The influence of MR field strength on the detection of focal liver lesions with superparamagnetic iron oxide

Isolated nerve segments may inherently contain all of the necessary factors required to support regeneration within a silicone tube conduit placed across a nerve gap. Thirty-six adult Lewis rats each weighing approximately 250 g were randomized into three groups. A sciatic nerve gap (13-15 mm in length) was bridged by an empty silicone tube (Group I), a silicone tube containing a short 2-mm interposed nerve segment (Group II), or a nerve autograft (Group III). At 16 weeks postoperatively, no regeneration was observed through the empty silicone tube. In contrast, regeneration across the silicone tube containing the isolated nerve segment was equivalent to that noted through nerve autografts as assessed by histological, electrophysiological, and functional criteria. Thus, an interposed nerve segment will extend the length of successful nerve regeneration through a silicone tube conduit.     

Nerve regeneration in a 'pseudo-nerve' graft created in a silicone tube

The pseudo-nerve, which contains longitudinal Schwann cell columns without axons and surrounded by perineurium-like tissue but no axons (Q. Zhao, L.B. Dahlin, M. Kanje, G. Lundborg, Brain Res. 592 (1992) 106-114), was applied as a graft to repair nerve defect in rats. Creation of the pseudo-nerve was accomplished by inserting the proximal and distal stumps of a cut sciatic nerve into a silicone tube. The proximal insert was cut far proximally to prevent axons from entering the tube. After 4 weeks, the pseudo-nerve was harvested, trimmed into a 10-mm long graft and transplanted into a corresponding defect of the contralateral sciatic nerve. Nerve regeneration through the pseudo-nerve was examined by pinch reflex test and neurofilament staining after 6 days or by morphology after 4, 6 or 8 weeks. The results showed that the pseudo-nerve could induce nerve regeneration to a similar extend as a real nerve graft. The neurobiological composition of the pseudo-nerve and the factors influencing its formation were also studied. By double staining of S-100 and laminin we found that the longitudinally organized Schwann cell columns in the pseudo-nerve were surrounded by basal laminae and ensheathed by a layer of vascularized perineurium-like tissue. Macrophages (ED1 and ED2) and their products interleukin-1beta (IL-1beta) and transforming growth factor-beta1 (TGF-beta1) were constantly present in the pseudo-nerve. Besides, the size of tube was a crucial factor in influencing pseudo-nerve formation, e.g. a thicker pseudo-nerve was formed in tubes with larger diameters or shorter gap lengths. No pseudo-nerve was formed when the gap was 15 mm long. When both proximal and distal inserts were isolated nerve segments the pseudo-nerve was still formed but thin, probably because of compromised vascular supply. Taken together, the results suggested that the pseudo-nerve contains the essential neurobiological elements to induce successful axonal elongation.     

Influence of collagen and laminin gels concentration on nerve regeneration after resection and tube repair

In order to assess the usefulness of collagen and laminin gels prefilling nerve chambers to enhance nerve regeneration, we compared reinnervation of target organs after sciatic nerve resection leaving gaps of 4 or 6 mm followed by repair with silicone tubes in different groups of mice. Tubes were prefilled with saline solution, collagen gels, or laminin-containing gels at different concentrations. Functional reinnervation was assessed by noninvasive methods to quantitate recovery of sweating, nociceptive, sensory, and motor functions in the hindpaw repeatedly during 4-5 months postoperation. The increase in gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved. Reinnervation started earlier and achieved slightly higher levels with collagen gel diluted at 1.28 mg/ml than with more concentrated (1.92 and 2.56 mg/ml) collagen gels and with saline-prefilled tubes bridging a 4-mm gap. Recovery was also better with diluted (4 mg/ml) than with concentrated (12 mg/ml) laminin-containing gel, although lower than with collagen gels and saline. By prefilling silicone tubes bridging a 6-mm gap, a length considered limiting for regeneration in the mouse sciatic nerve, with diluted collagen or laminin gels, both matrices allowed for higher levels of recovery and for successful regeneration in a higher proportion of mice than saline solution. The laminin gel performed slightly better than the collagen gel.     

Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.     

