Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus

Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I col-lagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve ifbers regenerated. Partial nerve ifber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the com-bination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats.     

The proteome of distal nerves:implication in delayed repair and poor functional recovery

Chronic denervation is one of the key factors that affect nerve regeneration.Chronic axotomy deteriorates the distal nerve stump,causes protein changes,and renders the microenvironment less permissive for regeneration.Some of these factors/proteins have been individually studied.To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect,we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model.Four rats that received immediate repair after sciatic nerve transection served as control,whereas four rats in the experimental group(chronic denervation)had their sciatic nerve repaired after a 12-week delay.All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins.Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels.NanoLC-MS/MS analysis was applied to the gels.Protein expression levels of nerves on the surgery side were compared to those on the contralateral side.Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed.All the differentially expressed proteins in both groups were further stratified according to the biological processes.A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration.Ingenuity Pathway Analysis(IPA)software was used for pathway analysis.The results showed that 709 differentially expressed proteins were identified in the delayed repair group,with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified.The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated.IPA revealed that protective pathways such as LXR/RXR,acute phase response,RAC,ERK/MAPK,CNTF,IL-6,and FGF signaling were inhibited in the delayed repair group,whereas three detrimental pathways,including the complement system,PTEN,and apoptosis signaling,were activated.An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types.The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins.The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.     

Effects of tacrolimus （FK506） on sciatic nerve regeneration in rats

BACKGROUND： Tacrolimus （FK506） protects peripheral nerves located in damaged regions by inhibiting T lymphocyte proliferation and activation. OBJECTIVE： To evaluate the effect of FK506 on promoting regeneration of rat sciatic nerve. DESIGN, TIME AND SETTING： A randomized, controlled, animal study was performed at the Laboratory of the Department of Orthopedic Surgery, Dalian Medical University, China, from September 2007 to September 2008. MATERIALS： A total of 60 adult, male, Sprague-Dawley rats were equally and randomly divided into model, local administration and systemic administration groups. All rats received a neurotomy of bilateral sciatic nerves to establish models of nerve regeneration chambers. The powder and injection of FK506 were supplied by Fujisawa Pharmaceutical, Japan. METHODS： The regeneration chambers of the model group were infused with 0.2 mL saline. The systemic group were injected with 0.2 mL saline, followed by daily subcutaneous injections of FK506 （1 mg/kg）, for 14 days. The local administration group was infused with 0.2 mL FK506 （1 μg/mL）. MAIN OUTCOME MEASURES： Local immune response was observed using hematoxylin-eosin staining. Myelinated nerve fiber number, myelin sheath and nerve fiber thickness were observed using toluidine blue staining. Wet weight of gastrocnemius was evaluated. Compound muscle action potential amplitude, latency, and conduction time were recorded, and motor nerve conduction velocity was calculated using electrophysiology. RESULTS： The total number of myelinated nerve fibers in the local and systemic administration groups was significantly higher than in the model group. The density of myelinated nerve fibers, myelin sheath thickness and mean axon diameter were significantly increased in the systemic administration group compared with the model group （P 〈 0.05）. Lymphocyte infiltration was decreased in the local and systemic administration groups compared with the model group. The wet weight of rat gastrocnemius in the local and systemic administration groups were significantly greater compared with the model group （P 〈 0.05）. Motor nerve conduction velocity was the fastest in the systemic administration group, and the slowest in the model group. Compound muscle action potential amplitude was larger in the systemic administration group compared with the local administration and model groups （P 〈 0.05）. CONCLUSION： Systemic administration of FK506 can promote regeneration of rat sciatic nerve and recovery of neural function. Systemic administration produced better regeneration and recovery of function than local administration of FK506.     

