Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

Single-chain variable domain fragment(scFv)637 is an antigen-specific scFv of myasthenia gravis.In this study,scFv and human serum albumin genes were conjugated and the fusion protein was expressed in Pichia pastoris.The affinity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunofluorescence staining.The ability of the fusion protein to block myasthenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA.The results showed that the inhibition rate was 2.0–77.4%,and the stability of fusion protein in static healthy sera was about 3 days.This approach suggests the scFv-human serum albumin is a potential candidate for specific immunosuppressive therapy of myasthenia gravis.     

Acrylamide inhibits nerve sprouting induced by botulinum toxin type A

Botulinum toxin type A is a potent muscle relaxant that blocks the transmission and release of acetylcholine at the neuromuscular junction. Intramuscular injection of botulinum toxin type A has served as an effective and safe therapy for strabismus and focal dystonia. However, muscular weakness is temporary and after 3–4 months, muscle strength usually recovers because functional recovery is mediated by nerve sprouting and reconstruction of the neuromuscular junction. Acrylamide may produce neurotoxic substances that cause retrograde necrotizing neuropathy and inhibit nerve sprouting caused by botulinum toxin type A. This study investigated whether acrylamide inhibits nerve sprouting after intramuscular injection of botulinum toxin type A. A tibial nerve sprouting model was established through local injection of botulinum toxin type A into the right gastrocnemius muscle of Sprague-Dawley rats. Following intramuscular injection, rats were given intraperitoneal injection of 3% acrylamide every 3 days for 21 days. Nerve sprouting appeared 2 weeks after intramuscular injection of botulinum toxin type A and single-fiber electromyography revealed abnormal conduction at the neuromuscular junction 1 week after intramuscular injection of botulinum toxin type A. Following intraperitoneal injection of acrylamide, the peak muscle fiber density decreased. Electromyography jitter value were restored to normal levels 6 weeks after injection. This indicates that the maximal decrease in fiber density and the time at which functional conduction of neuromuscular junction was restored were delayed. Additionally, the increase in tibial nerve fibers was reduced. Acrylamide inhibits nerve sprouting caused by botulinum toxin type A and may be used to prolong the clinical dosage of botulinum toxin type A.     

Phrenic nerve transfer to the musculocutaneous nerve for the repair of brachial plexus injury: electrophysiological characteristics

Phrenic nerve transfer is a major dynamic treatment used to repair brachial plexus root avulsion. We analyzed 72 relevant articles on phrenic nerve transfer to repair injured brachial plexus that were indexed by Science Citation Index. The keywords searched were brachial plexus injury, phrenic nerve, repair, surgery, protection, nerve transfer, and nerve graft. In addition, we performed neurophysiological analysis of the preoperative condition and prognosis of 10 patients undergoing ipsilateral phrenic nerve transfer to the musculocutaneous nerve in our hospital from 2008 to 201 3 and observed the electromyograms of the biceps brachii and motor conduction function of the musculocutaneous nerve. Clinically, approximately 28% of patients had brachial plexus injury combined with phrenic nerve injury, and injured phrenic nerve cannot be used as a nerve graft. After phrenic nerve transfer to the musculocutaneous nerve, the regenerated potentials first appeared at 3 months. Recovery of motor unit action potential occurred 6 months later and became more apparent at 12 months. The percent of patients recovering ‘excellent' and ‘good' muscle strength in the biceps brachii was 80% after 18 months. At 12 months after surgery, motor nerve conduction potential appeared in the musculocutaneous nerve in seven cases. These data suggest that preoperative evaluation of phrenic nerve function may help identify the most appropriate nerve graft in patients with an injured brachial plexus. The functional recovery of a transplanted nerve can be dynamically observed after the surgery.     