Tenascin-R inhibits the growth of optic fibers in vitro but is rapidly eliminated during nerve regeneration in the salamander Pleurodeles waltl

Tenascin-R is a multidomain molecule of the extracellular matrix in the CNS with neurite outgrowth inhibitory functions. Despite the fact that in amphibians spontaneous axonal regeneration of the optic nerve occurs, we show here that the molecule appears concomitantly with myelination during metamorphosis and is present in the adult optic nerve of the salamander Pleurodeles waltl by immunoblots and immunohistochemistry. In vitro, adult retinal ganglion cell axons were not able to grow from retinal explants on a tenascin-R substrate or to cross a sharp substrate border of tenascin-R in the presence of laminin, indicating that tenascin-R inhibits regrowth of retinal ganglion cell axons. After an optic nerve crush, immunoreactivity for tenascin-R was reduced to undetectable levels within 8 d. Immunoreactivity for the myelin-associated glycoprotein (MAG) was also diminished by that time. Myelin was removed by phagocytosing cells at 8-14 d after the lesion, as demonstrated by electron microscopy. Tenascin-R immunoreactivity was again detectable at 6 months after the lesion, correlated with remyelination as indicated by MAG immunohistochemistry. Regenerating axons began to repopulate the distal lesioned nerve at 9 d after a crush and grew in close contact with putative astrocytic processes in the periphery of the nerve, close to the pia, as demonstrated by anterograde tracing. Thus, the onset of axonal regrowth over the lesion site was correlated with the removal of inhibitory molecules in the optic nerve, which may be necessary for successful axonal regeneration in the CNS of amphibians.     

DNase I hypersensitive sites far upstream of the rat tryptophan oxygenase gene direct developmentally regulated transcription in livers of transgenic mice

An experimental study on the rat sciatic nerve was performed to evaluate nerve regeneration through a collagen guide and to study the effects of alpha-melanocytic stimulating hormone (alpha-MSH) and basic fibroblast growth factor (b-FGF) in accelerating axonal elongation. After transection, nerves were repaired over a 7 mm gap using a placental collagen type IV guide. The channel was filled with either a b-FGF solution or an alpha-MSH solution or was produced with b-FGF incorporated into the guide. Four weeks later, only groups in which b-FGF had been injected or incorporated displayed a significant somatosensory evoked potential response. Histological and quantitative analysis of nerve fibres confirmed the existence of nerve continuity in groups receiving an alpha-MSH solution or a channel containing b-FGF. These results demonstrate that alpha-MSH in solution and b-FGF incorporated into a collagen type IV channel enhance peripheral nerve regeneration. However, at 4 weeks, only b-FGF (3 ng) restores functional activity.     

Neurotoxic effects of sodium nitroprusside in rat hippocampal slices

The effect of a permanent transection on myelin gene expression in a regenerating sciatic nerve and in an adult sciatic nerve was compared to establish the degree of axonal control exerted upon Schwann cells in each population. First, the adult sciatic nerve was crushed, and the distal segment allowed to regenerate. At 12 days post-crush, the sciatic nerve was transected distal to the site of crush to disrupt the Schwann cell-axonal contacts that had reformed. Messenger RNA (mRNA) levels coding for five myelin proteins were assayed in the distal segment of the crush-transected nerve after 9 days and were compared to corresponding levels in the distal segments of sciatic nerves at 21 days post-crush and 21 days post-transection using Northern blot and slot-blot analysis. Levels of mRNAs found in the distal segment of the transected and crush-transected nerve suggested that Schwann cells in the regenerating nerve and in the mature adult nerve are equally responsive to axonal influences. The crush-transected model allowed the genes that were studied to be classified according to their response to Schwann cell-axonal contact. The levels of mRNAs were 1) down-regulated to basal levels (P0 and MBP mRNAs), 2) down-regulated to undetectable levels (myelin-associated glycoprotein mRNAs), 3) upregulated (mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase and beta-actin), or 4) not stringently controlled by the removal of Schwann cell-axonal contact (proteolipid protein mRNAs). This novel experimental model has thus provided evidence that the expression of some of the important myelin genes during peripheral nerve regeneration is dependent on continuous signals from the ingrowing axons.     

