4000/蛆虫匀浆物影响大鼠创面神经再生的实验研究

背景：蛆虫可以促进创面愈合,而创面愈合又与创面神经再生密切相关。 目的：研究蛆虫匀浆物与创面神经再生的关系。 设计、时间、单位：随机对照动物实验,于2008年8月—2009年4月在大连医科大学附属第一医院骨科实验室完成。 材料：48只体重为200—220克SD健康雄性大鼠由大连医科大学动物实验中心提供, 活体蛆虫在本实验室饲养繁殖。 方法：于每只大鼠背部制作2个圆形直径为1.5厘米的急性全层皮肤缺损创面。蛆虫经过两步消毒法消毒之后,在实验组大鼠创面中每平方厘米应用10只蛆虫的匀浆物;而在对照组中,只应用同等体积的生理盐水。在不同时间段,切除创面组织,应用组织学和免疫组织化学方法进行检测。 主要观察指标：在不同时间段计算创面的愈合率。应用HE染色和Masson三色法在组织水平上观察创面愈合情况。应用免疫组织化学方法观察P物质(SP)及蛋白基因产物9.5（PGP9.5）的表达,以评估创面神经再生情况。 结果：在第7、10、14天,实验组创面愈合率显著高于对照组,且实验组愈合质量好于对照组。在第3、7、10天,实验组SP在细胞和再生神经中的表达显著高于对照组。在第7、10天,可以在再生的神经中检测到PGP9.5的表达,且实验组的表达高于对照组。 结论：蛆虫匀浆物可以通过促进创面神经再生,进而促进创面的愈合。     

脊神经根离断对周围皮肤神经再生和创面愈合的影响

Burn wounds were produced on two sides on the backs of Wistar rats,in addition to denervation on one side.The skin neural regeneration at the injury site and burn wound healing were evaluated following spinal nerve root incision.No nerve regeneration was observed in the burn wound region post-denervation,and the degree of epithelization was significantly less than the control group.With increasing time,expression of type I collagen,which plays a supporting role,and collagen III,which exhibits elastic properties,were significantly increased in the two groups,but the expression was less in the denervation group compared with the control group,and the wound healing was faster in the control group.The ratio of type I collagen to type III collagen was significantly lower in the den-ervation group compared with the control group.The ratio gradually decreased with prolonged time in the denervation group,but remained unchanged in the control group.However,the elasticity of the tissues in the denervation group was better than the control group.During burn wound healing,innervations can promote wound healing,but denervation can improve the quality of wound re-modeling.     

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.     

Substance P,bombesin, and leucine-enkephalin immunoreactivities are restored in the frog tectum after optic nerve regeneration

Extensive regeneration of the optic nerve takes place in adult Amphibia. In this study, we have determined whether one aspect of retinotectal organisation, namely immunoreactive laminae in the retinorecipient layers of the optic tectum, is restored after optic nerve regeneration. To do so, the distributions of substance-P, bombesin, and leucine-enkephalin immunoreactivities were examined in the optic tectum of the frog Litoria (Hyla) moorei. Results of a normal series were compared with those at intervals up to 84 days and at 196 days after either unilateral deafferentation or optic nerve crush. In the normal series, distinct neuropeptide immunoreactive laminae were located within the retinorecipient tectal layers. There were two major laminae with substance-P, two with bombesin, and one with leucine-enkephalin immunoreactivities. Additional faint laminae of both substance-P and bombesin immunoreactivity were present in the tectal region that receives input from the visual streak. In addition, labelling of cell bodies and dendrites was seen elsewhere in the tectum. All except one immunoreactive lamina changed after deafferentation. The deeper of those with substance-P immunoreactivity, along with both bombesin laminae, were eventually lost the lamina with leucine-enkephalin immunoreactivity was halved in intensity. We assume that these laminae are wholely or, in the case of the leucine-enkephalin lamina, partially associated with primary optic input. By contrast, the more superficial lamina with substance-P immunoreactivity remained unchanged and is presumably not directly related to visual input. During nerve regeneration, the intensity of all laminae associated with optic input initially fell as in the deafferentation series but, in the long term, recovered to approximately 80% of normal intensities. We conclude that ganglion cells associated with each of the immunoreactivities tested had successfully regenerated. The reduced intensity of immunoreactivities after regeneration is due presumably in part to the cell loss from the ganglion cell population. Furthermore, we discuss the findings of similar studies for Rana pipiens (Kuljis and Karten [1983] J. Comp. Neurol. 217:239–251 and [1985] 240:1–15) in light of the present findings. We argue that some of the previous observations can be reinterpreted to indicate that regeneration was not limited to ganglion cells associated with substance-P immunoreactivity as first thought. © 1995 Wiley-Liss, Inc.     

