Effects of Broussonetia papyrifera (L.) L'Hér. ex Vent. fruits water extract on hippocampal neurogenesis in the treatment of APP/PS1 transgenic mice.

Background: Adult neurogenesis plays an important role in repairing damaged neurons and improving cognitive impairment in Alzheimer's disease (AD). B. Papyrifera (L.) L'Hér. ex Vent. fruits (BL), a traditional Chinese medicine for tonifying the kidney, has been reported to improve cognitive function in AD mice, but the underlying mechanisms have not been clearly illuminated. This study aimed to provide an overview of the differential compounds in the brain of APP/PS1 mice after BL water extract (BLWE) treatment through metabolomics technology and to elucidate whether the therapeutic effect and mechanism are through the enhancement of neurogenesis. Methods: APP/PS1 transgenic mice were treated with different doses of BLWE. After 6 weeks of intragastric injection, the therapeutic effects of BLWE on APP/PS1 transgenic mice were determined by the Morris water maze test, immunohistochemistry, hematoxylin & eosin and Nissl staining, enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Subsequently, metabolomics technology was used to analyze the regulatory effect of BLWE on differential compounds in the brain of APP/PS1 mice, and on this basis, its molecular mechanism of BLWE was screened. Finally, the protein expression of the Wnt/ß-catenin signaling pathway was detected by Western blotting. Results: After BLWE treatment, the learning and memory function of APP/PS1 mice were significantly improved, which was related to the increase in the number of Nestin+/BrdU+ and NeuN+/BrdU+ cells, and the decrease in the number of apoptotic cells in the hippocampus. BLWE treatment could also up-regulate the expression of synapse-associated proteins. Moreover, BLWE could modulate endogenous metabolic compounds in the brains of AD mice, including N-acetyl-aspartate, glutamine, etc. Furthermore, BLWE inhibited the phosphorylation of Tyr216-GSK-3ß and ß-catenin protein while increased CyclinD1 protein expression. Conclusion: We demonstrated that BLWE can enhance neural stem cells proliferation and improve neurogenesis, thereby efficiently repairing damaged neurons in the hippocampus and ameliorating cognitive impairment in APP/PS1 transgenic mice. The mechanism is at least partly through activating the Wnt/ß-catenin signaling pathway.

Osthole Enhances the Therapeutic Efficiency of Stem Cell Transplantation in Neuroendoscopy Caused Traumatic Brain Injury.

Neuroendoscopy processes can cause severe traumatic brain injury. Existing therapeutic methods, such as neural stem cell transplantation and osthole have not been proven effective. Therefore, there is an emerging need on the development of new techniques for the treatment of brain injuries. In this study we propose to combine the above stem cell based methods and then evaluate the efficiency and accuracy of the new method. Mice were randomly divided into four groups: group 1 (brain injury alone); group 2 (osthole); group 3 (stem cell transplantation); and group 4 (osthole combined with stem cell transplantation). We carried out water maze task to exam spatial memory. Immunocytochemistry was used to test the inflammatory condition of each group, and the differentiation of stem cells. To evaluate the condition of the damaged blood brain barrier restore, we detect the Evans blue (EB) extravasation across the blood brain barrier. The result shows that osthole and stem cell transplantation combined therapeutic method has a potent effect on improving the spatial memory. This combined method was more effective on inhibiting inflammation and preventing neuronal degeneration than the single treated ones. In addition, there was a distinct decline of EB extravasation in the combined treatment groups, which was not observed in single treatment groups. Most importantly, the combined usage of osthole and stem cell transplantation provide a better treatment for the traumatic brain injury caused by neuroendoscopy. The collective evidence indicates osthole combined with neural stem cell transplantation is superior than either method alone for the treatment of traumatic brain injury caused by neuroendoscopy.

Osthole Stimulated Neural Stem Cells Differentiation into Neurons in an Alzheimer's Disease Cell Model via Upregulation of MicroRNA-9 and Rescued the Functional Impairment of Hippocampal Neurons in APP/PS1 Transgenic Mice.