Low-intensity ultrasound for regeneration of injured peripheral nerve

BACKGROUND: Ultrasound is a kind of mechanical wave and characterized by mechanical effect, heat effect and physical and chemical effect. Ultrasound can promote regeneration of peripheral nerves after a slight injury based on its mechanical effect. However, whether it can promote regeneration of peripheral nerves after a severe injury or not is still unclear. OBJECTIVE: To study the effect of low-intensity ultrasound (LIU) on regeneration of injured peripheral nerve, through examining sciatic nerve function index, the sensory nerve conduction velocity and the thickness of myelin sheath. DESIGN: Single factor design of contrast observation. SETTING: Institute of Ultrasound Engineering, Chongqing Medical University. MATERIALS: A total of 64 female Wistar rats, of clean grade, aged 3 months, weighing 200－250 g, were provided by Experimental Animal Center of Chongqing Medical University. All rats were randomly divided into treatment group and control group with 32 in each group. In addition, rats were observed at 4 time points, including 2, 4, 6 and 8 weeks, with 8 at each time point. The main equipments were detailed as follows: forceps (Medical Treatment Apparatus Company, Chongqing), low-intensity ultrasound treatment instrument (Institute of Ultrasound Engineering in Medicine), the analysis instrument of diagram resembles and arithmetic figure (the United States Bio-RAD Company), ultrasound coupling agent (Xunde Image material factory, Hangzhou), Osmium Tetraoxide (Next Chimica, South Africa). METHODS: The experiment was carried out in Institute of Ultrasound Engineering of Chongqing Medical University from December 2003 to May 2004. The right sciatic nerves of 64 rats were crushed with forceps for 30 s to form the experimental animal models. Then they were treated at 3 days after operation. Rats in the treatment group received the LIU exposure. LIU was applied every other day to the crush site of rats, which had a spatial peak, time-averaged intensity of 0.25 W/cm2 operated at 1 MHz for 1 minute per application for total 8 weeks. At various stages after operation, the sciatic nerve function index(SFI), the sensory nerve conduction velocity and its histology were detected. Rats in the control received a sham exposure. MAIN OUTCOME MEASURES: SFI; sensory nerve conduction velocity; density of myelinated nerve fiber; velocity of nerve regeneration; histological examination. RESULTS: Among 64 Wistar rats, 2 were lost during the experiment and another 2 were supplied. ① Histological examination: Two weeks after treatment, degeneration of axis-cylinder and myelin sheath was obvious in treatment group as compared with that in control group. Within 4－8 weeks after treatment, regeneration of axis-cylinder and myelin sheath of nerve fiber was superior in treatment group to that in control group. At 8 weeks after treatment, axis-cylinder and myelin sheath in treatment group were closed to normal value. Quantity of nerve fiber was less in control group than that in treatment group and the arrangement was disorder. At 2, 4 and 6 weeks after treatment, proliferation of Schwann cells was superior in treatment group to that in control group. At 6 and 8 weeks after treatment, proliferation of fiber tissue in nerve was severer in control group than that in treatment group. ② SFI: At 4, 6 and 8 weeks after treatment, SFI was higher in treatment group than that in control group (t =8.00, 12.41, 15.13, P < 0.01). ③ Sensory nerve conduction velocity: At 2, 4, 6 and 8 weeks after treatment, sensory nerve conduction velocity was faster in treatment group than that in control group (t =11.74, 10.81, 3.51, P < 0.01). ④ Density of myelinated nerve fiber: At 2, 4, 6 and 8 weeks after treatment, density of myelinated nerve fiber was higher in treatment group than that in control group (t =2.16, P < 0.05; t =3.29, 3.52, 3.23, P < 0.01). ⑤ Velocity of nerve regeneration: Velocity of nerve regeneration was (1.50±0.08) mm/d and (1.22±0.10) mm/d of treatment group and control group, respectively. This suggested that velocity of nerve regeneration was faster in treatment group than that in control group (t =2.708, P < 0.05). CONCLUSION: LIU can promote the regeneration of injured peripheral nerve from the appearance, construction, function aspect and functional recovery. Its mechanism may be through the modulation and effect of many links of nerve regeneration process.     

