Schwann cells, acutely dissociated from a predegenerated nerve trunk, can be applied into a matrix used to bridge nerve defects in rats.

BACKGROUND: The gold standard to reconstruct a nerve defect is a conventional autologous nerve graft. There may be a lack of such grafts in severe nerve injuries. Alternatives to autologous nerve grafts are needed. METHODS: We have developed a technique where mainly Schwann cells are acutely dissociated from the ends of the severed nerve trunk after nerve injury. The technique does not require long-term cell culture procedures. The obtained cells, which can be dissociated within a few hours, are applied to a silicone tube or a tendon autograft used to bridge a nerve defect. FINDINGS: Dissociated cells from the ends of the severed nerve ends consist of more than 85% of Schwann cells. The remaining cells are ED1 stained macrophages. The cells survive transfer to a silicone tube or a tendon autograft which bridge the nerve defect. Axons do grow through such a graft filled with dissociated cells. CONCLUSION: Our novel model to obtain mainly Schwann cells by dissociation of the cells from the severed nerve ends after injury and add them to a matrix, thereby creating an artificial nerve graft, may be a new technique with potential clinical application in nerve reconstruction.

Functional recovery and mechanisms in end-to-side nerve repair in rats.

BACKGROUND: End-to-side nerve repair is attachment of a single distal nerve segment (recipient nerve) end-to-side to an intact donor nerve when there is a lack of proximal nerve segment after injury. The technique is currently used clinically but the mechanism(s) behind this technique are essentially unknown. METHODS: We have studied end-to-side nerve repair in the forelimb of rats, where a single distal radial nerve or an ulnar or a median, or both, nerves are attached end-to-side to an intact musculocutaneous nerve. We have studied functional recovery, origin of the regenerating axons and cell activation by the end-to-side nerve repair. FINDINGS: Functional recovery occurs after end-to-side nerve repair but is less sufficient than conventional end-to-end nerve repair or a nerve graft procedure. Sensory and motor axons grow from the musculocutaneous nerve out into the attached nerve segment(s). An injury is required to the musculocutaneous nerve to activate sensory and motor neurons as well as Schwann cells in the musculocutaneous nerve for initiation of regeneration. CONCLUSIONS: End-to-side nerve repair may be an alternative method in specific cases of complex nerve injuries to reconstruct nerve trunks when no other repair options are possible. Some functional recovery does occur but regeneration of sensory and motor axons require an injury to the neurons of the donor nerve.

Addition of cultured Schwann cells to tendon autografts and freeze-thawed muscle grafts improves peripheral nerve regeneration.

The effects of addition of Schwann cells on peripheral nerve regeneration through a novel graft material-the tendon autograft-and a conventional freeze-thawed muscle graft, were studied in the rat sciatic nerve. Adult Schwann cell cultures were established from predegenerated nerves. The Schwann cells were added to the autologous grafts by coculture (tendon autograft) or injection (freeze-thawed muscle graft). Both graft types supported adherence of the added Schwann cells. Addition of cultured Schwann cells to the two different graft models improved regeneration by increasing the rate of axonal outgrowth as compared with similar grafts without added cells.

Experimental nerve compression and upregulation of CPON in DRG.

Expression of C-terminal flanking peptide of neuropeptide Y (CPON) in DRG and cell proliferation (incorporation of BrdU) in sciatic nerve of rats following chronic nerve compression (silicone tubes with different internal diameters) was studied by immunocytochemistry. An increased number of CPON-positive neurons and cells incorporating BrdU was induced on the compressed side, most pronounced when a tight tube was used, while no cells expressed CPON or BrdU in intact nerves. The increase was transient and declined with time. Nerve compression induces transient cell proliferation in the nerve and expression of CPON in nerve cell bodies, but this is of a lesser magnitude than those following nerve transection.

C-terminal flanking peptide of neuropeptide Y in DRG following nerve compression.

The C-terminal flanking peptide of neuropeptide Y (CPON) was studied in dorsal root ganglia (DRG) by immunocytochemistry after different recovery periods (3, 6,14 and 28 days) following tourniquet compression of the rat hindlimb (sciatic nerve; 150 or 300 mmHg; 2 h). Compression induced a transient increase in the number of CPON-positive DRG-neurons (the contralateral uninjured side was devoid of CPON-positive cells). The compression-induced increase in CPON was less than that observed in separate rats subjected to sciatic nerve transection. The results show that compression induces regenerative changes in peripheral neurons and that such an injury of the nerve trunk is not limited to the site of the compression but results in the activation of the entire neuron.

