atorvastatin. After injury, regeneration-associated genes, including growth-associated protein-43, myelin basic protein, ciliary neurotrophic factor, and collagen, were upregulated by atorvastatin. The suppression of extracellular signal-regulated kinase, AKT, signal transducer and activators of transcription-1, and necrosis factor-kappaB and the elevated activation of c-Jun N-terminal kinase, Smad2/3, and activating protein-1 were associated with the neuroprotective action of atorvastatin. CONCLUSION These findings suggest that a short-term course of high-dose atorvastatin pretreatment can protect against sciatic nerve crush injury through modifying intracellular or extracellular environments, making it favorable for regeneration.

Human mesenchymal stromal cells (hMSC) play a crucial role in tissue engineering and regenerative medicine, and have important clinical potential for cell therapy. However, many hMSC studies have been restricted by limited cell numbers and difficult detection in vivo. To expand the lifespan, hMSC are usually immortalized by virus-mediated gene transfer. However, these genetically modified cells easily lose critical phenotypes and stable genotypes because of insertional mutagenesis.We used a non-viral transfection method to establish human telomerase reverse transcriptase-immortalized cord blood hMSC (hTERT-cbMSC). We also established red fluorescent protein (RFP)-expressing hTERT-cbMSC (hTERT/RFP-cbMSC) by the same non-viral transfection method, and these cells were injected into a rat model with traumatic brain injury for in vivo detection analysis.The hTERT-cbMSC could grow more than 200 population doublings with a stable doubling time and maintained differentiation capacities. hTERT/RFP-cbMSC could proliferate efficiently within 2 weeks at the injury location and could be detected easily under a fluorescent microscope. Importantly, both hTERT-cbMSC and hTERT/RFP-cbMSC showed no chromosomal abnormalities by karyotype analysis and no tumor formation in severe combined immunodeficient (SCID) mice by transplantation assay.We have developed immortalized cbMSC with hTERT expression and RFP expression, which will be useful tools for stem cell research and translational study.

Purpose Attenuation of pro-inflammatory cytokines and associated inflammatory cell deposits rescues human amniotic fluid mesenchymal stem cells (AFS) from apoptosis. Hyperbaric oxygen (HBO) suppressed stimulus-induced pro-inflammatory cytokine production in blood-derived monocyte-macrophages. Herein, we evaluate the beneficial effect of hyperbaric oxygen on transplanted AFS in a sciatic nerve injury model. Methods Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. Hyperbaric oxygen (100% oxygen, 2 ATA, 60 min/day) was administered 12 h after operation for seven consecutive days. Transplanted cell apoptosis, oxidative stress, inflammatory cell deposits and associated chemokines, pro-inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 and 28 days after injury. Results Crush injury induced an inflammatory response, disrupted nerve integrity, and impaired nerve function in the sciatic nerve. However, crush injury-provoked inflammatory cytokines, deposits of inflammatory cytokines, and associated macrophage migration chemokines were attenuated in groups receiving hyperbaric oxygen but not in the AFS-only group. No significant increase in oxidative stress was observed after administration of HBO. In transplanted AFS, marked apoptosis was detected and this event was reduced by HBO treatment. Increased nerve myelination and improved motor function were observed in AFS-transplant, HBO-administrated, and AFS/HBO-combined treatment groups. Significantly, the AFS/HBO combined treatment showed the most beneficial effect. Conclusion AFS in combination with HBO augment peripheral nerve regeneration, which may involve the suppression of apoptotic death in implanted AFS and the attenuation of an inflammatory response detrimental to peripheral nerve regeneration.

Granulocyte colony-stimulating factor (G-CSF) inhibits programmed cell death and stimulates neuronal progenitor differentiation. Neuronal stem cells transplanted into injured spinal cord can survive, differentiating into astroglia and oligodendroglia, and supporting axon growth and myelination. Herein, we evaluate the combined effects of G-CSF and neuronal stem cells on spinal cord injury. For 40 Sprague-Dawley rats (n=10 in each group) transverse spinal cord resections at the T8-9 level were carried out, leaving an approximately 2-mm gap between the distal and proximal ends of the cord. Neuronal stem cells embedded in fibrin glue treated with or without G-CSF (50 microg/kg x 5 days) (groups III and IV) or fibrin glue with or without G-CSF (50 microg/kg x 5 days) (groups I and II) were transplanted into the gap in the injured spinal cord. Spinal cord regeneration was assessed using a clinical locomotor rating scale scores and electrophysiological, histological and immunohistochemical analysis 3 months after injury. Regeneration was more advanced in group IV than in groups III or II according to the clinical motor score, motor evoked potential, and conduction latency. Most advanced cord regeneration across the gap was observed in group IV rats. Higher densities of bromodeoxyuridine in the injured area and higher expression levels of Neu-N and MAP-2 over the distal end of the injured spinal cord were observed in group IV compared with groups II or III, but there was no significant difference in expression of glial fibrillary acid protein. This synergy between G-CSF and neuronal stem cells may be due to increased proliferation of progenitor cells in the injured area and increased expression of neuronal stem cell markers extrinsically or intrinsically in the distal end of injured cord.

Amniotic fluid mesenchymal stem cells have the ability to secrete neurotrophic factors that are able to promote neuron survival in vitro. The purpose of this study was to evaluate the effects of neurotrophic factors secreted by rat amniotic fluid mesenchymal stem cells on regeneration of sciatic nerve after crush injury. Fifty Sprague-Dawley rats weighing 250-300 g were used. The left sciatic nerve was crushed with a vessel clamp. Rat amniotic fluid mesenchymal stem cells embedded in fibrin glue were delivered to the injured nerve. Enzyme-linked immunosorbent assay (ELISA) and immunocytochemistry were used to detect neurotrophic factors secreted by the amniotic fluid mesenchymal stem cells. Nerve regeneration was assessed by motor function, electrophysiology, histology, and immunocytochemistry studies. Positive CD29/44, and negative CD11b/45, as well as high levels of expression of brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, ciliary neurotrophic factor (CNTF), nerve growth factor, and neurotrophin-3 (NT-3) were demonstrated in amniotic fluid mesenchymal stem cells. Motor function recovery, the compound muscle action potential, and nerve conduction latency showed significant improvement in rats treated with amniotic fluid mesenchymal stem cells. ELISA measurement in retrieved nerves displayed statistically significant elevation of CNTF and NT-3. The immunocytochemical studies demonstrated positive staining for NT-3 and CNTF in transplanted cells. The histology and immunocytochemistry studies revealed less fibrosis and a high level of expression of S-100 and glial fibrillary acid protein at the crush site. Rat amniotic fluid mesenchymal stem cells may facilitate regeneration in the sciatic nerve after crush injury. The increased nerve regeneration found in this study may be due to the neurotrophic factors secreted by amniotic fluid mesenchymal stem cells.