Improved C3-4 transfer for treatment of root avulsion of the brachial plexus upper trunk Animal experiments and clinical application

Experimental rats with root avulsion of the brachial plexus upper trunk were treated with the improved C3-4 transfer for neurotization of C5-6.Results showed that Terzis grooming test scores were significantly increased at 6 months after treatment,the latency of C5-6 motor evoked potential was gradually shortened,and the amplitude was gradually increased.The rate of C3 instead of C5 and the C4 + phrenic nerve instead of C6 myelinated nerve fibers crossing through the anastomotic stoma was approximately 80%.Myelinated nerve fibers were arranged loosely but the thickness of the myelin sheath was similar to that of the healthy side.In clinical applications,39 patients with root avulsion of the brachial plexus upper trunk were followed for 6 months to 4.5 years after treatment using the improved C3 instead of C5 nerve root transfer and C4 nerve root and phrenic nerve instead of C6 nerve root transfer.Results showed that the strength of the brachial biceps and deltoid muscles recovered to level III-IV,scapular muscle to level III-IV,latissimus dorsi and pectoralis major muscles to above level III,and the brachial triceps muscle to level 0-III.Results showed that the improved C3-4 transfer for root avulsion of the brachial plexus upper trunk in animal models is similar to clinical findings and that C3-4 and the phrenic nerve transfer for neurotization of C5-6 can innervate the avulsed brachial plexus upper trunk and promote the recovery of nerve function in the upper extremity.     

Key changes in denervated muscles and their impact on regeneration and reinnervation

The neuromuscular junction becomes progressively less receptive to regenerating axons if nerve repair is delayed for a long period of time. It is difficult to ascertain the denervated muscle＇s residual receptivity by time alone. Other sensitive markers that closely correlate with the extent of denervation should be found. After a denervated muscle develops a fibrillation potential, muscle fiber conduction velocity, muscle fiber diameter, muscle wet weight, and maximal isometric force all decrease; remodeling increases neuromuscular junction fragmentation and plantar area, and expression of myogenesis-related genes is initially up-regulated and then down-regulated. All these changes correlate with both the time course and degree of denervation. The nature and time course of these denervation changes in muscle are reviewed from the literature to explore their roles in assessing both the degree of detrimental changes and the potential success of a nerve repair. Fibrillation potential amplitude, muscle fiber conduction velocity, muscle fiber diameter, mRNA expression levels of myogenic regulatory factors and nicotinic acetylcholine receptor could all reflect the severity and length of denervation and the receptiveness of denervated muscle to regenerating axons, which could possibly offer an important clue for surgical choices and predict the outcomes of delayed nerve repair.     

Galanin and its receptor system promote the repair of injured sciatic nerves in diabetic rats

Various studies have reported that galanin can promote axonal regeneration of dorsal root ganglion neuronsin vitro and inhibit neuropathic pain. However, little is known about its effects on diabetic peripheral neuropathy, andin vivo experimental data are lacking. We hypothesized that repeated applications of exogenous galanin over an extended time frame may also repair nerve damage in diabetic peripheral neuropathy, and relieve pain in vivo. We found that neuropathic pain occurred in streptozotocin-induced diabetic rats and was more severe after sciatic nerve pinch injury at 14 and 28 days than in diabetic sham-operated rats. Treatment with exogenous galanin alleviated the neuropathic pain and promoted sciatic nerve regeneration more effectively in diabetic rats than in non-diabetic rats after sciatic nerve pinch injury. This was accompanied by changes in the levels of endogenous galanin, and its receptors galanin receptor 1 and galanin receptor 2 in the dorsal root ganglia and the spinal dorsal horn when compared with nerve pinch normal rats. Our results show that application of exogenous galanin daily for 28 days can promote the regeneration of injured sciatic nerves, and alleviate neuropathic pain in diabetic rats.     

Progress in perisynaptic Schwann cell and neuromuscular junction research

The neuromuscular junction(NMJ)is widely studied for its utility in investigating synaptic properties and processes and neuromuscular changes in response to injury,aging,and disease.The NMJ consists of three essential anatomic components,the pre-synaptic motor axon terminal,the post-synaptic nicotinic acetylcholine receptors(AchRs)on the muscle,and the perisynaptic Schwann cell(PSC),also known as the terminal Schwann cell,that caps the synapse(Figure 1A).     

Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion

The present study found expressions of α7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibrillary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein 1α, RANTES, interleukin-1β, interleukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid β protein 1-42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1-42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated α7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1-42.     

Clinical significance of detection of antibodies to fetal and adult acetylcholine receptors in myasthenia gravis

Objective To evaluate the frequency, distribution and clinical significance of the antibodies to the fetal and/or adult acetylcholine receptor (AChR) in patients with myasthenia gravis (MG). Methods AChR antibodies were detected by cell-based assay in the serum of ocular MG (OMG) (n = 90) and generalized MG (GMG) patients (n = 110). The fetal-type (2α: β: γ: δ) and adult-type (2α: β: ε: δ) AChR were used as antigens, and their relevance to disease presentation was assessed. Results The overall frequencies of anti-adult and anti-fetal AChR antibodies were similar in all 200 patients examined, with 14 having serum specific to the AChR-γ subunit, and 22 to the AChR-ε subunit. The overall sensitivity when using the fetal and adult AChR antibodies was higher than that when using the fetal AChR antibody only (P = 0.015). Compared with OMG patients, the mean age at disease onset and the positive ratio of antibodies to both isoforms of the AChR were significantly higher in patients who subsequently progressed to GMG. Older patients and patients with both anti-fetal and anti-adult AChR antibodies had a greater risk for developing generalized disease [odds ratio (OR), 1.03; 95% confidence interval (CI), 1.01-1.06 and OR, 5.09; 95% CI, 2.23-11.62]. Conclusion Using both fetal-and adult-type AChRs as the antigens may be more sensitive than using either subtype. Patients with serum specific to both isoforms are at a greater risk of progressing to GMG. Patients with disease onset at an advanced age appear to have a higher frequency of GMG conversion.     

Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats

Edaravone has been shown to delay neuronal apoptosis, thereby improving nerve function and the microenvironment after spinal cord injury. Edaravone can provide a favorable environment for the treatment of spinal cord injury using Schwann cell transplantation. This study used rat models of complete spinal cord transection at T9. Six hours later, Schwann cells were transplanted in the head and tail ends of the injury site. Simultaneously, edaravone was injected through the caudal vein. Eight weeks later, the PKH-26-labeled Schwann cells had survived and migrated to the center of the spinal cord injury region in rats after combined treatment with edaravone and Schwann cells. Moreover, the number of PKH-26-labeled Schwann cells in the rat spinal cord was more than that in rats undergoing Schwann cell transplantation alone or rats without any treatment. Horseradish peroxidase retrograde tracing revealed that the number of horseradish peroxidase-positive nerve fibers was greater in rats treated with edaravone combined with Schwann cells than in rats with Schwann cell transplantation alone. The results demonstrated that lower extremity motor function and neurophysiological function were better in rats treated with edaravone and Schwann cells than in rats with Schwann cell transplantation only. These data confirmed that Schwann cell transplantation combined with edaravone injection promoted the regeneration of nerve fibers of rats with spinal cord injury and improved neurological function.     

Effectiveness of Qiangjijianli capsule on experimental autoimmune myasthenia gravis