5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglion: co-localization with calcitonin gene-related peptide, substance P and nitric oxide synthase.

5-Hydroxytryptamine (5-HT) is implicated in migraine and agonist directed against 5-HT(1B) and 5-HT(1D) receptors are commonly used as effective therapies. The antimigraine mechanisms involve the inhibition of intracranial sensory neuropeptide release. In order to determine which 5-HT(1) receptor subtypes are involved we have by immunocytochemistry examined the distribution of 5-HT(1B) and 5-HT(1D) receptors in the human trigeminal ganglia, and addressed which of them colocalize with calcitonin gene-related peptide (CGRP), substance P (SP) or nitric oxide synthase (NOS). We detected that 5-HT(1D) receptor immunoreactivity (i.r.) was predominantly expressed in medium-sized cells (86% of positive cells, 30-60 microm). About 9% of the 5-HT(1D) receptor i.r. cells were large in size (> 60 microm) and 5% were small in size (< 30 microm). In a similar pattern, 5-HT(1B) receptor i.r. was mainly expressed in medium-sized cells (81% in 30-60 microm, 15% in > 60 microm and 4% in < 30 microm). Double immunostaining was used to determine whether the 5-HT(1B) or 5-HT(1D) receptor immunoreactive cells co-localized with either CGRP, SP or NOS. Thus, 89% of the CGRP i.r. cells expressed 5-HT(1D) receptor i.r. and 65% of the CGRP positive cells were 5-HT(1B) receptor positive. Most of the 5-HT(1D) (95%) and the 5-HT(1B) (94%) receptor i.r. cells showed SP immunostaining and 83% of 5-HT(1D) receptor and 86% of 5-HT(1B) receptor i.r. cells contained NOS. In conclusion, both 5-HT(1B) and 5-HT(1D) receptors are expressed in the human trigeminal ganglion and they are mainly localized in medium-sized cells and they seem to colocalize with CGRP, SP and NOS.

Markedly reduced chronic nociceptive response in mice lacking the PAC1 receptor.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) has been proposed to have a role in nociception. Here we have used the formalin test, thermal laser stimulation and mechanical von Frey stimulation to investigate possible alteration of PAC1-/- mice nociceptive behaviour. Our finding, that PAC1-/- mice have a substantial, 75% decrease in nociceptive response during the late phase, provides clear evidence that the specific PACAP-receptor PAC1 is involved in the mediation of nociceptive responses during chronic conditions such as inflammation. PAC1-/- mice had small or no changes in the response to mechanical and thermal laser stimulation. This suggests a limited, if any, involvement of PAC1 in nociception after short-lasting stimuli. Injury-induced changes in DRG neuropeptide expression were more pronounced in PAC1-/- mice, implying neuroregulatory functions of PAC1.

Postnatal expression of VEGF and its receptor flk-1 in peripheral ganglia.

The postnatal (P0-P12) and adult expression of vascular endothelial growth factor and its receptor flk-1 was investigated in superior cervical (SCG) and dorsal root ganglia (DRG) in mice by immunocytochemistry. At P0 all neurons in SCG and DRG contained VEGF. The number of VEGF-immunoreactive neurons in DRG but not in SCG, decreased postnatally and reached adult levels (34%) at P12. At P0 flk-1 was found in virtually all neurons in the SCG and in roughly half of the neurons in DRG. The number of flk-1 positive neurons then decreased and reached adult levels at P12. The findings demonstrate temporal changes in VEGF and flk-1 expression, suggesting developmental regulation of VEGF activity in peripheral ganglia.

Neural regeneration along longitudinal polyglactin sutures across short and extended defects in the rat sciatic nerve.