Aim: To examine if magnesium lithospermate B (MLB) extracted from Danshen, the dried roots of Salvia miltiorrhiza , may act as an active component responsible for the cardiac therapeutic effect of this traditional Chinese herb via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Moreover, we wanted to test if MLB may provide neuroprotection against ischemic stroke as observed for cardiac glycosides. Methods: Similarity in the chemical structure and molecular configuration between MLB and ouabain was analyzed. The inhibition potency of MLB and ouabain on Na + ,K + -ATPase activity of a commercial product, as well as in purified membrane fractions from rat brain and heart tissues, was examined and compared. Neuroprotective effect of MLB against ischemic stroke was also evaluated using a cortical brain slice-based assay model. Results: Dose-dependent inhibition on the commercial Na + ,K + - ATPase equivalent to that for ouabain was observed for MLB of approximately half dosage by weight. This relative potency of ouabain and MLB was also observed for their inhibition on Na + ,K + -ATPase activity of plasma membrane purified from rat tissues, although these 2 inhibitors exhibited somewhat lower competence in these crude extracts. In ischemic gerbil brains, post-treatment with MLB significantly reduced the infarct size, visualized by 2,3,5-triphenyltetrazolium chloride staining, by approximately 55% when compared with the control group. Conclusion: These results evidently suggest that the cardiac therapeutic effect of Danshen should be at least partly attributed to the effective inhibition of Na + ,K + - ATPase by MLB, and that MLB provides anti-ischemic neuroprotection in gerbils subjected to focal ischemia and reperfusion.

Human neural stem cells (NSCs) are particularly valuable for the study of neurogenesis process and have a therapeutic potential in treating neurodegenerative disorders. However, current progress in the use of human NSCs is limited due to the available NSC sources and the complicated isolation and culture techniques. In this study, we describe an efficient method to isolate and propagate human NSCs from the amniotic fluid with diagnosed neural tube defects (NTDs), specifically, anencephaly. These amniotic fluid-derived NSCs (AF-NSCs) formed neurospheres and underwent long-term expansion in vitro. In addition, these cells showed normal karyotypes and telomerase activity and expressed NSC-specific markers, including Nestin, Sox2, Musashi-1, and the ATP-binding cassette G2 (ABCG2). AF-NSCs displayed typical morphological patterns and expressed specific markers that were consistent with neurons, astrocytes, oligodendrocytes, and dopaminergic neurons after proper induction conditions. Furthermore, grafted AF-NSCs improved the physiological functions in a rat stroke model. The ability to isolate and bank human NSCs from this novel source provides a unique opportunity for translational studies of neurological disorders.

Purpose The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. Materials and Methods Seventy two Sprague-Dawley rats weighing 200–250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. Results NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. Conclusion Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection furthermore improves the nerve regeneration in a long term nerve anastomosis model.

The electrical stimulation of meridian points in rats inhibits the withdrawal reflex of the nociceptive tail. Its pain mechanisms are well-documented. Moreover, electroacupuncture (EA) at special abdominal acupoints has been shown to induce a short-term hypoglycemia effect in streptozotocin diabetic rats. The Zusanli and Zhongwan acupoints have been widely used in traditional Chinese medicine to relieve symptoms of diabetes mellitus. It is still unclear whether they can affect extracellular glucose and lactate metabolites at the cellular level. The aim of this study is to evaluate these effects using a rat model for the analysis of extracellular neurochemicals. First, electrical stimulus of 2 ms 2 Hz square pulses (30 minutes) was applied to anesthetized intact rats ( n = 7) at the Zusanli points. One and a half hours later, a second electrical stimulus (2 Hz pulses, 30 minutes) was delivered to two of the rats at the same spot. Another two rats received a different stimulation (100 Hz pulses, 30 minutes) at the same location. In the final three rats, a second electrical stimulus of 2 Hz pulses was delivered to non-acupoints. An automated micro-blood sample collector was used to examine the glucose, pyruvate and lactate concentrations. The EA signal has an influence on the biologic process of energy metabolism by mediating dynamic extracellular neurochemical changes. The EA at limb acupoints of the lower limbs induces a decrease in glucose, an increase in lactate metabolites and a decrease in the lactate/glucose ratio. Moreover, the increased lactate/glucose ratio suggests that the cell has an increased anaerobic glucose metabolism.

Microglia-derived protection of brain cells (microglia, astrocytes, and neurons) during in vitro ischemic stress (deprivation of glucose, oxygen, and serum) was determined. Trypan blue exclusion assay, immunoblocking assay, Western blot analysis, and ELISA assay were used to determine the molecular mechanisms responsible for the microglia-derived protection. Results demonstrated that supernatants from the ischemic microglia protected all three cell-types from ischemia-induced damage by releasing the transforming growth factor-beta1 (TGF-beta1) and glial cell line-derived neurotrophic factor (GDNF). The protection of microglia was TGF-beta1 related, whereas astrocytes protection was GDNF-dependent. The protection of neurons was TGF-beta1 and GDNF independent, and the molecular nature responsible for their protection remains to be determined. These results indicate contribution from the surrounding cells and the types of receptors expressed on different brain cells probably also play an important role in determining their fate against ischemia.

The present study examined effects of the combined administration of naloxone (NX) and indomethacin (IM) on nigrostriatal catecholamines and locomotor activity after intranigral lipopolysaccharide (LPS) injection in Sprague-Dawley rats. NX plus IM was given 3 days after LPS injection; it significantly (P < .05) reversed LPS inflammation on nigrostriatal dopamine (DA) and nigral serotonin (5-HT) and nigral homovanillic acid (HVA)/DA ratio and nigrostriatal 5-hydroxyindoleacetic acid (5-HIAA)/5-HT ratio. It also tended to ameliorate the locomotor hyperactivity. However, NX plus IM given 30 min before LPS could not satisfactorily protect against LPS's damage both biochemically and behaviorally. These results reveal that NX plus IM may protect against LPS on DA, 5-HT, and motor function after LPS injection but not before. Thus it suggests that the combined treatment of NX and IM gives a potent therapy, but not prevention, of LPS-induced inflammation and also protect nigrostriatal dopaminergic and serotoninergic systems against LPS in rats.