Relationships between hemorrhage, angioarchitectural factors and collagen of arteriovenous malformations

Objective While associations between the angioarchitecture of arteriovenous malformations (AVMs) in the brain and pathological features have been described, here we investigated the relationship between the angioarchitecture, the pathological features of the vessel wall, and hemorrhagic events. Methods The study was conducted on 43 patients: 16 with ruptured AVM (rAVM), 15 with non-ruptured AVM (nrAVM), 6 with craniocerebral trauma (control) and 6 with epilepsy (control). The diagnosis of AVM was confirmed by preoperative digital subtraction angiography. Tissues were stained with hematoxylin and eosin and Masson’s trichrome (for collagen fibers) to evaluate the vessel wall structure and endothelial integrity. The content and distribution of collagen types I and III in the vessel wall were assessed by immunohistochemical staining. Results In the nrAVM group, the nidus had more draining veins than the rAVM group (P <0.05). Severely damaged endothelial cells, significantly fewer smooth muscle cells in the media, and hyperplasic type-I and -III collagen fibers were found in the rAVM group. The content of collagen types I and III in rAVMs was higher than that in the nrAVM (P <0.05) and control groups (P <0.01). Conclusion There is an association between angioarchitectural features such as the number of draining veins and the pathological structure of the AVM wall. These abnormalities may contribute to AVM rupture.     

Electrical stimulation does not enhance nerve regeneration if delayed after sciatic nerve injury: the role of fibrosis

Electrical stimulation has been shown to accelerate and enhance nerve regeneration in sensory and motor neurons after injury, but there is little evidence that focuses on the varying degrees of fibrosis in the delayed repair of peripheral nerve tissue. In this study, a rat model of sciatic nerve transection injury was repaired with a biodegradable conduit at 1 day, 1 week, 1 month and 2 months after injury, when the rats were divided into two subgroups. In the experimental group, rats were treated with electrical stimuli of frequency of 20 Hz, pulse width 100 ms and direct current voltage of 3 V; while rats in the control group received no electrical stimulation after the conduit operation. Histological results showed that stained collagen fibers comprised less than 20% of the total operated area in the two groups after delayed repair at both 1 day and 1 week but after longer delays, the collagen fiber area increased with the time after injury. Immunohistochemical staining revealed that the expression level of transforming growth factor β(an indicator of tissue fibrosis) decreased at both 1 day and 1 week after delayed repair but increased at both 1 and 2 months after delayed repair. These findings indicate that if the biodegradable conduit repair combined with electrical stimulation is delayed, it results in a poor outcome following sciatic nerve injury. One month after injury, tissue degeneration and distal fibrosis are apparent and are probably the main reason why electrical stimulation fails to promote nerve regeneration after delayed repair.     

Use of hybrid chitosan membranes and human mesenchymal stem cells from the Wharton jelly of umbilical cord for promoting nerve regeneration in an axonotmesis rat model

Many studies have been dedicated to the development of scaffolds for improving post-traumatic nerve regeneration. The goal of this study was to assess the effect on nerve regeneration, associating a hybrid chitosan membrane with non-differentiated human mesenchymal stem cells isolated from Wharton’s jelly of umbilical cord, in peripheral nerve reconstruction after crush injury. Chromosome analysis on human mesenchymal stem cell line from Wharton’s jelly was carried out and no structural alterations were found in metaphase. Chitosan membranes were previously tested in vitro, to assess their ability in supporting human mesenchymal stem cell survival, expansion, and differentiation. For the in vivo testing, Sasco Sprague adult rats were divided in 4 groups of 6 or 7 animals each:Group 1, sciatic axonotmesis injury without any other intervention (Group 1-Crush); Group 2, the axonotmesis lesion of 3 mm was infiltrated with a suspension of 1 250-1 500 human mesenchymal stem cells (total volume of 50 μL) (Group 2-CrushCell); Group 3, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane covered with a monolayer of non-differentiated human mesenchymal stem cells (Group 3-CrushChitIIICell) and Group 4, axonotmesis lesion of 3 mm was enwrapped with a chitosan type III membrane (Group 4-CrushChitIII). Motor and sensory functional recovery was evaluated throughout a healing period of 12 weeks using sciatic functional index, static sciatic index, extensor postural thrust, and withdrawal reflex latency. Stereological analysis was carried out on regenerated nerve fibers. Results showed that infiltration of human mesenchymal stem cells, or the combination of chitosan membrane enwrapment and human mesenchymal stem cell enrichment after nerve crush injury provide a slight advantage to post-traumatic nerve regeneration. Results obtained with chitosan type III membrane alone confirmed that they significantly improve post-traumatic axonal regrowth and may represent a very promising clinical tool in peripheral nerve reconstructive surgery. Yet, umbilical cord human mesenchymal stem cells, that can be expanded in culture and induced toform several different types of cells, may prove, in future experiments, to be a new source of cells for cell therapy, including targets such as peripheral nerve and muscle.     

