Regeneration of respiratory pathways within spinal peripheral nerve grafts

A region (NS1) that acts like an enhancer is located approximately 300 bp upstream of the larval cap site in the Adh gene of D. melanogaster. When this sequence is deleted (delta NS1), the gene fails to express ADH protein. Gene expression can be restored by placing a second Adh gene with an intact enhancer elsewhere on the same plasmid. In these circumstances, both genes are expressed equally regardless of their orientation on the plasmid. In this report we further characterize the interactions that occur when a single enhancer activates expression from a proximal and distant promoter. We have made the following observations: (1) While the two genes are expressed equivalently, their expression relative to a plasmid carrying two intact genes is reduced by a factor of 2 to 6 depending on the orientation of the two genes. (2) The single enhancer drives expression of both genes on any given plasmid molecule. (3) The enhancer does not interact with the Adh gene from which the NS7 region (which spans the larval TATA box) is removed. (4) Expression of the delta NS1 gene can be restored by an intact gene when both are inserted together into the Drosophila genome via P element-mediated transformation. (5) Increasing the separation between the two genes on a plasmid by up to 15 kbp does not prevent the restoration of expression of the delta NS1 gene. We propose a model that explains how a single enhancer can stimulate equal expression from two genes.     

Regeneration through long nerve grafts in the swine model

Salmonella is a Gram-negative bacterium that has been implicated in several food-borne poisoning outbreaks. Poultry products are considered to be the major vehicles of food-borne poisoning caused by Salmonella. The resistance of Salmonella, isolated from different stages in a local poultry abattoir, to hypochlorous acid (HOCl) was studied. Isolates cultured in quarter strength Tryptone Soya Broth were treated with 72 ppm HOCl, incubated at 30 degrees C with shaking, and absorbance was measured at 660 nm every 20 min. A number of resistant isolates, which carried on growing following HOCl addition, were obtained. All the isolates from the bleeding stage were sensitive to HOCl, whereas those from the scalding stage were resistant to HOCl. Other stages had a population of resistant and sensitive isolates. It is evident that some Salmonella will grow in the presence of concentrations of HOCl deemed to be inhibitory. Hence, an effective concentration of HOCl must be applied in poultry abattoirs to ensure that all the Salmonella are eradicated.     

Antioxidants in peripheral nerve

Oxidative stress and antioxidants have been related in a wide variety of ways with nervous tissue. This review attempts to gather the most relevant information related to a) the antioxidant status in non pathologic nervous tissue; b) the hypothesis and evidence for oxidative stress (considered as the disequilibrium between prooxidants and antioxidants in the cell) as the responsible mechanism of diverse neurological diseases; and c) the correlation between antioxidant alterations and neural function, in different experimental neuropathies. Decreased antioxidant availability has been observed in different neurological disorders in the central nervous system, for example, Parkinson's disease, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis, cerebral ischaemia, etc. Moreover, the experimental manipulation of the antioxidant defense has led in some cases to interesting experimental models in which electrophysiological alterations are associated with the metabolic modifications induced. In view of the electrophysiological and biochemical effects of some protein kinase C inhibitors on different neural experimental models, special attention is dedicated to the role of this kinase in peripheral nervous tissue. The nervous tissue, central as well as peripheral, has two main special features that are certainly related to its antioxidant metabolism: the lipid-enriched membrane and myelin sheaths, and cellular excitability. The former explains the importance of the glutathione (GSH)-conjugating activity towards 4-hydroxy-nonenal, a biologically active product of lipid peroxidation, present in nervous tissue and in charge of its inactivation. The impairment of the latter by oxidative damage or experimental manipulation of antioxidant metabolism is discussed. Work on different experimental neuropathies from author's laboratory has been primarily used to provide information about the involvement of free radical damage and antioxidants in peripheral nerve metabolic and functional impairment.     