The aim of this study was to develop a rapid and sensitive method for the simultaneous determination of unbound levofloxacin in rat blood and bile using high-performance liquid chromatography coupled with microdialysis for further pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of levofloxacin 3 mg/kg through the femoral vein. Levofloxacin and dialysates were separated using a Merck LiChrospher reversed-phase C18 column maintained at ambient temperature. The mobile phase was comprised of acetonitrile-1 mM 1-octanesulfonic acid (40:60, v/v, pH 3.0 adjusted with orthophosphoric acid). The fluorescence response for levofloxacin was observed at excitation and emission wavelengths of 292 and 494 nm, respectively. The detection limit of levofloxacin was 50 ng/ml. Intra-day and inter-day precision and accuracy of levofloxacin measurements fell well within the predefined limits of acceptability. The disposition of levofloxacin in the blood and bile fluid suggests that there was rapid exchange and equilibration between the blood and hepatobiliary systems, and the plasma level of levofloxacin was greater than that of the bile. Thus, levofloxacin undergoes hepatobiliary excretion but might not be related to the P-glycoprotein transport system.

Objective The objective of this study was to evaluate the relationship between the trabecular bone microarchitecture and cortical bone morphology by using micro-computed tomography (micro-CT) and dental cone-beam computed tomography (dental CT). Materials and Methods Sixteen femurs and eight fifth lumbar vertebrae were collected from eight male Sprague Dawley rats. Four trabecular bone microarchitecture parameters related to the fifth lumbar vertebral body (percent bone volume [BV/TV], trabecular thickness [TbTh], trabecular separation [TbSp], and trabecular number [TbN]) were calculated using micro-CT. In addition, the volumetric cancellous bone grayscale value (vCanGrayscale) of the fifth lumbar vertebral body was measured using dental CT. Furthermore, four cortical bone morphology parameters of the femoral diaphysis (total cross-sectional area [TtAr], cortical area [CtAr], cortical bone area fraction [CtAr/TtAr], and cortical thickness [CtTh]) were calculated using both micro-CT and dental CT. Pearson analysis was conducted to calculate the correlation coefficients (r) of the micro-CT and dental CT measurements. Paired-sample t tests were used to compare the differences between the measurements of the four cortical bone morphology parameters obtained using micro-CT and dental CT. Results High correlations between the vCanGrayscale measured using dental CT and the trabecular bone microarchitecture parameters (BV/TV [r = 0.84] and TbTh [r = 0.84]) measured using micro-CT were observed. The absolute value of the four cortical bone morphology parameters may be different between the dental CT and micro-CT approaches. However, high correlations (r ranged from 0.71 to 0.90) among these four cortical bone morphology parameters measured using the two approaches were obtained. Conclusion We observed high correlations between the vCanGrayscale measured using dental CT and the trabecular bone microarchitecture parameters (BV/TV and TbTh) measured using micro-CT, in addition to high correlations between the cortical bone morphology measured using micro-CT and dental CT. Further experiments are necessary to validate the use of dental CT on human bone.

A dual-probe microdialysis technique was developed for the simultaneous monitoring of glucose and related metabolite levels during testicular ischemia in rats. The determinations of lactate and pyruvate were achieved by liquid chromatography within 5 min. Glucose was determined by a microdialysis analyzer. A unilateral ligation was produced by occlusion of the right artery for 2 or 4 h in anesthetized rats. Microdialysis probes were inserted in both sides of the testis to simultaneously monitor glucose, lactate and pyruvate during basal, ischemia and reperfusion periods. Dynamic and comparative changes in these analytes in ipsilateral and contralateral testes were demonstrated. The present technique can be used as a tool for exploring energy related metabolites and their relationships in testicular ischemia.

A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2-2.0 microM with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6+/-5.6 and 607+/-143 microM, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.

Neuropathic pain is debilitating, and when chronic, it significantly affects the patient physically, psychologically, and socially. The neurobehavior of animals used as a model for chronic constriction injury seems analogous to the neurobehavior of humans with neuropathic pain. However, no data depicting the severity of histomorphological alterations of the nervous system associated with graded changes in neurobehavior are available. To determine the severity of histomorphological alteration related to neurobehavior, the authors created a model of chronic constrictive injury of varying intensity in rats and used the CatWalk XT system to evaluate neurobehavior.A total of 60 Sprague-Dawley rats, weighing 250-300 g each, were randomly assigned to 1 of 5 groups that would receive sham surgery or 1, 2, 3, or 4 ligatures of 3-0 chromic gut loosely ligated around the left sciatic nerve. Neurobehavior was assessed by CatWalk XT, thermal hyperalgesia, and mechanic allodynia before injury and periodically after injury. The nerve tissue from skin to dorsal spinal cord was obtained for histomorphological analysis 1 week after injury, and brain evoked potentials were analyzed 4 weeks after injury.Significant differences in expression of nerve growth factor existed in skin, and the differences were associated with the intensity of nerve injury. After injury, expression of cluster of differentiation 68 and tumor necrosis factor-α was increased, and expression of S100 protein in the middle of the injured nerve was decreased. Increased expression of synaptophysin in the dorsal root ganglion and dorsal spinal cord correlated with the intensity of injury. The amplitude of sensory evoked potential increased with greater severity of nerve damage. Mechanical allodynia and thermal hyperalgesia did not differ significantly among treatment groups at various time points. CatWalk XT gait analysis indicated significant differences for print areas, maximum contact maximum intensity, stand phase, swing phase, single stance, and regular index, with sham and/or intragroup comparisons.Histomorphological and electrophysiological alterations were associated with severity of nerve damage. Subtle neurobehavioral differences were detected by the CatWalk XT system but not by mechanical allodynia or thermal hyperalgesia. Thus, the CatWalk XT system should be a useful tool for monitoring changes in neuropathic pain, especially subtle alterations.

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 microl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C18, 150x1 mm I.D., 5 microm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).

On-line microdialysis coupled with microbore liquid chromatography was used to investigate the pharmacokinetics of chloramphenicol and its glucuronide in rat blood. A microdialysis probe was inserted into a jugular vein of male Sprague-Dawley rats. Chloramphenicol succinate (20 mg/kg, intravenously) was then administered via a femoral vein. Dialysates were automatically injected onto a LC system, via an on-line injector. Samples were eluted with a mobile phase containing acetonitrile-10 mM monochloroacetic acid (30:70, v/v, pH 3.0). The UV detector wavelength was set at 278 nm. The limit of quantitation for chloramphenicol was 10 ng/ml. The in vitro recoveries of chloramphenicol and chloramphenicol glucuronide at 500 ng/ml were 32.2+/-0.3% and 11.4+/-0.7%, respectively (n = 6). Intra- and inter-assay accuracy and precision of the analyses wereor =10% in the range of 0.01 to 5.0 microg/ml.