BACKGROUND: Experimental autoimmune myasthenia gravis (EAMG) and anti-AchR antibody of human myasthenia gravis are the same immune globulin. This antibody restricts the activity of nicotinic acetylcholine receptor and the amount of receptor binding sites is decreased, so myasthenia gravis occurs. OBJECTIVE: To observe the therapeutic effect and mechanism of action of Qiangjijianli capsule on EAMG rats. DESIGN: A randomized controlled animal experiment. SETTING: Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine. MATERIALS: Acetylcholine receptors (AchR) were extracted from electric skate's electric organ which lives in the sea near Guangzhou. It had high biological activity and the protein content was 1.63 g/L. Qiangjijianli capsule (Astragalus mongholicus, Codonopsis pilosula, Atractylodes macrocephala, Angelica sinensis, Bupleurum chinense, Cairo morningglory root or leaf, Glycyrrhiza uralensis, etc. 0.5 g crude drug per capsule) was bought from the Manufacturing Laboratory of Guangzhou University of Traditional Chinese Medicine with the Batch No. 89-11-1. METHODS: This experiment was carried out in the Experimental Animal Center of Guangzhou University of Traditional Chinese Medicine from May to August 1990. ①Adult female SD rats were immunized with AchR. The animals' movement condition was observed and recorded everyday. Ten rats were chosen as normal control group, and they were not given any treatment and raised normally. After modeled, 20 successful rats were randomly assigned into 2 groups: treatmental group and model group. 2 mL Qiangjijianli capsule suspension (1 g) was intragastrically administrated into each rat of treatmental group for 30 days; The same amount of clean water was intragastrically administrated into the rats of model group for 30 days. ② Serum AchR antibody was measured with ABC-ELISA method. After administration, the rats were sacrificed. The complete diaphragmatic muscle was extracted for in vitro receptor binding test. The constitution of N-AchR per milligram of tissue was measured with FT-603 well-type γ scintillation detector and FH408 calibrater. ③ t test was used for the comparison of difference of mean value of two samples. MAIN OUTCOME MEASURES: The effect of Qiangjijianli capsule on the amount of binding site of serum AchR antibody and nicotine-like acetylcholinergic receptor(N-AChR). RESULTS: Before Qiangjijianli capsule treatment, serum AchR antibody titre was close between model group and treatmental group (0.82±0.15 vs. 0.79±0.12, P > 0.05); After Qiangjijianli capsule treatment, serum AchR antibody titre of treatmental group was significantly decreased (0.45±0.11,t =6.602,P < 0.01), and that in the treatmental group did not change significantly (0.71±0.13,P > 0.05). So, serum AchR antibody titre of treatmental group was significantly lower than that of model group (t =5.780, P < 0.01). ③ The number of N-AchR of normal control group was significantly more than that of model group [(1 503±156) mg·min-1 vs. (1 118±128) mg·min-1, t = 6.034,P < 0.01]. The number of N-AchR of treatmental group was significantly more than that of model group [(1 277±143) mg·min-1 vs. (1 118±128) mg·min-1, t =2.619,P < 0.05]. ③After the treatment of Qiangjijianli capsule, all of the six rats which had clinical symptoms in treatmental group did well more or less, while in model group, the six rats had no change except one. CONCLUSION: Qiangjijianli capsule can improve the symptoms of myasthenia gravis by immunological regulation and pharmacological effect of N-AchR.     

Acupuncture at Baihui and Dazhui reduces brain cell apoptosis in heroin readdicts简

Acupuncture at Baihui （GV20） and Dazhui （GV14） reduces neuronal loss and attenuates ultra- structural damage in cerebral ischemic rats. However, whether acupuncture can treat addiction and prevent readdiction through changes to brain cell ultrastructure remains unknown. In this study, cell apoptosis was observed in the hippocampus and frontal lobe of heroin readdicted rats by electron microscopy. Immunohistochemical staining displayed a reduction in Bcl-2 ex- pression and an increase in Bax expression in the hippocampus and frontal lobe. After rats were given acupuncture at Baihui and Dazhui, the pathological damage in the hippocampus and frontal lobe was significantly reduced, Bcl-2 expression was upregulated and Bax expression was downregulated. Acupuncture exerted a similar effect with methadone, a commonly used drug for clinical treatment of drug addiction. Experimental findings suggest that acupuncture at Dazhui and Baihui can prevent brain cell apoptosis in heroin readdicted rats.     