Adipose-derived stem cells modified by BDNF gene rescue erectile dysfunction after cavernous nerve injury

Cavernous nerve injury is the main cause of erectile dysfunction following radical prostatectomy.The recovery of erectile function following radical prostatectomy remains challenging.Our previous studies found that injecting adipose-derived stem cells(ADSCs)into the cavernosa could repair the damaged cavernous nerves,but the erectile function of the treated rats could not be restored to a normal level.We evaluated the efficacy of ADSCs infected with a lentiviral vector encoding rat brain-derived neurotrophic factor(lenti-rBDNF)in a rat model of cavernous nerve injury.The rats were equally and randomly divided into four groups.In the control group,bilateral cavernous nerves were isolated but not injured.In the bilateral cavernous nerve injury group,bilateral cavernous nerves were isolated and injured with a hemostat clamp for 2 minutes.In the ADSCGFP and ADSCrBDNF groups,after injury with a hemostat clamp for 2 minutes,rats were injected with ADSCs infected with lenti-GFP(1×106 in 20μL)and lenti-rBDNF(1×106 in 20μL),respectively.Erectile function was assessed 4 weeks after injury by measuring intracavernosal pressures.Then,penile tissues were collected for histological detection and western blot assay.Results demonstrated that compared with the bilateral cavernous nerve injury group,erectile function was significantly recovered in the ADSCGFP and ADSCrBDNF groups,and to a greater degree in the ADSCrBDNF group.Neuronal nitric oxide synthase content in the dorsal nerves and the ratio of smooth muscle/collagen were significantly higher in the ADSCrBDNF and ADSCGFP groups than in the bilateral cavernous nerve injury group.Neuronal nitric oxide synthase expression was obviously higher in the ADSCrBDNF group than in the ADSCGFP group.These findings confirm that intracavernous injection with ADSCs infected with lenti-rBDNF can effectively improve erectile dysfunction caused by cavernous nerve injury.This study was approved by the Medical Animal Care and Welfare Committee of Wuhan University,China(approval No.2017-1638)on June 20,2017.     

Reduction in nerve root compression by the nucleus pulposus after Feng's Spinal Manipulation

Ninety-four patients with lumbar intervertebral disc herniation were enrolled in this study. Of these, 48 were treated with Feng’s Spinal Manipulation, hot fomentation, and bed rest (treatment group). The remaining 46 patients were treated with hot fomentation and bed rest only (control group). After 3 weeks of treatment, clinical parameters including the angle of straight-leg raising, visual analogue scale pain score, and Japanese Orthopaedic Association score for low back pain were improved. The treatment group had significantly better improvement in scores than the control group. Magnetic resonance myelography three-dimensional reconstruction imaging of the vertebral canal demonstrated that filling of the compressed nerve root sleeve with cerebrospinal fluid increased significantly in the treatment group. The diameter of the nerve root sleeve was significantly larger in the treatment group than in the control group. However, the sagittal diameter index of the herniated nucleus pulposus and the angle between the nerve root sleeve and the thecal sac did not change significantly in either the treatment or control groups. The effectiveness of Feng’s Spinal Manipulation for the treatment of symptoms associated with lumbar intervertebral disc herniation may be attributable to the relief of nerve root compression, without affecting the herniated nucleus pulposus or changing the morphology or position of the nerve root.     