An isocratic high-performance liquid chromatographic method with ultraviolet detection was utilized for the investigation of the pharmacokinetics of naringenin and its glucuronide conjugate in rat plasma and brain tissue. Plasma and brain tissue were deproteinized by acetonitrile, then centrifuged for sample clean-up. The drugs were separated by a reversed-phase C18 column with a mobile phase consisting of acetonitrile-orthophosphoric acid solution (pH 2.5-2.8) (36:64, v/v). The detection limits of naringenin in rat plasma and brain tissue were 50 ng/ml and 0.4 microg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme beta-glucuronidase. The naringenin conjugation ratios in rat plasma and brain tissue were 0.86 and 0.22, respectively, 10 min after naringenin (20 mg/kg, i.v.) administration. The mean naringenin conjugation ratio in plasma was approximately four fold that in brain tissue.

Glutathione (GSH), present in a high concentration in the liver, serves important protective functions. We investigated the effect of lowered tissue GSH content, accomplished by diethylmaleate (DEM) administration, on liver extracellular GSH levels before and after global ischemia in anesthetized rats. Liver extracellular GSH levels were determined by microdialysis perfusion and an on-line high performance liquid chromatography system. Global liver ischemia was induced by ligation of the hepatic pedicles including the hepatic artery, portal vein, and bile duct. DEM (4 mmol/kg) significantly lowered both the liver tissue GSH levels (1.36 +/- 0.26 micromol/g wet wt vs 9.50 +/- 0.55 micromol/g wet wt for the untreated) and the liver extracellular GSH levels (4.3 +/- 2.4 microM vs 25.2 +/- 8.7 microM for the untreated). Global liver ischemia induced a dramatic increase in the liver extracellular GSH level. Although the liver tissue GSH level was lowered following DEM treatment, DEM administration did not affect significantly ischemia-induced elevation of extracellular GSH (when presented as fold increase relative to basal value). In conclusion, DEM showed a direct effect on liver extracellular GSH content in anesthetized rats. However, DEM treatment did not affect the relative release of GSH following global liver ischemia.

Glucose mobilization and utilization in the periphery and central nervous system are important during exercise and are responsible for exercise efficacy. Magnesium (Mg) is involved in energy production and plays a role in exercise performance. This study aimed to explore the effects of Mg on the dynamic changes in glucose and lactate levels in the muscle, blood and brain of exercising rats using a combination of auto-blood sampling and microdialysis. Sprague-Dawley rats were pretreated with saline or magnesium sulfate (MgSO4, 90 mg/kg, i.p.) 30 min before treadmill exercise (20 m/min for 60 min). Our results indicated that the muscle, blood, and brain glucose levels immediately increased during exercise, and then gradually decreased to near basal levels in the recovery periods of both groups. These glucose levels were significantly enhanced to approximately two-fold (P

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise ratio=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.

An improved high performance liquid chromatographic assay for plasma-unbound atenolol is described. The assay has a wide range (10–5000 ng ml−1) of linearity and a detection limit of 5 ng ml−1 (or 0.1 ng per injection) with acceptable intra- and inter-assay reproducibilities using small volumes of plasma (100 μl). Following administration of a single dose of atenolol to the rat, nine blood samples were collected over a period of 8 h. These samples were analyzed for atenolol concentrations by a sensitive and specific microbore high performance liquid chromatograph with a photodiode-array detector. This multi-channel detector was used to acquire spectral information on atenolol and demonstrated a superior performance in comparison to all other techniques in that both qualitative and quantitative information were acquired with the system. Because of its sensitivity and applicability to plasma analysis, the assay can be used for pharmacokinetic studies and is valuable in therapeutic drug monitoring.

Object Mobilization of hematopoietic progenitor cells (HPCs) from bone marrow involved in the process of peripheral nerve regeneration occurs mostly through deposits of CD34(+) cells. Treadmill exercise, with either differing intensity or duration, has been shown to increase axon regeneration and sprouting, but the effect of mobilization of HPCs on peripheral nerve regeneration due to treadmill exercise has not yet been elucidated. Methods Peripheral nerve injury was induced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were categorized into 2 groups: those with and without treadmill exercise (20 m/min for 60 minutes per day for 7 days). Cytospin and flow cytometry were used to determine bone marrow progenitor cell density and distribution. Neurobehavioral analysis, electrophysiological study, and regeneration marker expression were investigated at 1 and 3 weeks after exercise. The accumulation of HPCs, immune cells, and angiogenesis factors in injured nerves was determined. A separate chimeric mice study was conducted to assess CD34(+) cell distribution according to treadmill exercise group. Results Treadmill exercise significantly promoted nerve regeneration. Increased Schwann cell proliferation, increased neurofilament expression, and decreased Schwann cell apoptosis were observed 7 days after treadmill exercise. Elevated expression of S100 and Luxol fast blue, as well as decreased numbers of vacuoles, were identified in the crushed nerve 3 weeks after treadmill exercise. Significantly increased numbers of mononuclear cells, particularly CD34(+) cells, were induced in bone marrow after treadmill exercise. The deposition of CD34(+) cells was abolished by bone marrow irradiation. In addition, deposits of CD34(+) cells in crushed nerves paralleled the elevated expressions of von Willebrand factor, isolectin B4, and vascular endothelial growth factor. Conclusions Bone marrow HPCs, especially CD34(+) cells, were able to be mobilized by low-intensity treadmill exercise, and this effect paralleled the significant expression of angiogenesis factors. Treadmill exercise stimulation of HPC mobilization during peripheral nerve regeneration could be used as a therapy in human beings.