Effect of wound healing and tissue transplantation on the navigation of axons in organ-cultured embryonic chick eyes

Wound closure and repair of embryonic neuroepithelium were studied in organ-cultured embryonic retinae. Eyes from 3 to 4-day-old embryos were cultured after removing pieces of retinal tissue. During the subsequent 24 hours of incubation, the 150 to 200 μm wide holes in the retina closed completely. Histological studies showed that the wound closure was not accomplished by cell migration or cell proliferation, but by an approximation of the wound edges mediated by extracellular matrix fibrils of the vitreous body. The wound contraction facilitated the integration of transplants into the retinal neuroepithelium with a perfect alignment of the implants with the host at the vitreal surface. Within 24 hours, a continuous inner limiting membrane between transplant and host retina was established. The effect of wound healing and tissue transplantation on the navigation of optic axons in the retina was investigated. The wound contraction in the retina caused the optic axons near the lesion site to grow to the wound center, where the axons traversed the retina and formed a neuroma at the ventricular side, resembling the organization of axons at the optic disc. In the transplantation paradigm, axons from the host retina migrated into the transplant and vice versa. However, due to the wound contraction around the transplant, most axons grew into the interface between the transplant and host tissue. 1993 Wiley-Liss, Inc.     

Rhesus monkey neural stem cell transplantation promotes neural regeneration in rats with hippocampal lesions

Rhesus monkey neural stem cells are capable of differentiating into neurons and glial cells.Therefore,neural stem cell transplantation can be used to promote functional recovery of the nervous system.Rhesus monkey neural stem cells(1×105 cells/μL) were injected into bilateral hippocampi of rats with hippocampal lesions.Confocal laser scanning microscopy demonstrated that green fluorescent protein-labeled transplanted cells survived and grew well.Transplanted cells were detected at the lesion site,but also in the nerve fiber-rich region of the cerebral cortex and corpus callosum.Some transplanted cells differentiated into neurons and glial cells clustering along the ventricular wall,and integrated into the recipient brain.Behavioral tests revealed that spatial learning and memory ability improved,indicating that rhesus monkey neural stem cells noticeably improve spatial learning and memory abilities in rats with hippocampal lesions.     

Perioperative complications of scoliosis surgery in patients with Duchenne muscular dystrophy and spinal muscular atrophy,focussing on wound healing disorders

Purpose: Patients with Duchenne muscular dystrophy (DMD) or spinal muscular atrophy (SMA), both neuromuscular diseases, sustain spinal scoliosis in the course of their disease. To reduce the concomitant major morbidity and to improve their quality of life, patients require surgical spine stabilization. This can lead to complications like respiratory, cardiac or neurological complications or wound healing disorders (WHD). To find out the different complexities and risk factors increasing the chance to develop a WHD, the inpatient database was analyzed. Methods: We performed a retrospective statistical study. Therefore, we analyzed the inpatient database of 180 patients (142 DMD and 38 SMA patients). The focus was on WHD. To figure out the risk factors leading to WHD, we conducted a logistic regression. Results: Cardiac complications occurred most frequently, followed by pulmonary complications and neurological lesions. Fifty-seven out of 180 patients developed a WHD. In 23 cases the WHD was aseptic, in the other 34 cases dermal organisms, Pseudomonas species and intestinal organisms were responsible. By means of the logistic regression, we were able to identify two more risk factors, in addition to diagnosis and gender, for developing a WHD in our patients: the year of surgery and the direction of pelvic tilt. Conclusions: Most common complications following scoliosis surgery are respiratory and cardiac complications. WHD is a severe complication that implies a prolonged therapy. Some risk factors for developing WHD could be identified in this analysis. Specifically, these were the date of surgery and the direction of pelvic tilt.     