Skeletal muscle-derived cells repair peripheral nerve defects in mice

Skeletal muscle-derived cells have strong secretory function,while skeletal muscle-derived stem cells,which are included in muscle-derived cells,can differentiate into Schwann cell-like cells and other cell types.However,the effect of muscle-derived cells on peripheral nerve defects has not been reported.In this study,5-mm-long nerve defects were created in the right sciatic nerves of mice to construct a peripheral nerve defect model.Adult female C57BL/6 mice were randomly divided into four groups.For the muscle-derived cell group,muscle-derived cells were injected into the catheter after the cut nerve ends were bridged with a polyurethane catheter.For external oblique muscle-fabricated nerve conduit and polyurethane groups,an external oblique muscle-fabricated nerve conduit or polyurethane catheter was used to bridge the cut nerve ends,respectively.For the sham group,the sciatic nerves on the right side were separated but not excised.At 8 and 12 weeks post-surgery,distributions of axons and myelin sheaths were observed,and the nerve diameter was calculated using immunofluorescence staining.The number,diameter,and thickness of myelinated nerve fibers were detected by toluidine blue staining and transmission electron microscopy.Muscle fiber area ratios were calculated by Masson’s trichrome staining of gastrocnemius muscle sections.Sciatic functional index was recorded using walking footprint analysis at 4,8,and 12 weeks after operation.The results showed that,at 8 and 12 weeks after surgery,myelin sheaths and axons of regenerating nerves were evenly distributed in the muscle-derived cell group.The number,diameter,and myelin sheath thickness of myelinated nerve fibers,as well as gastrocnemius muscle wet weight and muscle area ratio,were significantly higher in the muscle-derived cell group compared with the polyurethane group.At 4,8,and 12 weeks post-surgery,sciatic functional index was notably increased in the muscle-derived cell group compared with the polyurethane group.These criteria of the muscle-derived cell group were not significantly different from the external oblique muscle-fabricated nerve conduit group.Collectively,these data suggest that muscle-derived cells effectively accelerated peripheral nerve regeneration.This study was approved by the Animal Ethics Committee of Plastic Surgery Hospital,Chinese Academy of Medical Sciences(approval No.040)on September 28,2016.     

烧伤愈合过程中皮肤神经存在的重构

背景：组织重塑早在2002年提出,与创伤的愈合有关,多数研究表明烧伤愈合过程中涉及皮肤神经的再生。目前少有研究对皮肤神经的重塑进行描述。 目的：观察创伤愈合过程中神经再生的动态变化。 方法：采取伤区组织及瘢痕组织标本,改良三色法染色观察胶原;利用免疫荧光技术以神经丝蛋白作为标记物,免疫荧光显微镜、激光共聚焦扫描显微镜（LSCM）下观察神经纤维的二、三维结构。利用胶原容积分数CVF、神经纤维容积分数NVF进行半定量分析。 结果：伤后胶原比例逐渐增大,到增生期CVF达到最高,成熟期趋于正常。伤后神经持续再生,瘢痕增生期NVF达到最高,到成熟期NVF则低于正常。三维重建发现增生期神经纤维存在扭曲变形,崩解断裂现象,成熟期则趋于正常。 结论：烧（创）伤愈合过程中神经纤维经历数目逐渐增多、形态出现扭曲粗大,然后崩解断裂、数目减少、形态规则的重构过程。     

Expression of type I and III collagen and laminin beta1 after rat sciatic nerve crush injury