Background A new version of the CatWalk XT system was evaluated as a tool for detecting very subtle alteration in gait based on higher speed sample rate; the system could also demonstrate minor changes in neurological function. In this study, we evaluated the neurological outcome of sciatic nerve injury intervened by local injection of hyaluronic acid. Using the CatWalk XT system, we looked for differences between treated and untreated groups and differences within the same group as a function of time so as to assess the power of the Catwalk XT system for detecting subtle neurological change. Methods Peripheral nerve injury was induced in 36 Sprague–Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into one of two groups: Group I: crush injury as the control; Group II: crush injury and local application with hyaluronic acid. These animals were subjected to neurobehavior assessment, histomorphology evaluation, and electrophysiology study periodically. These data were retrieved for statistical analysis. Results The density of neurofilament and S-100 over the distal end of crushed nerve showed significant differences either in inter-group comparison at various time points or intra-group comparison from 7 to 28 days. Neuronal structure architecture, axon counts, intensity of myelination, electrophysiology, and collagen deposition demonstrate significant differences between the two groups. There was significant difference of SFI and angle of ankle in inter- group analysis from 7 to 28 days, but there were no significant differences in SFI and angle of ankle at time points of 7 and 14 days. In the Cat Walk XT analysis, the intensity, print area, stance duration, and swing duration all showed detectable differences at 7, 14, 21, and 28 days, whereas there were no significant difference at 7 and 14 days with CatWalk 7 testing. In addition, there were no significant differences of step sequence or regularity index between the two versions. Conclusion Hyaluronic acid augmented nerve regeneration as early as 7 days after crush injury. This subtle neurological alteration could be detected through the CatWalk XT gait analysis but not the SFI, angle of ankle, or CatWalk 7 methods.

Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated allogeneic mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress and apoptotic death in the rat PC12 cells. The remaining cells exhibited a greater capability of neurite outgrowth. Furthermore, allogeneic and xenogeneic transplantation of peptide-labeled mitochondria after 3 months improved the locomotive activity in the PD rats. This increase was accompanied by a marked decrease in dopaminergic neuron loss in the substantia nigra pars compacta (SNc) and consistent enhancement of tyrosine hydroxylase-positive immunoreaction of dopaminergic neurons in the SNc and striatum. We also observed that in the SN dopaminergic neuron in the treated PD rats, mitochondrial complex I protein and mitochondrial dynamics were restored, thus ameliorating the oxidative DNA damage. Moreover, we determined signal translocation of graft allogeneic mitochondria from the MFB to the calbindin-positive SN neuron, which demonstrated the regulatory role of mitochondrial transport in alleviating 6-OHDA-induced degeneration of dopaminergic neurons.

Human amniotic fluid-derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration. The expression of stromal cell-derived factor-1α (SDF-1α) in the injured nerve exerts a trophic effect by recruiting progenitor cells that promote nerve regeneration. In this study, the authors investigated the feasibility of intravenous administration of AFMSCs according to SDF-1α expression time profiles to facilitate neural regeneration in a sciatic nerve crush injury model.Peripheral nerve injury was induced in 63 Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. The animals were randomized into 1 of 3 groups: Group I, crush injury as the control; Group II, crush injury and intravenous administration of AFMSCs (5 × 10(6) cells for 3 days) immediately after injury (early administration); and Group III, crush injury and intravenous administration of AFMSCs (5 × 10(6) cells for 3 days) 7 days after injury (late administration). Evaluation of neurobehavior, electrophysiological study, and assessment of regeneration markers were conducted every week after injury. The expression of SDF-1α and neurotrophic factors and the distribution of AFMSCs in various time profiles were also assessed.Stromal cell-derived factor-1α increased the migration and wound healing of AFMSCs in vitro, and the migration ability was dose dependent. Crush injury induced the expression of SDF-1α at a peak of 10-14 days either in nerve or muscle, and this increased expression paralleled the expression of its receptor, chemokine receptor type-4 (CXCR-4). Most AFMSCs were distributed to the lung during early or late administration. Significant deposition of AFMSCs in nerve and muscle only occurred in the late administration group. Significantly enhanced neurobehavior, electrophysiological function, nerve myelination, and expression of neurotrophic factors and acetylcholine receptor were demonstrated in the late administration group.Amniotic fluid-derived mesenchymal stem cells can be recruited by expression of SDF-1α in muscle and nerve after nerve crush injury. The increased deposition of AFMSCs paralleled the expression profiles of SDF-1α and its receptor CXCR-4 in either muscle or nerve. Administration of AFMSCs led to improvements in neurobehavior and expression of regeneration markers. Intravenous administration of AFMSCs may be a promising alternative treatment strategy in peripheral nerve disorder.

ObjectiveThis study compared the capabilities of dual-energy X-ray absorptiometry (DXA) and dental cone-beam computed tomography (CBCT) for predicting the cortical bone strength of rat femurs and tibias.Materials and methodsSpecimens of femurs and tibias obtained from 14 rats were first scanned with DXA to obtain the areal bone mineral density (BMD) of the midshaft cortical portion of the bones. The bones were then scanned using dental CBCT to measure the volumetric cortical bone mineral density (vCtBMD) and the cross-sectional moment of inertia (CSMI) for calculating the bone strength index (BSI). A three-point bending test was conducted to measure the fracture load of each femur and tibia. Bivariate linear Pearson analysis was used to calculate the correlation coefficients (r values) among the CBCT measurements, DXA measurements, and three-point bending parameters.ResultsThe correlation coefficients for the associations of the fracture load with areal BMD (measured using DXA), vCtBMD (measured using CBCT), CSMI (measured using CBCT), and BSI were 0.585 (p = 0.028) and 0.532 (p = 0.050) (for the femur and tibia, respectively), 0.638 (p = 0.014) and 0.762 (p = 0.002), 0.778 (p = 0.001) and 0.792 (pConclusionsCBCT was found to be superior to DXA for predicting cortical bone fracture loads in rat femurs and tibias. The BSI, which is a combined index of densitometric and geometric parameters, was especially useful. Further clinical studies are needed to validate the predictive value of BSI obtained from CBCT and should include testing on human cadaver specimens.

A method for the simultaneous measurement of serotonin, catecholamines, and their metabolites, 5-hydroxy-indoleacetic acid, homovanillic acid, and 3, 4-dihydroxyphenylacetic acid, by ultrafiltration and microbore high-performance liquid chromatography with dual electrochemical detection in small plasma volumes was established. Unlike the traditional assays which require at least 1-2 ml of plasma for each measurement, the present method uses only a 20-microliters sample volume. Since blood loss is minimal, repeated blood sampling from a single animal as a rat becomes practicable. The present microassay provides low detection limits (signal-to-noise ratio = 3) for all analytes (0.2-0.5 pg per 5-microliters injection or 50-120 pg/ml plasma). Isocratic separation of these analytes on a microbore column is achieved within 15 min. This rapid and sensitive method can be used as a routine research tool in various physiological or pharmacokinetic studies especially in small animals.