Necrotizing peripheral nerve vasculitis

Human exposure to nonsteroidal estrogens in the environment has recently been proposed as a risk factor for endocrine disruption and the development of cancers of the breast and reproductive tract. Certain polycyclic aromatic hydrocarbons (PAHs), which closely resemble steroid hormones, are ubiquitous environmental contaminants whose carcinogenicity has been extensively studied. This review examines the available evidence regarding the actions of PAHs on estrogen receptor activity, estrogen metabolism and the hypothalamo-pituitary axis. In most studies, PAHs exhibited either weakly estrogenic or antiestrogenic responses. The possibility is raised that the endocrine toxicology of certain PAHs reflects both genotoxic and non-genotoxic components which may be interrelated, particularly with regard to carcinogenesis.     

Long-term patency of vein grafts preserved in liquid nitrogen in dimethyl sulfoxide

Autogenous canine jugular veins were stored in 15% dimethyl sulfoxide (DMSO) in liquid nitrogen vapor for one to 28 days and then implanted in the carotid artery as autografts. The patency rate at one year was 62.5-87.5%. The patency rate of fresh jugular vein autografts placed in the carotid artery for one year was 75%. Similar autografts stored in liquid nitrogen vapor for one to 28 days without the cryopreservative DMSO exhibited a zero to 12.5% patency rate at one year. Scanning electron microscope studies revealed preservation of theendothelium in DMSO protected veins and a damaged or sloughed endothelium in veins frozen without DMSO cryopreservation.     

Enhancement of axon growth by detergent-extracted nerve grafts

BACKGROUND: The immunogenicity of nerve allografts is responsible for their rejection. We have developed a method for preparing cell-free nerve grafts using lysophosphatidylcholine to remove cells, axons, and myelin sheaths. METHODS: The remaining intact nerve extracellular matrix is the extracted nerve graft (eNG). Cultured neonatal Schwann cells were micro-injected into the eNG to form recellularized nerve grafts (rNG). eNG, rNG, and normal isografts (15 mm long) were implanted in the peroneal nerves of F-344 rats. Ten rats were given an eNG on the right, and an isograft on the left. Ten rats were given an rNG on the right, and a sham operation on the left. Sham operation was used as the control and the isograft was used as the benchmark procedure. Walking track analysis was performed every 15 days after surgery to determine the peroneal functional index. Morphometric analysis of the distal peroneal nerve and extensor digitorum muscle weight were analyzed 3 months after surgery. RESULTS: The three types of grafted legs had the classical effect observed after peripheral nerve repair, with decreased functional ability, decreased target muscle weight, fewer large nerve fibers, and more small nerve fibers. Isografts, eNG, and rNG all had similar patterns of peroneal functional index improvement after implantation. The extensor digitorum longus muscle weight and axon counts for the three types of graft were not statistically different. Hence, eNG and rNG can enhance nerve regeneration in the same way as isografts. The host Schwann cells that invaded the implanted eNG probably acted in the same fashion as the cultured Schwann cells injected into the rNG and the resident cells of isografts. CONCLUSIONS: The great permeability of the longitudinally oriented matrix of eNG to cells is, therefore, a major advantage over the reported poor permeability of freeze-thawed nerve grafts.     

Impairment of peripheral nerve excitability

Functional abnormalities, especially the excitability changes of axon in the peripheral nerve involvement, were reviewed. In GBS and CIDP, the correlation between conduction block and anti-ganglioside antibodies have been discussed. Using anti GM1 antibody positive sera, the suppression of voltage-gated sodium channels (VGSC) has been reported. Although this findings have not been confirmed, the involvement of VGSC may be an important mechanism for eliciting conduction block. In Isaacs' syndrome, voltage-gated potassium channels (VGKC) were suppressed by autoantibodies to VGKC. Furthermore, in generalized myokymia syndrome which shows only myokymia and muscle cramp without grip myotonia, VGKCs are also suppressed in some cases. These findings suggest that some patients with myokymia and neuromyotonia are induced by anti-VGKC antibodies. For evaluating the axonal excitability in vivo, the threshold electrotonus method have been developed and applied for the involvement of peripheral nerves. In ALS, impairment of potassium conductance was shown and was speculated to have the possible rrelation with fasciculation. Thus threshold electrotonus method will be an important method for evaluating axonal excitability in human. The accumulated knowledge about the involvement of axonal ion channels will expand and will be categorized as axonal channelopathies.     