Extracellular matrix changes are thought to be essential to the regeneration of peripheral nerves. The production of this matrix is believed to be regulated by interactions between axons and their supporting cells. In this study matrix production and cell proliferation were studied during rat sciatic nerve regeneration after a crush injury, and compared to that after rat sciatic nerve transection. Expression of proalpha1(I) and proalpha1(III) collagen and laminin beta1 mRNAs was followed in isolated endoneuria by Northern and in situ hybridization both proximally and distally to the site of either a crush injury or transection of rat sciatic nerve up to 18 weeks. Changes in the Schwann cell and fibroblast populations were monitored by morphometric analysis of endoneurial cross-sections immunostained for S-100 protein. The process of axonal regeneration was followed by Bielschowsky's silver staining. A crush injury initially resulted in increased expression of all mRNAs studied in the endoneurial cells. However, with progressing axonal regeneration the amount of collagen mRNAs returned to control levels, whereas the amount of laminin beta1 mRNA in the distal site of the crush remained elevated throughout the study period. The expression of type I collagen mRNA was enhanced after nerve transection injury compared to that after the crush injury. The epineurial fibroblasts actively expressed both type I and III collagen mRNAs after the injury. The proliferation of Schwann cells and the expression of collagen mRNAs are not, at least directly, related to the axonal regeneration. However, the long-lasting and strong expression of laminin beta1 mRNA after a nerve crush injury may be related to good axonal regeneration. The expression of type I collagen in the epineurium may lead to clinically well-recognized epineurial scarring and thus impede axonal regeneration.     

Maggot homogenate is associated with neural regeneration and wound healing in the rat

BACKGROUND:Live delivery limits the clinical application of maggot therapy. To date in China, there are no in vivo reports regarding wound healing mechanisms of maggot therapy or the effects of maggot homogenate on wound nerve regeneration.OBJECTIVE:To avoid complications due to the use of live maggots, an aseptic maggot homogenate was applied. Substance P (SP) and gene protein product 9.5 expression in a cutaneous wound was analyzed to explore possible mechanisms of neural regeneration and wound healing in the rat.DESIGN, TIME AND SETTING:A random grouping and controlled animal study was performed at the laboratory of the Department of Orthopedic Surgery, First Affiliated Hospital, Dalian Medical University from August 2008 to April 2009.MATERIALS:Live maggots were cultured and provided by the laboratory of the Department of Orthopedic Surgery of the First Affiliated Hospital, Dalian Medical University, China.METHODS:A total of 48 adult rats were selected and two acute, full-thickness wounds (round, 1.5 cm diameter) were created on the back of each rat. The two wounds were randomly assigned to homogenate product and control groups. Following two-step disinfection of maggots, a homogenate was produced from 10 maggots and applied to the wound area in the homogenate product group, while the wounds in the control group were treated with normal saline alone.MAIN OUTCOME MEASURES:On days 1,3, 7, 10, 14, and 21 following injury, the wound tissue was excised. Histological examination of the wound was observed by hematoxylin and eosin staining or Masson's Trichrome staining. SP and protein gene product 9.5 expressions were examined by immunohistochemistry to evaluate wound neural regeneration.RESULTS:On days 7, 10, and 14, the rate of wound healing was significantly greater in the homogenate product group compared with the control group (P < 0.05), and homogenate healing was better than that seen in the control group. On days 3, 7, and 10, SP expression in cells and regenerative nerves was significantly greater in the homogenate product group compared with the control group (P < 0.05). On days 7 and 10, protein gene product 9.5 expression was detected in the regenerative nerve, and expression level was significantly greater in the homogenate product group compared with the control group (P < 0.05).CONCLUSION:Maggot homogenate resulted in upregulated SP and protein gene product 9.5 expressions, thereby promoting neural regeneration and wound healing.     

Chondrotinase ABC enhances axonal regeneration across nerve gaps.

We evaluated the effects of chondroitinase ABC on axonal regeneration across peripheral nerve gaps. We compared axonal regeneration after 15-mm tibial nerve resection and repair with a silicone tube filled with type I collagen gel (negative control group), with a silicone tube filled with type I collagen gel containing chondroitinase ABC at three different concentrations (2.5 units/mL, 5 units/mL, 10 units/mL) (chondroitinase ABC groups), and with an autologous nerve segment (nerve autograft group). Electrophysiological and histological assessments were carried out 12 weeks after surgery. In the electrophysiological study, compound muscle action potentials (CMAPs) and nerve conduction velocities (NCVs) were recorded in all groups except the negative control group. Although both CMAPs and NCVs were highest in the nerve autograft group, there were no significant differences among the three chondroitinase ABC groups in either parameter. Histological findings were consistent with electrophysiological results. Based on these findings, we conclude that topical injection of chondroitinase ABC can significantly increase the critical length of nerve gap repair by tubulization or artificial nerve placement.     