A method for the simultaneous measurement of serotonin catecholamines, and their metabolites in rat plasma by ultrafiltration and microbore liquid chromatography with electrochemical detection (LC-ED) in small volumes is established. Prior to the LC assay, sixteen plasma ultrafiltrates are readily prepared within 30 min in the present study. The present method, applying a dual-electrode detection technique, provides an additional reliable assignment or measurement of peaks by identifying the peaks on the basis of their redox ratios. In addition, the important early-eluting peaks and interfering peaks are eliminated in the cathodic chromatogram resulting in a reliable measurement of norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Isocratic separation of serotonin, catecholamines, and their metabolites by a microbore column is achieved within 15 min. Hence, theoretically, over 90 analyses can be performed in a working day. The limit of detection (signal-to-noise ratio = 3) of this method is ca. 0.2-0.5 pg per injection for all analytes. The required volume of the plasma samples can be less than 100 microliters. Hence, the remainder of the plasma sample can be analysed for other substances. This rapid, simple, and sensitive method can thus be used as a research tool in the simultaneous measurement of rat plasma serotonin, catecholamines, and their metabolites.

Increased integration of CD34(+) cells in injured nerve significantly promotes nerve regeneration, but this effect can be counteracted by limited migration and short survival of CD34(+) cells. SDF-1α and its receptor mediate the recruitment of CD34(+) cells involved in the repair mechanism of several neurological diseases. In this study, the authors investigate the potentiation of CD34(+) cell recruitment triggered by SDF-1α and the involvement of CD34(+) cells in peripheral nerve regeneration.Peripheral nerve injury was induced in 147 Sprague-Dawley rats by crushing the left sciatic nerve with a vessel clamp. The animals were allocated to 3 groups: Group 1, crush injury (controls); Group 2, crush injury and local application of SDF-1α recombinant proteins; and Group 3, crush injury and local application of SDF-1α antibody. Electrophysiological studies and assessment of regeneration markers were conducted at 4 weeks after injury; neurobehavioral studies were conducted at 1, 2, 3, and 4 weeks after injury. The expression of SDF-1α, accumulation of CD34(+) cells, immune cells, and angiogenesis factors in injured nerves were evaluated at 1, 3, 7, 10, 14, 21, and 28 days after injury.Application of SDF-1α increased the migration of CD34(+) cells in vitro, and this effect was dose dependent. Crush injury induced the expression of SDF-1α, with a peak of 10-14 days postinjury, and this increased expression of SDF-1α paralleled the deposition of CD34(+) cells, expression of VEGF, and expression of neurofilament. These effects were further enhanced by the administration of SDF-1α recombinant protein and abolished by administration of SDF-1α antibody. Furthermore, these effects were consistent with improvement in measures of neurological function such as sciatic function index, electrophysiological parameters, muscle weight, and myelination of regenerative nerve.Expression of SDF-1α facilitates recruitment of CD34(+) cells in peripheral nerve injury. The increased deposition of CD34(+) cells paralleled significant expression of angiogenesis factors and was consistent with improvement of neurological function. Utilization of SDF-1α for enhancing the recruitment of CD34(+) cells involved in peripheral nerve regeneration may be considered as an alternative treatment strategy in peripheral nerve disorders.

The developing neural cell must form a highly organized architecture to properly receive and transmit nerve signals. Neural formation from embryonic stem (ES) cells provides a novel system for studying axonogenesis, which are orchestrated by polarity-regulating molecules. Here the ES-derived motoneurons, identified by HB9 promoter-driven green fluorescent protein (GFP) expression, showed characteristics of motoneuron-specific gene expression. In the majority of motoneurons, one of the bilateral neurites developed into an axon that featured with axonal markers, including Tau 1, vesicle acetylcholine transporter, and synaptophysin. Interestingly, one third of the motoneurons developed bi-axonal processes but no multiple axonal GFP cell was found. The neuronal polarity-regulating proteins, including the phosphorylated AKT and ERK, were compartmentalized into both of the bilateral axonal tips. Importantly, this aberrant axon morphology was still present after the engraftment of GFP+ neurons into the spinal cord, suggesting that even a mature neural environment fails to provide a proper niche to guide normal axon formation. These findings underscore the necessity for evaluating the morphogenesis and functionality of neurons before the clinical trials using ES or somatic stem cells.

Although gait change is considered a useful indicator of severity in animal models of Parkinson's disease, systematic and extensive gait analysis in animal models of neurological deficits is not well established. The CatWalk-assisted automated gait analysis system provides a comprehensive way to assess a number of dynamic and static gait parameters simultaneously. In this study, we used the Catwalk system to investigate changes in gait parameters in adult rats with unilateral 6-OHDA-induced lesions and the rescue effect of dopaminergic neuron transplantation on gait function. Four weeks after 6-OHDA injection, the intensity and maximal area of contact were significantly decreased in the affected paws and the swing speed significantly decreased in all four paws. The relative distance between the hind paws also increased, suggesting that animals with unilateral 6-OHDA-induced lesions required all four paws to compensate for loss of balance function. At 8 weeks post-transplantation, engrafted dopaminergic neurons expressed tyrosine hydroxylase. In addition, the intensity, contact area, and swing speed of the four limbs increased and the distance between the hind paws decreased. Partial recovery of methamphetamine-induced rotational response was also noted.

Human amniotic fluid-derived mesenchymal stem cells (AFMSCs) have been shown to promote peripheral nerve regeneration, and the local delivery of neurotrophic factors may additionally enhance nerve regeneration capacity. The present study evaluates whether the transplantation of glia cell line-derived neurotrophic factor (GDNF)-modified human AFMSCs may enhance regeneration of sciatic nerve after a crush injury.Peripheral nerve injury was produced in Sprague-Dawley rats by crushing the left sciatic nerve using a vessel clamp. Either GDNF-modified human AFMSCs or human AFMSCs were embedded in Matrigel and delivered to the injured nerve. Motor function and electrophysiological studies were conducted after 1 and 4 weeks. Early or later nerve regeneration markers were used to evaluate nerve regeneration. The expression of GDNF in the transplanted human AFMSCs and GDNF-modified human AFMSCs was monitored at 7-day intervals.Human AFMSCs were successfully transfected with adenovirus, and a significant amount of GDNF was detected in human AFMSCs or the culture medium supernatant. Increases in the sciatic nerve function index, the compound muscle action potential ratio, conduction latency, and muscle weight were found in the groups treated with human AFMSCs or GDNF-modified human AFMSCs. Importantly, the GDNF-modified human AFMSCs induced the greatest improvement. Expression of markers of early nerve regeneration, such as increased expression of neurofilament and BrdU and reduced Schwann cell apoptosis, as well as late regeneration markers, consisting of reduced vacuole counts, increased expression of Luxol fast blue and S100 protein, paralleled the results of motor function. The expression of GDNF in GDNF-modified human AFMSCs was demonstrated up to 4 weeks; however, the expression decreased over time.The GDNF-modified human AFMSCs appeared to promote nerve regeneration. The consecutive expression of GDNF was demonstrated in GDNF-modified human AFMSCs up to 4 weeks. These findings support a nerve regeneration scenario involving cell transplantation with additional neurotrophic factor secretion.