Morphology and function of preserved microvascular arterial grafts: an experimental study in rats

The aim of this study is to examine the morphology and function and small-caliber, arterial grafts after preservation in the University of Wisconsin solution (UW). Rat carotid arteries were stored in UW (n = 10) or in phosphate-buffered saline (PBS) (n = 10) for 1, 3, 7, and 14 days and were examined with light microscopy (LM) and scanning electron microscopy (SEM). Rat aortic preparations were stored in UW or PBS for 1 hour, 24 hours, 72 hours, 7 days, and 14 days and assessed for functional responses (stimulated contraction and endothelium-dependent relaxation). Segments (5 mm) of rat carotid arteries were stored in UW or PBS for 3 days, 7 days, and 14 days and orthotopically implanted as autografts and allografts. No immunosuppressive or anticoagulant agents were used. After 28 days of implantation, the grafts were assessed for patency and excised for LM and SEM. In UW, the endothelial layer remained intact up to 9 days of storage. In PBS, the endothelial layer showed deterioration after 1 day and was completely lost after 3 days. Functional responses were demonstrated to exist for as long as 7 days storage in UW. In PBS, no responses could be evoked after 24 hours storage. Autografts preserved in UW for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 83.3%, 66.6%, and 66.6%, respectively, whereas patency rates of allografts were 66.6%, 33.3%, and 33.3%, respectively. Autografts stored in PBS for 3 days (n = 6), 7 days (n = 6), and 14 days (n = 6) showed patency rates of 33.3%, 33.3%, and 50%, respectively, whereas patency rates of allografts were 16.7%, 0%, and 33.3%, respectively. The UW preserved autografts showed normal morphology. All other groups showed vessel wall degeneration which in the allograft groups, were accompanied by lymphocellular infiltration. In conclusion, the endothelial layer and vessel wall of arteries are adequately preserved in UW. Functional responses are retained up to 14 days storage in UW, but, are lost after 24 hours storage in PBS. Autograft implantation studies accordingly show good performance of arterial segments preserved in UW, whereas allografts are subject to degradation as a result of rejection.     

Regeneration of cochlear efferent nerve terminals after gentamycin damage

Chickens recover auditory function after hair cell loss caused by ototoxic drug damage or acoustic overstimulation, indicating that mechanisms exist to reestablish appropriate neuronal connections to regenerated hair cells. However, despite similar hair cell regeneration times, hearing recovery takes substantially longer after aminoglycoside than after sound damage. We have therefore begun examining damage and regeneration of efferent nerve terminals by immunolabeling whole-mount cochleae for differentially localized synaptic proteins and by visualizing the distribution of label with confocal microscopy. In undamaged cochleae, the synaptic proteins synapsin and syntaxin show similar distribution patterns corresponding to the large cup-like terminals on short hair cells. After gentamycin administration, these terminals are disrupted as hair cells are lost, leaving smaller, more numerous synapsin-reactive structures in the sensory epithelium. Syntaxin reactivity remains associated with the extruded hair cells, indicating that the presynaptic membrane is still attached to the postsynaptic site. In contrast, after sound damage, both synapsin and syntaxin reactivity are lost from the epithelium with extruded hair cells. As regenerated hair cells differentiate after gentamycin treatment, the synapsin labeling associated with cup-like efferent endings reappears but is not completely restored even after 60 d of recovery. Thus, efferent terminals are reestablished much more slowly than after sound damage (), consistent with the prolonged loss of hearing function. This in vivo model system allows comparison of axonal reconnection after either complete loss (sound damage) or partial disruption (gentamycin treatment) of axon terminals. Elucidating the differences in recovery between these injuries can provide insights into reinnervation mechanisms.     