Endoneurial fibrosis following nerve transection

Summary Indirect immunofluorescent techniques with antibodies to type I, III, and V collagens and fibronectin were used to study rat sciatic nerve tributaries after transection with intact contralateral nerves as controls.Codistribution of type I and III collagens characterized the epineurium of normal nerve. In the perineurium, however, type I collagen was absent, but type III and V collagens and fibronectin were detected. Type I and III collagens were codistributed in the endoneurial stroma where a homogeneous staining by antibodies to fibronectin was also observed.During the 4-week observation period after transection the perineurium reacted by slight thickening which was most clearly demonstrated by staining with antibodies to fibronectin and to type V collagen. A widening of the type I-negative cleft also occurred. Endoneurial, type V collagen-positive cuffs around the nerve fibers became disorganized, and a concomitant increase of the stroma containing type I and III collagens and fibronectin was observed.The codistribution of the fibrous collagen types appeared similar in normal epineurium and endoneurium. Type V collagen was locatd in the perineurium and in endoneurial cuffs surrounding the nerve fibers. The present data indicate that collagen accumulation takes place in the perineurium and endoneurium of transected nerve. The cell type responsible for the synthesis of the connective tissue material is discussed.Financially supported by a grant (to V. S.) from the Research and Science Foundation of Lääke Oy and by institutional grants from the Turku University Foundation and the Sigrid Jusélius Foundation     

Role of extracellular matrix components in facial nerve regeneration: an experimental study

OBJECTIVE: The aim of this study is to evaluate the role of extracellular matrix components in nerve regeneration. Matrigel, a complex of extracellular matrix components such as laminin (the most abundant) heparan sulphate proteoglycans (HSPG), type IV collagen and fibronectin, was used. METHODS: Forty male rabbits, which had undergone section of the right facial nerve, were later treated by reinnervation through an artificial graft of expanded polytetrafluoroethylene (ePTFE). In 20 animals the tubes of ePTFE were filled with Matrigel; in 20 control animals the tubes were filled with saline solution. RESULTS: The Matrigel group showed a better axonal organization and a significantly higher number of regenerated axons in the early phases (at days 15 and 30 respectively) than the control group, whereas the difference of the axons number at day 60 was less significant; besides, the axon diameter and the myelin thickness were not significantly improved by Matrigel. DISCUSSION: Our data suggest that Matrigel is an important factor in promoting and enhancing the early phases of the regeneration after nerve injuries. Tree neurite promoting agents, such as laminin, fibronectin and collagen, allow a more systematic and agonized regeneration. Extracellular matrix components may represent a direction guidance for axonal pathway.     

Saikosaponin a increases interleukin-10 expression and inhibits scar formation after sciatic nerve injury

Nerve scarring after peripheral nerve injury can severely hamper nerve regeneration and functional recovery.Further,the anti-inflammatory cytokine,interleukin-10,can inhibit nerve scar formation.Saikosaponin a(SSa) is a monomer molecule extracted from the Chinese medicine,Bupleurum.SSa can exert anti-inflammatory effects in spinal cord injury and traumatic brain injury.However,it has not been shown whether SSa can play a role in peripheral nerve injury.In this study,rats were randomly assigned to three groups.In the sham group,the left sciatic nerve was directly sutured after exposure.In the sciatic nerve injury(SNI) + SSa and SNI groups,the left sciatic nerve was sutured and continuously injected daily with SSa(10 mg/kg) or an equivalent volume of saline for 7 days.Enzyme linked immunosorbent assay results demonstrated that at 7 days after injury,interleukin-10 level was considerably higher in the SNI + SSa group than in the SNI group.Masson staining and western blot assay demonstrated that at 8 weeks after injury,type I and III collagen content was lower and nerve scar formation was visibly less in the SNI + SSa group compared with the SNI group.Simultaneously,sciatic functional index and nerve conduction velocity were improved in the SNI + SSa group compared with the SNI group.These results confirm that SSa can increase the expression of the anti-inflammatory factor,interleukin-10,and reduce nerve scar formation to promote functional recovery of injured sciatic nerve.     