Clearance of fibrin and associated inflammatory cytokines by tissue-type plasminogen activator (t-PA) is related to improved regeneration in neurological disorder. The biological activity of fermented soybean (natto) is very similar to that of t-PA. We investigated the effect of the dietary supplement of natto on peripheral nerve regeneration. The peripheral nerve injury was produced by crushing the left sciatic nerve with a vessel clamp in Sprague-Dawley rats. The injured animals were fed orally either with saline or natto (16 mg/day) for seven consecutive days after injury. Increased functional outcome such as sciatic nerve functional index, angle of ankle, compound muscle action potential and conduction latency were observed in natto-treated group. Histological examination demonstrated that natto treatment improved injury-induced vacuole formation, S-100 and vessel immunoreactivities and axon loss. Oral intake of natto prolonged prothrombin time and reduced fibrinogen but did not change activated partial thromboplastin time and bleeding time. Furthermore, natto decreased injury-induced fibrin deposition, indicating a tolerant fibrinolytic activity. The treatment of natto significantly improved injury-induced disruption of blood-nerve barrier and loss of matrix component such as laminin and fibronectin. Sciatic nerve crush injury induced elevation of tumor necrosis factor alpha (TNF-alpha) production and caused apoptosis. The increased production of TNF-alpha and apoptosis were attenuated by natto treatment. These findings indicate that oral intake of natto has the potential to augment regeneration in peripheral nerve injury, possibly mediated by the clearance of fibrin and decreased production of TNF-alpha.

Background Anastomosis of the nerve especially at narrow surgical field and presence of surgical tension is not easily accessible. DuraSeal demonstrates strong adhesive power without producing neurotoxicity. Herein, we evaluate the possibility of DuraSeal as a substitute in the repair of sciatic nerve gap injury. Materials and Methods The nerve gap model was constructed by excising the sciatic nerve (5 mm in length) in Sprague Dawley rats leaving a 5 mm nerve defect between nerve stumps. Animals were categorized into four groups: Group I: no treatment; Group II: 4 stitches suture; Group III: nerve approximation fixed by tissue glue; Group IV: nerve approximation fixed by DuraSeal. The motor function assessment included the CatWalk and SFI as well as electrophysiological studies. Nerve continuity and regeneration was examined at 1 and 8 wk after injury. The inflammatory cells, Schwann cell apoptosis, and Schwann cell proliferation were also investigated 1 wk after injury. Results The achievement of nerve continuity and myelination by DuraSeal approached that of suture demonstrated by crystal violet and Luxol Fast Blue staining at 1 and 8 wk, respectively. Motor function and electrophysiological parameters were restored in DuraSeal and suture group. Early expression of neurofilament and bromodeoxyuridine (BrdU) was also observed in these two groups. There was no statistically significant difference in deposits of macrophages and neutrophil cells or cell apoptosis among these four groups. Conclusions DuraSeal achieved the same nerve regeneration compared with that of suture and produced better regeneration than that of the tissue glue or without any treatment. The accomplishment of nerve regeneration and continuity without causing neurotoxicity justifies using DuraSeal as a ligature in the anastomosis of nerve gap injury.

Amniotic fluid mesenchymal stem cells (AFS) harbor the potential to improve peripheral nerve injury by inherited neurotrophic factor secretion, but present the drawback of the short-term survival after transplantation. Granulocyte-colony stimulating factor (G-CSF) has a diversity of functions, including anti-inflammatory and anti-apoptotic effects. This study was conducted to evaluate whether G-CSF could augment the neuroprotective effect of transplanted AFS against peripheral nerve injury. The potential involvement of anti-inflammation/anti-apoptosis effect was also investigated. Peripheral nerve injury was produced in Sprauge-Dawley rats by crushing left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. G-CSF (50 microg/kg) was administrated by intra-peritoneal injection for 7 consecutive days. Cell apoptosis, inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. Crush injury induced inflammatory response, disrupted nerve integrity, and impaired nerve function in sciatic nerve. Crush injury-provoked inflammation was attenuated in groups receiving G-CSF but not in AFS only group. In transplanted AFS, marked apoptosis was detected and this event was reduced by G-CSF treatment. Increased nerve myelination and improved motor function were observed in AFS transplanted, G-CSF administrated, and AFS/G-CSF combined treatment groups. Significantly, the combined treatment showed the most beneficial effect. In conclusion, the concomitant treatment of AFS with G-CSF augments peripheral nerve regeneration which may involve the suppression of apoptotic death in implanted AFS and the attenuation of inflammatory response.

We tested the herbal extract 2,3,5,6-tetramethylpyrazine (TMP) for possible therapeutic efficacy against a glioma cell line and against gliomas transplanted into rat brains. In the cultured glioma cells, 50 muM TMP significantly inhibited glutamate-induced increase in intracellular calcium. Significant cell damage (30%) and proliferation suppression (10%), however, occurred only at higher concentrations (200-400 microM). Gliomaneuronal co-culturing resulted in significant neuronal damage and higher proliferation of the glioma cells (140%) compared with single cultures. Low concentrations of TMP (< or =200 microM) attenuated the neuronal damage, suppressed glioma migration, and decreased glioma proliferation in the neuronal-glioma co-culture. Gliomas transplanted into the frontal cortical area exhibited high proliferation, with untreated rats dying 10-23 days later. TMP treatment inhibited tumor growth and significantly extended survival time. The results indicate that TMP can suppress glioma activity, including growth, and protect neurons against glioma-induced excitotoxicity, suggesting that TMP may have therapeutic potential in the treatment of malignant gliomas.