Electroacupuncture diminishes P2X2 and P2X3 purinergic receptor expression in dorsal root ganglia of rats with visceral hypersensitivity

Electroacupuncture at Shangjuxu (ST37) and Tianshu (ST25) can improve visceral hypersensitivity in rats. Colorectal distension was used to establish a rat model of chronic visceral hypersensitivity. Immunohistochemistry was used to detect P2X2 and P2X3 receptor expression in dorsal root ganglia from rats with chronic visceral hypersensitivity. Results demonstrated that abdominal withdrawal reflex scores obviously increased following establishment of the model, indicating visceral hypersensitivity. Simultaneously, P2X2 and P2X3 receptor expression increased in dorsal root ganglia. After bilateral electroacupuncture at Shangjuxu and Tianshu, abdominal withdrawal reflex scores and P2X2 and P2X3 receptor expression decreased in rats with visceral hypersensitivity. These results indicated that electroacupuncture treatment improved visceral hypersensitivity in rats with irritable bowel syndrome by reducing P2X2 and P2X3 receptor expression in dorsal root ganglia.     

Suspended moxibustion at Tianshu (ST25) inhibits prokineticin 1 and prokineticin receptor 1 expression in the spinal cord of rats with chronic visceral hypersensitivity

Suspended moxibustion can decrease the expression of prokineticin 1 and its receptor in colonic tissue from rats modeling chronic visceral hyperalgesia. This study aimed to verify if rat spinal cord prokineticin 1 and its receptor contribute to the analgesic effect of suspended moxibustion in a rat model of irritable bowel syndrome where rats display chronic visceral hypersensitivity. Results showed that suspended moxibustion at Tianshu (ST25) point significantly decreased visceral sensitivity to colorectal distention in a chronic visceral hyperalgesia rat model; also protein and mRNA expression of prokineticin 1 and prokineticin receptor 1 in the spinal cord of rats was significantly decreased. Experimental findings indicate that prokineticin 1 and prokineticin receptor 1 are involved in the analgesia using suspended moxibustion in rats with chronic visceral hyperalgesia.     

Dynamic expression of nerve growth factor and its receptor TrkA after subarachnoid hemorrhage in rat brain

Delayed ischemic neurologic deifcit after subarachnoid hemorrhage results from loss of neural cells. Nerve growth factor and its receptor TrkA may promote regeneration of neural cells, but their expression after subarachnoid hemorrhage remains unclear. In the present study, a rat model of subarachnoid hemorrhage was established using two injections of autologous blood into the cistern magna. Immunohisto-chemical staining suggested that the expression of nerve growth factor and TrkA in the cerebral cortex and brainstem increased at 6 hours, peaked at 12 hours and decreased 1 day after induction of subarachnoid hemorrhage, whereas the expression in the hippocampus increased at 6 hours, peaked on day 1, and decreased 3 days later. Compared with those for the rats in the sham and saline groups, neurobehavioral scores decreased signiifcantly 12 hours and 3 days after subarachnoid hemorrhage (P<0.05). These results suggest that the expression of nerve growth factor and its receptor TrkA is dynamically changed in the rat brain and may thus participate in neuronal survival and nerve regeneration after subarachnoid hemorrhage.     

Electroacupuncture atBaihui (DU20) acupoint up-regulates mRNA expression of NeuroD molecules in the brains of newborn rats sufferingin utero fetal distress

Neuro D plays a key regulatory effect on differentiation of neural stem cells into mature neurons in the brain.Thus,we assumed that electroacupuncture at Baihui(DU20) acupoint in newborn rats exposed to in utero fetal distress would influence expression of Neuro D.Electroacupuncture at Baihui was performed for 20 minutes on 3-day-old(Day 3) newborn Sprague-Dawley rats exposed to in utero fetal distress;electroacupuncture parameters consisted of sparse and dense waves at a frequency of 2–10 Hz.Real-time fluorescent quantitative PCR results demonstrated that m RNA expression of Neuro D,a molecule that indicates Neuro D,increased with prolonged time in brains of newborn rats,and peaked on Day 22.The level of m RNA expression was similar between Day 16 and Day 35.These findings suggest that electro acupuncture at Baihui acupoint could effectively increase m RNA expression of molecules involved in Neuro D in the brains of newborn rats exposed to in utero fetal distress.     