Alzheimer's disease (AD) is the most serious neurodegenerative disease worldwide and is characterized by progressive cognitive impairment and multiple neurological changes, including neuronal loss in the brain. However, there are no available drugs to delay or cure this disease. Consequently, neuronal replacement therapy may be a strategy to treat AD. Osthole (Ost), a natural coumarin derivative, crosses the blood-brain barrier and exerts strong neuroprotective effects against AD in vitro and in vivo. Recently, microRNAs (miRNAs) have demonstrated a crucial role in pathological processes of AD, implying that targeting miRNAs could be a therapeutic approach to AD. In the present study, we investigated whether Ost could enhance cell viability and prevent cell death in amyloid precursor protein (APP)-expressing neural stem cells (NSCs) as well as promote APP-expressing NSCs differentiation into more neurons by upregulating microRNA (miR)-9 and inhibiting the Notch signaling pathway in vitro. In addition, Ost treatment in APP/PS1 double transgenic (Tg) mice markedly restored cognitive functions, reduced Aß plague production and rescued functional impairment of hippocampal neurons. The results of the present study provides evidence of the neurogenesis effects and neurobiological mechanisms of Ost against AD, suggesting that Ost is a promising drug for treatment of AD or other neurodegenerative diseases.

Osthole Protects Bone Marrow-Derived Neural Stem Cells from Oxidative Damage through PI3K/Akt-1 Pathway.

In recent years, neural stem cell (NSC) transplantation has been widely explored as a treatment for neurodegenerative diseases. NSCs are special cells that have some capacity for self-renewal and the potential to differentiate into multiple cell types. However, the inflammatory environment of diseased tissue is not conducive to the survival of transplanted cells. Osthole (Ost) is a principal bioactive component of Fructus Cnidii, Radix Angelicae Pubescentis and other traditional Chinese medicines. Ost has a wide range of pharmacological activities, such as anti-inflammation, immunomodulation, and neuroprotection. In the present study, we assessed the protective effects of Ost on bone marrow-derived-NSCs (BM-NSCs) against injury induced by hydrogen peroxide (H2O2). BM-NSCs were pre-treated with different doses of Ost and treated with H2O2. The cell counting kit-8 (CCK-8) method and lactate dehydrogenase (LDH) leakage assay were used to determine cell viability. Using the TUNEL assay and RT-PCR, we evaluated the effect of Ost on cell apoptosis. The results showed that Ost had protective effects against H2O2-induced cell damage, and the number of apoptotic cells was significantly decreased in the Ost pre-treated groups compared to the H2O2 group. The expression ratio of Bax/Bcl-2 mRNA was also decreased. Furthermore, western blotting was used to analyze levels of proteins related to PI3K/Akt-1 signaling pathway, and results indicated that ost can increase p-Akt and PI3K. Our findings suggested that Ost protects BM-NSCs against oxidative stress injury, and it can be used to improve the inflammatory environment of neurodegenerative diseases so and promote the survival rate of transplanted NSCs.

Neurotrophin-3 promotes proliferation and cholinergic neuronal differentiation of bone marrow- derived neural stem cells via notch signaling pathway.

AIMS: Recently, the potential for neural stem cells (NSCs) to be used in the treatment of Alzheimer's disease (AD) has been reported; however, the therapeutic effects are modest by virtue of the low neural differentiation rate. In our study, we transfected bone marrow-derived NSCs (BM-NSCs) with Neurotrophin-3 (NT-3), a superactive neurotrophic factor that promotes neuronal survival, differentiation, and migration of neuronal cells, to investigate the effects of NT-3 gene overexpression on the proliferation and differentiation into cholinergic neuron of BM-NSCs in vitro and its possible molecular mechanism. MAIN METHODS: BM-NSCs were generated from BM mesenchymal cells of adult C57BL/6 mice and cultured in vitro. After transfected with NT-3 gene, immunofluorescence and RT-PCR method were used to determine the ability of BM-NSCs on proliferation and differentiation into cholinergic neuron; Acetylcholine Assay Kit was used for acetylcholine (Ach). RT-PCR and WB analysis were used to characterize mRNA and protein level related to the Notch signaling pathway. KEY FINDINGS: We found that NT-3 can promote the proliferation and differentiation of BM-NSCs into cholinergic neurons and elevate the levels of acetylcholine (ACh) in the supernatant. Furthermore, NT-3 gene overexpression increase the expression of Hes1, decreased the expression of Mash1 and Ngn1 during proliferation of BM-NSCs. Whereas, the expression of Hes1 was down-regulated, and Mash1 and Ngn1 expression were up-regulated during differentiation of BM-NSCs. SIGNIFICANCE: Our findings support the prospect of using NT-3-transduced BM-NSCs in developing therapies for AD due to their equivalent therapeutic potential as subventricular zone-derived NSCs (SVZ-NSCs), greater accessibility, and autogenous attributes.