Typing of methicillin-resistant Staphylococcus aureus isolates from Düsseldorf by six genotypic methods

This in vivo double-blind study evaluated the effect of recombinant human glial growth factor 2 (rhGGF2), a Schwann cell mitogen, on the recovery of motor function of rat sciatic nerve following crush injury. Seventy three rats were divided into three groups. Group I (n=5), sham operated; Groups II (n=34) and III (n=34) received a 100 g crush load for 2 h over a 5 mm segment of the sciatic nerve. Group III was treated with 1 mg/kg rhGGF2, via subcutaneous injection one day before nerve crush and daily for the following four days. Group II received an equivalent volume of saline as a control. Motor functional recovery was assessed by calculating the sciatic functional index (SFI) and the recovery rate of tetanic contractile force of the extensor digitorum longus (EDL) muscle. Recovery of nerve function was evident at day 11 after crush in the rhGGF2-treated animals, whereas the nerves in controls were still paralyzed. The rhGGF2-treated animals showed a significant improvement of the SFI between days 11-21 postoperatively when compared to controls. The isometric tetanic contractile force was stronger in the rhGGF2-treated group than in controls, with a significant difference at 40 to 70 Hz stimulus frequencies on day 4. Correlation analysis showed that tetanic contractile force had a linear correlation with the SFI. Histologic assessment indicated that the rhGGF2-treated animals showed less severe degeneration and earlier robust remyelination of axons than controls. The results suggest that treatment with rhGGF2 is effective in promoting nerve regeneration as seen in measurements of functional recovery and qualitative assessment of nerve morphology. The mechanism of GGF's protective effect may be related to its direct action on Schwann cells, stimulating their mitosis as well as inducing neurotrophic factors essential to neuronal maintenance and repair.     

Procurement, preservation, and transport of cadaver kidneys

Numerous findings support the possibility that highly sulfated proteoglycans are inhibitory molecules which, at high concentration relative to growth-promoting signals, may regulate or guide axonal growth. Although most studies implicate sulfated proteoglycans in the poor regenerative capacity of the central nervous system, inhibitory proteoglycans also may play an important role in the successful regeneration of axons within peripheral nerve. Cultured rat schwannoma and Schwann cells produce chondroitin sulfate proteoglycan (CSPG) which binds to and inhibits the neurite-promoting activity of laminin [Muir et al. (1989) J. Cell Biol. 109:2353]. In the present study, we found a similar neurite-inhibiting activity associated with CSPG isolated from normal adult rat sciatic nerve. Following nerve crush injury, this inhibitory activity was increased sevenfold in regenerating nerve distal to the injury. This increase was largely attenuated by in vivo administration of the proteoglycan synthesis inhibitor beta-D-xyloside. In normal adult nerve, immunolabeling for CSPG core protein was concentrated in slender bands surrounding axon-Schwann cell units and within nodes of Ranvier. Following nerve crush injury, immunolabeling of CSPG and laminin became more intense in distal nerve and CSPG increased within endoneurium and surrounding nerve sheaths. Embryonic dorsal root ganglionic neurons cultured on longitudinal nerve sections extended neurites along the exposed surfaces of Schwann cell basal lamina. The length of neurites was increased 58% on normal nerve sections pretreated with chondroitinase. Even though laminin levels were elevated in basal lamina of injured nerve, neuritic growth on sections of injured nerve was not significant increased unless sections were pretreated with chondroitinase. These results indicate that inhibitory CSPG is up-regulated in injured nerve and plays a role in regulating axonal regeneration.     

Selective reinnervation of regenerating mixed nerve fibres across a silicone tube gap. Further experimental evidence of neurotropism

This study investigated specific regeneration of a mixed motor and sensory nerve by the method of spinal dorsal root ganglions resection. A 10 mm segment of tibial nerve was resected and the nerve ends inserted in a silicone tube. Fourteen weeks later, dorsal root ganglia from L6 to S1 were resected on the experiment side. Twenty weeks later, the regenerating motor nerve fibres of mixed nerves selectively grew into motor branches. The rate of misdirected growth in mixed nerves was less than 6%. These results suggest that regenerating motor and sensory axons of mixed nerves are able to select their distal target organs accurately. Better results may be obtained using the entubulation repair method.     

Auditory processing and dyslexia: evidence for a specific speech processing deficit

Following transection of a peripheral nerve in mice, a newly synthesized neurotropic pyrimidine compound, MS-818 was administered intraperitoneally at a dose of 1 mg kg-1 b.wt. day-1. The film model experiments for analyzing the early growth of axonal regeneration suggested that MS-818 activated Schwann cells which migrate from the proximal stump, inducing axonal elongation in vivo.     