Wallerian degeneration of peripheral nerve. Age-dependent loss of nerve lipids

The age-dependent loss of the major peripheral nerve lipids (cholesterol, phospholipid, and total galactolipid) was quantitated over a period of 9 weeks of Wallerian degeneration induced by surgical transection of rabbit sciatic nerves in animals of several ages. Proportionate losses of these lipids were determined by calculating the content of each lipid on a per nerve and on a per gram fresh weight basis remaining after a given period of Wallerian degeneration as a percent of original normal values at several time following surgery. The proportionate loss of each lipid from the distal stump was the most prompt and the most complete in nerves transected at 2 weeks of age, and the least in nerves transected at 20 weeks of age. The prompter clearance of these lipids from younger than older degenerating nerve gives convincing evidence that the suggestion from light-microscopic studies of faster clearance of neural debris in younger than in older animals is correct. A possible relationship between these biochemical findings and the phenomenon of greater functional recovery from peripheral nerve injury in younger than in older subjects is discussed.     

A tissue-engineered conduit for peripheral nerve repair

BACKGROUND: Peripheral nerve repair using autograft material has several shortcomings, including donor site morbidity, inadequate return of function, and aberrant regeneration. Recently, peripheral nerve research has focused on the generation of synthetic nerve guidance conduits that might overcome these phenomena to improve regeneration. In our laboratory, we use the unique chemical and physical properties of synthetic polymers in conjunction with the biological properties of Schwann cells to create a superior prosthesis for the repair of multiply branched peripheral nerves, such as the facial nerve. OBJECTIVES: To create a polymeric facial nerve analog approximating the fascicular architecture of the extratemporal facial nerve, to introduce a population of Schwann cells into the analog, and to implant the prosthesis into an animal model for assessment of regeneration. RESULTS: Tubes of poly-L-lactic acid (molecular weight, 100000) or polylactic-co-glycolic acid copolymer were formed using a dip-molding technique. They were created containing 1, 2, 4, or 5 sublumina, or "fascicular analogs." Populations of Schwann cells were isolated, expanded in culture, and plated onto these polymer films, where they demonstrated excellent adherence to the polymer surfaces. Regeneration was demonstrated through several constructs. CONCLUSIONS: A tubular nerve guidance conduit possessing the macroarchitecture of a polyfascicular peripheral nerve was created. The establishment of resident Schwann cells onto poly-L-lactic acid and polylactic-co-glycolic acid surfaces was demonstrated, and the feasibility of in vivo regeneration through the conduit was shown. It is hypothesized that these tissue-engineered devices, composed of widely used biocompatible, biodegradable polymer materials and adherent Schwann cells, will be useful in promoting both more robust and more precisely directed peripheral nerve regeneration.     

"Hitch-hike" grafts for limb salvage in peripheral arterial disease

A technique of arterial reconstruction suitable for patients with extensive arterial occlusions and for previous failed arterial surgery is described. The "hitch-hike" graft consists of a proximal limb of 6-mm velour Dacron prosthesis and a distal limb of autogenous vein. The intermediate prosthesis-to-vein anastomosis is made into an endarterectomized segment of upper popliteal artery. The results in the first 16 limbs are described. Eleven grafts are functioning from 2 to 14 months after operation and 5 of these have functioned for more than 1 year. The advantages of the technique are: long arterial occlusions may be bridged; autogenous vein is used to cross the knee joint; good measured blood flows may be demonstrated at operation; acceptable patency rates may be obtained up to 1 year.     

Quantitative study of muscle fibre atrophy and restitution after nerve grafts

Our comparative experimental studies on rabbits using clinical, electromyographical, and quantitative histological examinations of long autologous and homologous nerve grafts in 35 rabbits, paying special attention to quantitative histological changes in the gastrocnemius muscles, allow the statement that, compared to the short holografts, the long homografts showed worse results. Moreover, it became obvious that the regeneration rate of autografts was not influenced by increasing the lengths of the grafts. The answer to the question of how far even longer grafts may influence the quality of regeneration will need further investigations.