Arctigenin Confers Neuroprotection Against Mechanical Trauma Injury in Human Neuroblastoma SH-SY5Y Cells by Regulating miRNA-16 and miRNA-199a Expression to Alleviate Inflammation.

Mechanical trauma injury is a severe insult to neural cells. Subsequent secondary injury involves the release of inflammatory factors that have dramatic consequences for undamaged cells, leading to normal cell death after the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary effects and evaluated the mechanism underlying the action of microRNA (miRNA)-199a and miRNA-16 in a mechanical trauma injury (MTI) model using SH-SY5Y cells in vitro. SH-SY5Y cells are often applied to in vitro models of neuronal function and differentiation. Recently, miRNAs have been demonstrated to play a crucial role in NF-κB and cholinergic signaling, which can regulate inflammation. The cell model was established by scratch-induced injury of human SH-SY5Y cells, which mimics the characteristics of MTI. A cell counting kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunocytochemistry were used to measure cell viability. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the inflammatory cytokine and cholinesterase (CHE) content. The lactate dehydrogenase (LDH) content was measured to assess the degree of cell injury. The mRNA levels were measured by RT-PCR to analyze ARC's mechanism of action. miRNA inhibitors and mimics were used to inhibit and strengthen the expression of miRNAs. Protein expression was detected by western blotting analysis. ARC treatment reduced the TNF-α and IL-6 levels as well as the number of TUNEL+ apoptotic SH-SY5Y cells surrounding the scratch and increased the IL-10 level compared to the controls. ARC attenuated the increase of the cell damage degree and LDH content induced by scratching, indicating increased cell survival. Mechanistic studies showed that ARC upregulated the miRNA-16 and miRNA-199a levels to reduce upstream protein (IKKα and IKKß) expression and inhibit NF-κB signaling pathway activity; moreover, the increased miRNA-199a suppresses cholinesterases to increase cholinergic signaling, resulting in decreased expression of proinflammatory cytokines. ARC treatment confers protection for SH-SY5Y cells through positive regulation of miRNA expression, thereby reducing the inflammatory response. In turn, these effects accelerate injury repair in the scratch-induced injury model. These results might provide insights into the pharmacological role of ARC in anti-inflammation and neuroprotection in neural cells.

Arctigenin Treatment Protects against Brain Damage through an Anti-Inflammatory and Anti-Apoptotic Mechanism after Needle Insertion.