Acupuncture at the “Huatuojiaji” point affects nerve root regional interleukin-1 level in a rat model of lumbar nerve root compression

BACKGROUND：It has been shown that interleukin-1 （IL-1） may cause inflammatory reactions, which stimulate the nerve root of patients with lumbar intervertebral disc protrusion and leads to pain. Whether the clinical curative effects of acupuncture in the treatment of lumbar and leg pain are linked to an inhibition of local IL-1 secretion is unknown. OBJECTIVE： To assess the influence of acupuncture on IL-1, this study was designed to verify the effects of acupuncture at the ＂Huatuojiaji （Extra）＂ point on the nerve root in a rat model of lumbar nerve root compression, compared with administration of meloxicam, a non-steroidal anti-inflammatory drug. DESIGN, TIME AND SETTING： Randomized, controlled, molecular biology experiment, performed at the Experimental Center, Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University between September 2005 and April 2006. MATERIALS： Forty healthy adult Sprague Dawley rats of either gender were included in this study. The rats were randomly and evenly divided into the following four groups： normal control, model, acupuncture and meloxicam groups. Lumbar nerve root compression was induced in rats in the model, acupuncture, and meloxicam groups by inserting a specially made silicon rubber slice at the juncture of the L5 nerve root and the dural sac. The acupuncture needle （pattern number N3030, 30#, 1.5 inch） was purchased from Suzhou Medical Appliance Factory, China. IL-1 enzyme linked immunosorbent assay （ELISA） kit was purchased from Santa Cruz Biotechnology, Inc., USA. METHODS： The acupuncture group was acupunctured at the ＂Huatuojiaji＂ point, which is lateral to the compressed L5-6 nerve root, with an acupuncture depth of 0.5 cm. There were two treatment courses, each of involved seven 20-minute acupuncture sessions, one session a day. The meloxicam group was administered intragastrically 3.75 mg/kg meloxicam （5 mg meloxicam /10 mL physiological saline）. Rats in the normal control group and model group received an intragast     

Perlecan and synaptophysin changes in denervated skeletal muscle

The present study observed sciatic nerve and gastrocnemius muscle changes in denervated rats using morphology methods,and assessed expression of perlecan,an extracellular matrix com-ponent,which is located at the skeletal muscle cell surface as acetylcholine esterase,as well as synaptophysin,a synaptic marker.Results showed degeneration and inflammation following transection of the sciatic nerve.In addition,the sciatic nerve-dominated skeletal muscle degen-erated with mild inflammation,indicating that skeletal muscle atrophy primarily contributed to denervation-induced nutritional disturbances.With prolonged injury time(1-4 weeks post-injury),perlecan expression gradually decreased and reached the lowest level at 4 weeks,but synap-tophysin expression remained unchanged after denervation.Results suggested that perlecan expression was more sensitive to denervation and reflected regional extracellular matrix changes following denervation.     

Temporal and spatial distribution of metabotropic glutamate receptor 5 during development in the rat cortex and hippocampus

Metabotropic glutamate receptor 5 (mGluR5) is expressed by neurons in zones of active neurogenesis and is involved in the development of neural stem cells in vivo and in vitro.We examined the expression of mGluR5 in the cortex and hippocampus of rats during various prenatal and postnatal periods using immunohistochemistry.During prenatal development,mGluR5 was primarily localized to neuronal somas in the forebrain.During early postnatal periods,the receptor was mainly present on somas in the cortex.mGluR5 immunostaining was visible in apical dendrites and in the neuropil of neurons and persisted throughout postnatal development.During this period,pyramidal neurons were strongly labeled for the receptor.In the hippocampal CA1 region,mGluR5 immunoreactivity was more intense in the stratum oriens,stratum radiatum,and lacunosum moleculare at P0,P5 and P10 relative to P60.mGluR5 expression increased significantly in the molecular layer and decreased significantly in the granule cell layer of the dentate gyrus at P5,P10 and P60 in comparison with P0.Furthermore,some mGluR5-positive cells were also bromodeoxyuridine-or NeuroD-positive in the dentate gyrus at P14.These results demonstrate that mGluR5 has a differential expression pattern in the cortex and hippocampus during early growth,suggesting a role for this receptor in the control of domain specific brain developmental events.