Transplantation of olfactory ensheathing cells promotes axonal regeneration in a rat model of spinal cord injury

BACKGROUND:Transplantation of olfactory ensheathing cells (OECs) into the injured spinal cord has been shown to promote axonal regeneration and functional recovery.However,the mechanisms underlying the effects of OEC transplantation remain controversial.OBJECTIVE:To observe fibrotic scar formation and axonal regeneration in the damaged spinal cord following OEC transplantation,and to determine whether OEC transplantation promotes neural regeneration by attenuating fibrotic scar formation.DESIGN,TIME AND SETTING:A randomized,controlled animal experiment was performed at the Department of Developmental Morphology,Tokyo Metropolitan Institute for Neuroscience,Fuchu,Japan and at the Department of Human Anatomy,College of Basic Medical Sciences,China Medical University,China between April 2007 and May 2009.MATERIALS:OECs were obtained from olfactory nerves and olfactory bulbs of male,4-week-old,Sprague Dawley rats.Rabbit anti-serotonin polyclonal antibody,rabbit anti-calcitonin gene-related peptide polyclonal antibody,rabbit anti-glial fibrillary acidic protein polyclonal antibody,rabbit anti-type IV collagen polyclonal antibody,and mouse anti-rat endothelial cell antigen-1 monoclonal antibody were used.METHODS:Male,Sprague Dawley rats aged 8 weeks were randomly divided into three groups:sham-surgery (n = 3),surgery (n = 9),and OEC transplantation (n = 11).Spinal cord transection at the T9-10 level was performed and the rats were transplanted with a 2-μL (1 × 105 cells) cell suspension.MAIN OUTCOME MEASURES:Formation of glial and fibrotic scars was examined using immunohistochemistry for glial fibrillary acidic protein and type IV collagen.Serotonin-positive and calcitonin gene-related peptide-positive axons were visualized by immunohistochemistry,respectively.Double immunofluorescence for type IV collagen and rat endothelial cell antigen-1 was also performed to determine co-localization of type IV collagen deposition and blood vessels.RESULTS:At 1 week after spinal cord injury,numerous glial cells were observed around the lesion site.Formation of fibrotic scar was determined by a large amount of type IV collagen deposition in the lesion center,and descending serotonin- or ascending calcitonin gene-related peptideconiaining axons stopped at the fibrotic scar that was formed in the lesion site.At week after transplantation,the formation of fibrotic scar was significantly inhibited.In addition,the fibrotic structure was partly formed and centralized in the blood vessel,and serotonergic and calcitonin gene-related peptide-containing axons were regenerated across the lesion site.CONCLUSION:OEC transplantation into the injured spinal cord attenuated fibrotic scar formation and promoted axon regeneration.     

Three morphologically distinct types of interface develop between adult host and fetal brain transplants: implications for scar formation in the adult central nervous system

The development of the host/graft interface of cerebellar and cerebral transplants was studied 1-60 days after operation. Grafts from fetal Wistar rats were transplanted to a cavity over the superior colliculus of adult rats by removing parts of the overlying cortex and hippocampus according to the Bj?rklund/Stenevi technique. In sham-operated control rats, in which a cavity was made in the brain but no graft was implanted, the parenchyma bordering the entire cavity developed a complete glial-meningeal scar within 2 weeks after operation consisting of multilayered glial processes, a basal lamina, and fibroblasts (meningeal cells). A similar interface also developed between graft and host in the most superficial parts of the transplantation cavity. In the basal parts of the transplantation cavity, the host/graft interface consisted either of an incomplete sheet of astrocyte processes aligned in parallel to each other but without a covering basal lamina or of completely fused neuropil without any morphological signs of separation between host and transplant. It is concluded that these three zones of host/graft interface are established by differential interaction between the growing transplant and the host cicatrix. At the basal host/graft parenchymatous interface the fetal transplant interferes with the normal adult cicatrization process of the host, possibly by either releasing inhibitory factors or by preventing contact between the astroglia of the host and fibroblasts (meningeal cells). In white matter regions of the transplantation cavity, voluminous cysts developed, both in sham-operated controls and in graft recipients, which were invaded by transplanted neurons.     

Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen Neu N, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ? anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.     

Optogenetics and its application in neural degeneration and regeneration

Neural degeneration and regeneration are important topics in neurological diseases. There are limited options for therapeutic interventions in neurological diseases that provide simultaneous spatial and tem-poral control of neurons. This drawback increases side effects due to non-specific targeting. Optogenetics is a technology that allows precise spatial and temporal control of cells. Therefore, this technique has high potential as a therapeutic strategy for neurological diseases. Even though the application of optogenetics in understanding brain functional organization and complex behaviour states have been elaborated, reviews of its therapeutic potential especially in neurodegeneration and regeneration are still limited. This short review presents representative work in optogenetics in disease models such as spinal cord injury, multiple sclerosis, epilepsy, Alzheimer's disease and Parkinson's disease. It is aimed to provide a broader perspective on opto-genetic therapeutic potential in neurodegeneration and neural regeneration.     

Influence of exogenous triiodothyronine on axonal regeneration and wound healing in the brain of the rat

Rats with parasagittal incised wounds of the telencephalon were treated for 1, 7, 21 or 56 days with triiodothyronine (T₃), 0.5 μg/100 g body weight, injected once daily. Controls were injected with the aqueous solution in which the T₃ was dissolved. The brains were examined histologically and quantitative assessments were made of the regeneration of axons into and across the lesions and of the healing of the wounds. T₃, when administered over an 8-week period, stimulated axonal regeneration in the dorsal cortex and corpus callosum and promoted healing of the wound in the corpus callosum. The results of this investigation suggest that the use of T₃ in the clinical treatment of injury to the central nervous system may be of less value than the work of earlier authors had indicated.     

Spontaneous longitudinally orientated axonal regeneration is associated with the Schwann cell framework within the lesion site following spinal cord compression injury of the rat

Spontaneous cellular reorganisation at the lesion site has been investigated following massive spinal cord compression injury in adult rats. By 2 days post operation (p.o.), haemorrhagic necrosis, widespread axonal degeneration, and infiltration by polymorphnuclear granulocytes and OX42-positive macrophages were observed in the lesion site. By 7 days p.o., low affinity nerve growth factor receptor-positive Schwann cells, from activated spinal roots, were identified as they migrated far into the lesion. Between 7 and 14 days p.o., the overlapping processes of Schwann cells within the macrophage-filled lesion formed a glial framework which was associated with extensive longitudinally orientated ingrowth by many neurofilament-positive axons. Relatively few of these axons were calcitonin gene-related peptide (CGRP)-, substance P (SP)-, or serotonin (5HT)-positive; however, many were glycinergic or gamma aminobutyric acid (GABA)ergic. At 21 and 28 days p.o. (the longest survival times studied), a reduced but still substantial amount of orientated Schwann cells and axons could be detected at distances of up to 5 mm within the lesion. Glial fibrillary acidic protein (GFAP) immunoreactivity demonstrated the slow formation of astrocytic scarring which only became apparent at the lesion interface between 21 and 28 days p.o. The current data suggest the possibility of developing future therapeutic strategies designed to maintain or even enhance these spontaneous and orientated regenerative events. J. Neurosci. Res. 53:51–65, 1998. © 1998 Wiley-Liss, Inc.     

Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson’s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson’s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson’s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson’s disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were signiifcantly higher than the Parkinson’s model group. These ifndings suggest that the mechanism by which verbascoside treats Parkinson’s disease is related to the regeneration of tyrosine hy-droxylase-immunoreactive neurons in the substantia nigra.     

Axonal regeneration is associated with glial migration: Comparison between the injured optic nerves of fish and rats

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted a the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve. In contrast, the site of injury in the rat remained devoid of glial fibrillary acidic protein immunoreactive cells, and the expression of GAP-43 by growing axons was still restricted to the proximal part of the nerve. Double-labeling experiments and transmission electron microscopy performed 2 weeks after crush injury of the fish optic nerve revealed that the frontier of axonal growth (i.e., the leading growth cones) appeared to be 200–300 μm ahead of the nearest vimentin-positive glial cells. The leading growth cones were associated with other cells, presumably glial precursors, that seemed to have a high migratory potential. We suggest that the ability of fish glial precursor cells to migrate into the injured area may contribute to the potential of growing axons to traverse this area. The failure of glia and glial precursor cells to migrate into the injured area in the rat may partially account for the failure of rat axons to enter and traverse the injured area.     