UNLABELLED: Convection enhanced delivery (CED) infuses drugs directly into brain tissue. Needle insertion is required and results in a stab wound injury (SWI). Subsequent secondary injury involves the release of inflammatory and apoptotic cytokines, which have dramatic consequences on the integrity of damaged tissue, leading to the evolution of a pericontusional-damaged area minutes to days after in the initial injury. The present study investigated the capacity for arctigenin (ARC) to prevent secondary brain injury and the determination of the underlying mechanism of action in a mouse model of SWI that mimics the process of CED. After CED, mice received a gavage of ARC from 30 min to 14 days. Neurological severity scores (NSS) and wound closure degree were assessed after the injury. Histological analysis and immunocytochemistry were used to evaluated the extent of brain damage and neuroinflammation. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used to detect universal apoptosis. Enzyme-linked immunosorbent assays (ELISA) was used to test the inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10) and lactate dehydrogenase (LDH) content. Gene levels of inflammation (TNF-α, IL-6, and IL-10) and apoptosis (Caspase-3, Bax and Bcl-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these, we analyzed ARC's efficacy and mechanism of action. RESULTS: ARC treatment improved neurological function by reducing brain water content and hematoma and accelerating wound closure relative to untreated mice. ARC treatment reduced the levels of TNF-α and IL-6 and the number of allograft inflammatory factor (IBA)- and myeloperoxidase (MPO)-positive cells and increased the levels of IL-10. ARC-treated mice had fewer TUNEL+ apoptotic neurons and activated caspase-3-positive neurons surrounding the lesion than controls, indicating increased neuronal survival. CONCLUSIONS: ARC treatment confers neuroprotection of brain tissue through anti-inflammatory and anti-apoptotic effects in a mouse model of SWI. These results suggest a new strategy for promoting neuronal survival and function after CED to improve long-term patient outcome.

The effect of calcium modulating agents on peripheral nerve recovery after crush.

After a nerve injury, calcium concentration in the intra-nerve fiber drastically increases. The purpose of our study was to test an implantable micro-osmotic pump to deliver medications to accelerate calcium absorption, thereby greatly improving nerve regeneration. Twenty-four SD rats were divided into four groups of six each: (1) Sham control: crush injury to sciatic nerve only; (2) Crush injury with a Nifedipine pump; (3) Crush injury with a Calcitonin pump; (4) Crush injury with a Saline pump. Each rat's right sciatic nerve was crushed. The micro-osmotic pump was implanted in the neck, and the dripping tube was routed to the injured nerve. After four weeks of survival time, compound muscle action potential (CMAP), tetanic muscle force (TMF), myelinated nerve fiber area (NFA), nerve calcium concentration (NCC), and calcified spots (CS) were evaluated. The calcium absorption rate (CAR) was also determined. The order from highest to lowest recovery rate was Nifedipine>Calcitonin>Sham control>Saline. Differences among the groups were statistically significant (P<0.001, ANOVA test), and the difference between Nifedipine/Calcitonin and Saline/Sham control were all statistically significant (P<0.001, t-test). The correlation rate of NCC with CMAP/TMF and with NFA/CS and CAR were calculated to be 0.99 (all P<0.001, Pearson's Correlation). We conclude from this study that nerve regeneration strongly correlated with calcium absorption; our new data has shown greatly improved nerve functional recovery, and this can potentially be translated into clinical applications.

Helicoid end-to-side and oblique attachment technique in repair of the musculocutaneous nerve injury with the phrenic nerve as a donor: an experimental study in rats.

The purpose of this study was to identify if a modified end-to-side repair can achieve equal results of nerve regeneration compared to an end-to-end repair using donor phrenic nerves in repair of the musculocutaneous nerve and also pulmonary protection. Eighteen rats were divided into three groups of six each comparing two nerve graft techniques: helicoid end-to-side plus distal oblique repair vs. traditional end-to-end repair, using a donor phrenic nerve. The saphenous nerve was used as a graft between the phrenic nerve and the musculocutaneous nerve. The third group was used as control; the musculocutaneous nerve was transected without any repair. Three months postoperatively, electrophysiology, tetanic force, moist muscle weight, histology, nerve fiber counting, and chest X-ray were evaluated. All results have shown that this modified end-to-side repair was superior to the end-to-end repair. The former did not compromise the diaphragm function, but the latter showed an elevation of the diaphragm. Little recovery was seen in the third group. The conclusion is that this modified end-to-side repair can replace the traditional end-to-end repair using donor phrenic nerves with better results of nerve regeneration without diaphragm compromise.

Intraoperative electrophysiological studies to predict the efficacy of neurolysis after nerve injury-experiment in rats.