Temporal relationships between the pharmacokinetics of methylphenidate in the human brain and its behavioral and cardiovascular effects

We compared reinnervation of target organs after sciatic nerve section leaving gaps of 2, 4, 6, or 8 mm or gaps repaired with silicone tubes in different groups of mice. Functional reinnervation was assessed by noninvasive methods to determine recovery of sweating, nociceptive, and muscular functions in the hindpaw repeatedly during 3 months postoperation. The increase of gap length between nerve stumps delayed the beginning and reduced the degree of functional recovery achieved either with or without repair. When lesions were left unrepaired, functional reinnervation was only noticeable with a 2-mm gap and practically absent with longer gaps. With tube repair, reinnervation started earlier and achieved higher values than in the corresponding unrepaired groups. Tubulization was most effective with 4-mm gaps and comparatively less with shorter and longer gaps. With 4-mm gaps, recovery was higher when the silicone tube had a cross-sectional area 2.5 times that of the sciatic nerve than with narrower or wider tubes and when the wall was the thinnest available. In all cases muscle reinnervation showed a lower progression than sweating and nociceptive recovery.     

Peripheral component in the enhanced antinociceptive effect of systemic U-69,593, a kappa-opioid receptor agonist in mononeuropathic rats

To evaluate the ability of cilostazol, an antiplatelet and vasodilating agent, to promote axonal regeneration in streptozotocin-induced diabetic rats, the time until beginning of regeneration (initial delay) and the axonal regeneration rate of the sciatic nerve were estimated using the pinch test, and ornithine decarboxylase activity was measured in dorsal root ganglia. At 5 weeks of diabetes, axonal regeneration rate remained unchanged but the initial delay was prolonged and ornithine decarboxylase induction was delayed in diabetic rats compared with those in normal rats. Cilostazol had little effect on these parameters in normal or diabetic rats. At 10 weeks of diabetes, diabetic rats showed both prolongation of initial delay and a decrease in axonal regeneration rate. Cilostazol markedly increased axonal regeneration rate in diabetic rats. Ornithine decarboxylase induction following nerve injury disappeared almost completely in diabetic rats but was maintained by cilostazol treatment. The effect of cilostazol in diabetic rats is thought to be mediated through its preventive effect on circulatory disorders. The active site of the drug appears to be early processes in nerve regeneration before ornithine decarboxylase induction. Further, the results suggest that the both axonal regeneration and this induction are sensitive to circulatory defects in diabetes.     

Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions

To examine the question of whether or not prevention of axonal regrowth after injury affects the molecular responses of neurons to axotomy, Northern blotting and in situ hybridization were used to study changes in the mRNA levels of neurofilament (NF) proteins and tubulins in rat dorsal root ganglion (DRG) cells. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration-permissive condition) or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration-prohibitive condition). At 14 days post-injury, the relative levels of the low (NF-L) and middle (NF-M) molecular weight NF protein mRNAs, as well as those of beta II- and beta III-tubulin, were examined in the L4 and L5 DRG. The data showed that the levels of NF-L and NF-M mRNAs decreased while beta II- and beta III-tubulin mRNA levels increased in the DRG after either crush axotomy or cut/ligation axotomy of the sciatic nerve, suggesting that the elicitation of these molecular changes by axon disconnection is independent of the ultimate success or failure of the axonal regrowth process. However, cut/ligation axotomy had a more pronounced effect than did crush injury on the mRNA changes. This result suggests that feedback mechanisms from regrowing axons are important in regulating the extent of the cytoskeletal mRNA changes in injured neurons.     

Regeneration of the node of Ranvier: a light and electron microscope study

Regeneration of the node of Ranvier was investigated in the rat peroneal nerve 10-60 days after nerve crush, by light and electron microscopy. At 10 and 20 days after crush nodes of Ranvier were clearly identifiable by electron microscopy but had a relatively simple structure. At 40 days after crush however nodes were highly differentiated showing specialised features such as paranodal bulbs, nodal constriction of the axon, paranodal Schwann cell mitochondria, nodal Schwann cell microvilli, and nodal gap substance. By light microscopy some nodes were identifiable as early as 20 days after crush. At both 30 and 60 days after crush regenerated internodes were uniformly short (means of 275 micronm and 339 micronm respectively).     