Environmental enrichment promotes neural remodeling in newborn rats with hypoxic-ischemic brain damage

We evaluated the effect of hypoxic-ischemic brain damage and treatment with early environmental enrichment intervention on development of newborn rats,as evaluated by light and electron mi-croscopy and morphometry.Early intervention with environmental enrichment intelligence training attenuated brain edema and neuronal injury,promoted neuronal repair,and increased neuronal plasticity in the frontal lobe cortex of the newborn rats with hypoxic-ischemic brain damage.     

Dysfunction of the cortico-basal ganglia-cortical loop in a rat model of early parkinsonism is reversed by metabotropic glutamate receptor 5 antagonism

This study examined the cellular correlates of the akinetic deficits produced in Wistar rats by discrete bilateral 6-hydroxydopamine (6-OHDA) striatal infusions in the dorsolateral striatum, mimicking the preferential denervation of the motor striatal territory in early symptomatic stage of Parkinson's disease (PD). Intraneuronal gene expression of cytochrome oxidase subunit I (COI), a metabolic index of neuronal activity, was increased in the subthalamic nucleus, substantia nigra pars reticulata and decreased in frontal cortical areas, but paradoxically unchanged in the striatum, globus pallidus, entopeduncular nucleus and ventrolateral thalamic nucleus. Neither preproenkephalin A nor preprotachykinin mRNA expression, markers of striatal projection neurons, were modified in the denervated striatal area despite 90% loss of dopamine (DA) terminals. Preproenkephalin A mRNA expression was however, decreased in the nondepleted striatal region, suggesting compensatory increase of dopamine tone from those spared areas. A chronic treatment with the metabotropic glutamate receptor 5 (mGluR5) antagonist 2-methyl-6-(phenylethylnyl)-pyridine (MPEP), which alleviated the akinetic disorders produced by the lesion, reversed the lesion-induced variations of COI gene expression, moderately increased this marker in the structures unaffected by the lesion and did not modify the striatal neuropeptides gene expression. These data suggest that the expression of akinetic deficits in early parkinsonism is associated with focused metabolic changes in the cortico-basal ganglia-cortical loop downstream of the striatum and pallidal complex.     

Erythropoietin upregulates growth associated protein-43 expression and promotes retinal ganglion cell axonal regeneration in vivo after optic nerve crush

In this study, we established a rat model of optic nerve crush to explore the effects of erythropoietin on retinal ganglion cell axonal regeneration. At 15 days after injury in erythropoietin treated rats, retinal ganglion cell densities in regions corresponding to the 1/6, 3/6 and 5/6 ratios of the retinal radius were significantly increased. In addition, the number of growth associated protein-43 positive axons was significantly increased at different distances (50, 250 and 500 ?m) from the crush site after erythropoietin treatment. Erythropoietin significantly increased growth associated protein-43 protein levels in the retina after crush injury, as determined by western blot and immunofluorescence analysis. These results demonstrate that erythropoietin protects injured retinal ganglion cells and promotes axonal regeneration.     

Bumetanide promotes neural precursor cell regeneration and dendritic development in the hippocampal dentate gyrus in the chronic stage of cerebral ischemia

Bumetanide has been shown to lessen cerebral edema and reduce the infarct area in the acute stage of cerebral ischemia. Few studies focus on the effects of bumetanide on neuroprotection and neurogenesis in the chronic stage of cerebral ischemia. We established a rat model of cerebral ischemia by injecting endothelin-1 in the left cortical motor area and left corpus striatum. Seven days later, bumetanide 200 μg/kg/day was injected into the lateral ventricle for 21 consecutive days with a mini-osmotic pump. Results demonstrated that the number of neuroblasts cells and the total length of dendrites increased, escape latency reduced, and the number of platform crossings increased in the rat hippocampal dentate gyrus in the chronic stage of cerebral ischemia. These findings suggest that bumetanide promoted neural precursor cell regeneration, dendritic development and the recovery of cognitive function, and protected brain tissue in the chronic stage of ischemia.