Compound muscle action potentials (CMAPs) can be used to analyze injury and recovery of nerve. This standardized study evaluates the value of CMAP analysis in predicting the long-term efficacy of neurolysis. CMAP amplitude is also used to determine the optimal extent of neurolysis. The left peroneal nerves of 30 rats were crushed. CMAPs were recorded for both crushed (left) and control (right) nerves. Fifteen rats underwent neurolysis 3 months post crush injury; the remaining 15 were sham controls and did not undergo neurolysis. CMAP measurements were taken after: (1) release of the nerve from the fascia, (2) opening the epineurium, and (3) opening the perineurium. At 3 months post crush injury, opening the epineurium resulted in a statistically significant increase in CMAP. CMAP increase with perineurial neurolysis was greater than with fascial release of the nerve but was not statistically different from that of epineurial release. At 5 months post crush injury, recovery of crushed nerves that underwent neurolysis was 90% and significantly less at 70.5% in rats not treated with neurolysis, according to CMAP analysis. Two conclusions can be made from this study. First, intraoperative neurophysiologic studies can monitor the immediate results of neurolysis and predict long-term results in the injured nerve. Second, epineurotomy is important in neurolysis, improves the function of the nerve, less invasive, and a slightly more effective technique than perineurotomy.

Pathophysiological process of traumatic vascular spasm in multiple crush injury.

Despite recent advances in microsurgery, the incidence of thrombosis of repaired digital vessels following crush injury is higher than that of uncrushed arteries. The purpose of this study was to evaluate the effects of traumatic crush injury on uncrushed segments of artery adjacent to crushed segments. Sprague-Dawley rat-tail arteries were crushed for 1 hour by 30 pounds of force at two separate 3-mm-wide segments, with 10 mm of uncrushed artery between segments. Luminal size of the uncrushed segments decreased progressively over the first 12 hours, with a significant reduction by 4 hours postcrush. The condition of the arteries continued to deteriorate until, by 24 hours postinjury, they had been denuded of endothelium, and smooth muscle damage had occurred. Untreated vasospasm following a multiple-level crush injury therefore results in progressive endothelial and intimal damage and thrombosis in the intervening healthy uncrushed segments of artery. This article discusses the implications of these findings.

Effects of cellular phone emissions on sperm motility in rats.

OBJECTIVE: To evaluate the effects of cellular phone emissions on rat sperm cells. DESIGN: Classic experimental. SETTING: Animal research laboratory. SUBJECTS: Sixteen 3-month-old male Sprague-Dawley rats, weighing 250-300 g. INTERVENTION(S): Rats in the experimental group were exposed to two 3-hour periods of daily cellular phone emissions for 18 weeks; sperm samples were then collected for evaluation. MAIN OUTCOME MEASURE(S): Evaluation of sperm motility, sperm cell morphology, total sperm cell number, and mRNA levels for two cell surface adhesion proteins. RESULT(S): Rats exposed to 6 hours of daily cellular phone emissions for 18 weeks exhibited a significantly higher incidence of sperm cell death than control group rats through chi-squared analysis. In addition, abnormal clumping of sperm cells was present in rats exposed to cellular phone emissions and was not present in control group rats. CONCLUSION(S): These results suggest that carrying cell phones near reproductive organs could negatively affect male fertility.

Nerve repair at different angles of attachment: experiment in rats.

Sixteen rats underwent two procedures to test the efficacy of oblique vs. transverse nerve repair. On the left, the peroneal nerve was transected at an oblique angle (30-degree) with subsequent oblique end-to-end repair performed. On the right, the peroneal nerve was transected at a transverse angle (90-degree) with subsequent end-to-end repair. At 3.5 months postoperatively, the moist muscle weight of the extensor digitorum longus (EDL) and tetanic force were measured, and axons were counted in donor and recipient nerves. Muscle volume, tetanic force, and moist weight of EDL muscles were statistically significantly higher on the left side (oblique) than on the right (transverse). Histologic analysis with nerve axon counting of the recipient peroneal nerve revealed significantly more regenerated nerve fibers on the left than on the right. The authors concluded that oblique nerve repair enhances nerve-fiber sprouting from the proximal donor nerve by increasing the coapted surface area, thus improving muscle recovery.