Long-distance axonal regeneration in the transected adult rat spinal cord is promoted by olfactory ensheathing glia transplants

The lack of axonal regeneration in the injured adult mammalian spinal cord leads to permanent functional impairment. To induce axonal regeneration in the transected adult rat spinal cord, we have used the axonal growth-promoting properties of adult olfactory bulb ensheathing glia (EG). Schwann cell (SC)-filled guidance channels were grafted to bridge both cord stumps, and suspensions of pure (98%) Hoechst-labeled EG were stereotaxically injected into the midline of both stumps, 1 mm from the edges of the channel. In EG-transplanted animals, numerous neurofilament-, GAP-43-, anti-calcitonin gene-related peptide (CGRP)-, and serotonin-immunoreactive fibers traversed the glial scars formed at both cord-graft interfaces. Supraspinal serotonergic axons crossed the transection gap through connective tissue bridges formed on the exterior of the channels, avoiding the channel interior. Strikingly, after crossing the distal glial scar, these fibers elongated in white and periaqueductal gray matter, reaching the farthest distance analyzed (1.5 cm). Tracer-labeled axons present in SC grafts were found to extend across the distal interface and up to 800 microm beyond in the distal cord. Long-distance regeneration (at least 2.5 cm) of injured ascending propriospinal axons was observed in the rostral spinal cord. Transplanted EG migrated longitudinally and laterally from the injection sites, reaching the farthest distance analyzed (1.5 cm). They moved through white matter tracts, gray matter, and glial scars, overcoming the inhibitory nature of the CNS environment, and invaded SC and connective tissue bridges and the dorsal and ventral roots adjacent to the transection site. Transplanted EG and regenerating axons were found in the same locations. Because EG seem to provide injured spinal axons with appropriate factors for long-distance elongation, these cells offer new possibilities for treatment of CNS conditions that require axonal regeneration.     

Identification of peptides inhibiting enzyme I of the bacterial phosphotransferase system using combinatorial cellulose-bound peptide libraries

The effect of hyperbaric oxygen (HBO) treatment on regeneration of the rat sciatic nerve was studied. The sciatic nerve was crushed with a pair of pliers and the animals were either left untreated or subjected to a series of 45-min exposures to 100% O2 at 3.3 atm absolute pressure at 0, 4, and 8 h postoperatively and then every 8 h. Regeneration was evaluated using the pinch-reflex test at 3, 4, or 5 days following surgery and with neurofilament staining at 4 days. The regeneration distances at all time points were significantly longer in animals exposed to hyperbaric oxygen treatment independent of the evaluation procedure. A short initial period of the same HBO treatment schedule, with no more treatments after 25 h, appeared as effective as when treatments were maintained being given every 8 h until evaluation. We conclude that HBO treatment stimulates axonal outgrowth following a nerve crush lesion.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Antibody to transforming growth factor beta reduces collagen production in injured peripheral nerve

Epineurial scarring in peripheral nerve after injury inhibits normal axonal regeneration primarily due to fibroblast deposition of type I collagen. The transforming growth factor beta (TGF-beta) family is an important class of signaling molecules that has been shown to stimulate fibroblasts to produce collagen. The aim of this study was to design a prototypic therapeutic system in which the neutralization of TGF-beta in crushed rat sciatic nerve would decrease collagen formation. A total of 45 experimental Lewis rats were used. Group 1 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate buffer solution. Group 2 animals (20 rats) sustained a unilateral crush injury to the sciatic nerve with injection of phosphate-buffered saline and goat, anti-rat, panspecific TGF-beta antibody. Group 3 control animals (five rats) underwent only exposure of sciatic nerve with injection of antibody. All animals were killed at 14 days and sciatic nerve specimens were harvested at that time. Slides of experimental tissue were processed using a 35S-labeled oligomer for procollagen alpha-1 mRNA, then dipped in photographic emulsion and examined by darkfield autoradiography. Morphometric analysis of pixel counts was then performed. A significant reduction in total pixel count per high-power field and in total number of fibroblasts per high-power field was found in crushed rat sciatic nerve treated with anti-TGF-beta antibody when compared with those treated only with phosphate-buffered saline. These findings are consistent with successful reduction in procollagen induction after a crush injury by topical administration of blocking antibody against transforming growth factor beta. The concept of growth factor blockade for therapeutic collagen reduction is attractive in the context of nerve injury, and the current article provides a model for future clinical application.