深Ⅱ度烧伤创面磨痂治疗后残留皮肤组织中表皮干细胞标记物

背景：大面积烧伤往往需要削痂清除痂皮,但削痂手术往往会导致人为因素除去过多的残留再生皮肤组织。 目的：观察磨痂治疗深Ⅱ度烧伤创面对残留皮肤组织中表皮干细胞标记物角化蛋白19表达的影响。 设计、时间及地点：随机分组对比观察,于2002-10/2004-01在广西医科大学完成。 对象：烧伤整形外热烧伤科患者40例,年龄18~37岁,平均烧伤面积为15%~45%,深Ⅱ度烧伤面积为15%~30%。随机分组方法分为磨痂组(n=20)、削痂组(n=20)。 方法：磨痂组采用电动磨痂仪对创面进行磨痂,由浅入深磨去坏死组织,至创面基底呈现红色充血,有珠状的小出血点为止。削痂组采用滚轴削痂刀对创面进行削痂,削至创面基底呈瓷白色、组织致密,湿润面有光泽,无网状血管栓塞和呈灰暗棕色的无光泽组织,放松止血带后可见密集点状出血较均匀,但有时因操作原因削痂过深,基底露出脂肪组织。两组创面术后用辐照猪皮覆盖。 主要观察指标：取术前、术后小块创面组织以免疫组织化学SP法检测标皮肤再生组织中标记物角化蛋白19的表达,在100倍光学显微镜下,任意选取5个视野计数细胞团的表达数;观察两组创面愈合时间,记录超过4周不愈合的肉芽创面,需要再次手术植皮修复创面。 结果：磨痂保留了较多的真皮组织,毛囊,汗腺,皮脂腺等皮肤附件。削痂后创面基底组织有薄层网状组织残留及部分毛囊及汗腺,有些标本视野中可见无真皮组织,为脂肪组织。细胞团的表达数比较结果显示,两组前及术后创面均有创面残留皮肤组织中标记物角化蛋白19表达,磨痂组术前、术后数量无明显变化(P > 0.05),削痂组术后较术前数量减少(P < 0.05);磨痂组创面较削痂组提前愈合(P < 0.05)。需要手术植皮创面磨痂组2处,削痂组8处(P < 0.05)。 结论：应用磨痂术治疗烧伤深Ⅱ度创面能有效掌握磨痂深度,对组织损伤小,与削痂比较保留更多的再生皮肤组织,通过表皮干细胞的再生,利于创面再上皮修复,缩短创面愈合时间。     

FK506 promotes functional recovery in crushed rat sciatic nerve

In this study we examine whether the systemic administration of FK506 or Cyclosporin A (CsA) expedited functional recovery following an axonotmetic nerve injury, and compared their effects in a rat model. Seventy-five adult Buffalo rats received a crush injury to the right posterior tibial nerve and subsequently underwent either no treatment (group I), daily injections of FK506 (group II), or daily injections of CsA (group III). Walking track analysis demonstrated return of hindlimb function by 20 days postoperatively in group I, 14 days in group II, and 18 days in group III. The blood-nerve barrier (BNB) was reconstituted by postoperative day (POD) 7 in both FK506- and CsA-treated animals and by POD 13 in control animals. These results suggest that recovery of function is more rapid with daily administration of FK506 than with CsA or no treatment, perhaps because of earlier restoration of the blood-nerve barrier. Agents that facilitate nerve regeneration have the potential to limit the extent of motor endplate loss and muscle atrophy seen with prolonged denervation, thereby limiting permanent functional loss.