The time window in the treatment of ischemic stroke with tissue plasminogen activator (tPA) is narrow, arbitrarily within 3 hours after the onset of symptom. Hemorrhagic transformation resulting from cerebral ischemia may be related to damage of the microvascular basal lamina of the brain, which may in turn cause microvascular fibrin deposition and aggravate cerebral ischemia. Here, we investigated the effect of tPA on the microvascular tissue changes during cerebral ischemia/reperfusion. Sprague-Dawley rats were subjected to focal cerebral ischemia by ligation of the right middle cerebral artery and bilateral common carotid arteries for 90 minutes. Sixty minutes after the onset of ischemia, escalated dosages of tPA from 2.5 to 10 mg/kg or saline were intravenously infused for 60 minutes. Twenty-four hours after reperfusion, the animals were allowed to be killed for examination. Low dosage of tPA (2.5-7.5 mg/kg) reduced post-ischemic brain infarction, suppressed metalloproteinase 2 (MMP-2) activity and restored blood-brain barrier (BBB) integrity. In contrast, high dose of tPA (10 mg/kg) aggravated brain infarction, increased MMP-2 activity and exacerbated BBB disruption. Cerebral ischemia/reperfusion decreased the immunoreactivity of both collagen type IV- and laminin-positive microvessels, whereas the low dosage of tPA (2.5-7.5 mg/kg) attenuated the reduction. When these molecules in whole cortical tissues were analysed, tPA dosage-dependently decreased the total content of collagen type IV, laminin and fibronectin. Although the detailed mechanisms regarding the action of tPA are yet to be investigated, our findings demonstrate that the detrimental effect of tPA was mediated, at least in part, through the destruction of the basal lamina in the cerebral microvessels by activating MMP-2.

An in vitro ischemia model (oxygen, glucose, and serum deprivation) is used to investigate the possible cellular and molecular mechanisms responsible for cerebral ischemia. We have previously demonstrated that supernatants derived from ischemic microglia can protect ischemic brain cells by releasing GDNF and TGF-beta1. In the present study, we investigate whether products of ischemic astrocytes can also protect ischemic microglia, astrocytes, and neurons in a similar manner. Supernatants from ischemic astrocytes were collected after various periods of ischemia and incubated with microglia, astrocytes, or neurons individually, under in vitro ischemic conditions. The components responsible for the protective effects of astrocyte-derived supernatants were then identified by Western blot, ELISA, trypan blue dye exclusion, and immunoblocking assays. Results showed that under conditions of in vitro ischemia the number of surviving microglia, astrocytes, and neurons was significantly increased by the incorporation of the astrocyte-derived supernatants. Astrocyte supernatant-mediated protection of ischemic microglia was dependent on TGF-beta1 and NT-3, ischemic astrocytes were protected by GDNF, and ischemic neurons were protected by NT-3. In addition, protein expression of TGF-beta1 and NT-3 receptors in microglia, GDNF receptors in astrocytes, and NT-3 receptors in neurons was increased by in vitro ischemia. These results suggest that astrocyte-derived protection of ischemic brain cells is dependent not only on factors released from the ischemic astrocytes, but also on the type of receptor present on the responding cells. Therapeutic potential of TGF-beta1, GDNF, and NT-3 in the control of cerebral ischemia is further suggested.

An in vitro ischemia model was used to determine the molecular mechanisms responsible for the ischemia-induced neuronal cell death. Additionally, the neuronal protective mechanisms of anti-apoptotic drugs against ischemia were also evaluated. In this study, the primary neuronal cultures were incubated in an anoxic chamber with 95% of N2 and 5% of CO2 for various times. The death rate, degree of the apoptotic damage, reduction of mitochondrial membrane potential, translocation of Bax, release of cytochrome C and activation of caspase-9 and -3 were determined at each time point. Results showed that a Bax-regulated mitochondria- mediated apoptosis is responsible for the in vitro ischemia-induced neuronal death. Reduction in mitochondrial membrane potential plays no role in triggering this apoptosis. Furthermore, the anti-apoptotic drugs: furosemide (a Bax blocker) and ZVAD-fmk (caspase inhibitor) but not cyclosporine A (a MPT pore blocker), significantly protected the neurons against ischemia-induced damage. This provides an additional consideration in the future selection of new anti-ischemic drugs.

Objective Amniotic fluid mesenchymal stem cells (MSCs) have the potential to differentiate into neuronal stem cells in vitro. We evaluated using amniotic fluid MSCs to support or enhance the ability of the injured sciatic nerve to cross a nerve gap. Materials and methods We created a 5 mm nerve defect in Sprague Dawley rats. One group received therapy with MSCs embedded into woven oxidised regenerated cellulose gauze (Surgical; Ethicon, Somerville, NJ) and fibrin glue, while a control group received woven Surgicel and fibrin glue only. Evaluation methods included behavioural, electrophysiological and immunohistochemical studies. Results In gait analysis, the angle of the ankles in the treatment and control group were 46.4° (standard deviation [SD] = 15°) and 36° (SD = 8.2°), respectively, which was statistically significant ( p = 0.045). Five of 10 treated rats (50%) demonstrated partial foot movement, while none of the control group had any movement. The percentage amplitude of muscle compound action potential in the experimental group was 43% (SD = 12.5%) compared to 29% (SD = 8.8%) in the control group ( p = 0.038). The conduction latencies in the control and experimental groups was 2.5 ms (SD = 0.45) and 1.7 ms (SD = 0.47), respectively ( p = 0.005). Histological examination demonstrated that 70% of the treatment group achieved a maximum axon diameter percentage across the nerve gap of greater than 50%, compared with 0% in the control group. There were no differences in direction of fibre growth and fibrotic reaction between the two groups. Conclusion Amniotic fluid MSC can augment growth of injured nerve across a nerve gap. This effect may be due to neurotrophic or induction effects of the MSC interacting with Schwann cells. Further study is required to determine the underlying mechanism of this effect.

The disposition and biliary excretion of omeprazole was investigated following i.v. administration to rats at 10 mg/kg. We used a microdialysis technique coupled to a validated microbore HPLC system to monitor the levels of protein-unbound omeprazole in rat blood, brain and bile, constructing the relationship of the time course of the presence of omeprazole. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium, the brain striatum and the bile duct of the male Sprague-Dawley rats for biological fluid sampling after the administration of omeprazole (10 mg/kg) through the femoral vein. The concentration-response relationship from the present method indicated linearity (r2>0.995) over a concentration range of 0.01-50 microg/ml for omeprazole. Intra-assay and inter-assay precision and accuracy of omeprazole fell well within the predefined limits of acceptability. Following omeprazole administration, the blood-to-brain coefficient of distribution was 0.15, which was calculated as the area under the concentration versus time curve (AUC) in the brain divided by the AUC in blood (k=AUCbrain/AUCblood). The blood-to-bile coefficient of distribution (k=AUCbile/AUCblood) was 0.58. The decline of unbound omeprazole in the brain striatum, blood and bile fluid suggests that there was rapid exchange and equilibration between the compartments of the peripheral and central nervous systems. In addition, the results indicated that omeprazole was able to penetrate the blood-brain barrier and undergo hepatobiliary excretion.