OBJECT: The authors have previously shown that longitudinal sutures without artificial tube support regeneration across a 7-mm gap in the rat sciatic nerve. In the present study, the authors compared this new approach with the use of autologous nerve grafts across short defects and examined whether the approach could be used to support regeneration across extended gaps and whether the interposition of a short nerve segment (the stepping-stone procedure) was applicable in this model. METHODS: Longitudinal sutures were used to bridge 7- and 15-mm gaps in the rat sciatic nerve. Contralateral comparisons were made to nerve autografts in the 7-mm group and to sutures plus a short interposed nerve segment in the 15-mm group. Regeneration was evaluated at 2, 4, and 12 weeks by using immunocytochemical analysis for Schwann cells, neurofilament protein, and macrophages and at 12 weeks also by using histological examination, including morphometry in the distal tibial trunk and tetanic force measurements in the gastrocnemius muscle. CONCLUSIONS: The authors found that the results of regeneration after repair with longitudinal polyglactin sutures across short defects were not significantly different from those produced by the use of autologous nerve grafts. Regeneration, although poor, occurred along sutures across extended gaps and was significantly enhanced by an interposed nerve segment acting as a Schwann cell resource in this model.

Use of chemically extracted muscle grafts to repair extended nerve defects in rats.

Nerve regeneration, measured as axonal outgrowth, Schwann cell migration, macrophage invasion, and neovascularisation, was compared after repair of a 15 mm gap in rats' sciatic nerves using autologous muscle grafts made acellular either by freezing and thawing or by chemical extraction. Both extracted and freeze-thawed acellular muscle grafts could be used to bridge the defect. However, axons and Schwann cells, as shown by immunohistochemical staining for neurofilaments and S-100 protein, respectively, grew faster into the extracted muscle grafts than into the freeze-thawed acellular muscle grafts and somewhat more axons were observed in the former graft. There were no significant differences between the two graft types with respect to neovascularisation as showed by staining for endothelial alkaline phosphatase, and limited differences concerning invasion of macrophages (ED1 and ED2) as detected by immunocytochemistry. The results showed that chemically extracted muscle grafts could be used to bridge an extended nerve defect and that such grafts in some aspects were superior to freeze-thawed muscle grafts for extended gaps.

Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor.

Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates axonal outgrowth. Here we used in situ hybridization and immunocytochemistry to study the VEGF receptor flk-1 in cultured superior cervical ganglia (SCG) and dorsal root ganglia (DRG) from adult mice, and also the effects of VEGF on regeneration in vitro. Neurons in both ganglia contained the flk-1 receptor and showed an increased mRNA expression and immunoreactivity for flk-1 after 48 h in culture. In SCG, but not in DRG, double immunostaining for flk-1 and VEGF revealed coexpression in many neurons, implying that VEGF may exert both autocrine and paracrine actions. One proportion of the flk-1-positive neurons in DRG stained positive for the large neuron marker RT97 and another proportion expressed calcitonin gene-related peptide (CGRP). Small IB4-positive neurons were devoid of flk-1 immunoreactivity. Most flk-1-positive neurons in the DRG, but not in the SCG, were also immunoreactive to neuropilin-1. VEGF was found to stimulate axonal outgrowth from DRG, both by an action on the growing axons and the nerve cell bodies. The latter effect could be mediated by retrograde axonal transport as revealed by the use of a two compartment system to assay axonal outgrowth. We also found that the VEGF-induced axonal outgrowth was blocked by the flk-1 inhibitor SU5416. The results strongly suggest that VEGF acts as a neurotrophic factor and plays an important role during the regeneration of peripheral nerves.

CGMP increases in satellite cells of nitric oxide synthase-containing sensory ganglia.

cGMP and the enzymes, nitric oxide synthase (NOS) and heme oxygenase-1 (HO-1), the products of which stimulate soluble guanylyl cyclase activity, were investigated in cultured dorsal root ganglia (DRG), and nodose ganglia of adult rats. A dramatic increase of cGMP-positive satellite cells in ganglia cultured for 24 or 48 h was observed, particularly in Th8-L2 DRG and in nodose ganglia. These ganglia also contained most NOS-positive neurones, as reflected by NADPH-diaphorase histochemistry. HO-1 immunoreactivity increased in satellite cells, but in different cells to those in which cGMP increased. These results suggest that both NO and CO could be involved in signalling between neurones and satellite cells in sensory ganglia during regeneration.

Collateral sprouting from sensory and motor axons into an end to side attached nerve segment.

A nerve segment, sutured end-to-side (ETS) to an intact rat sciatic nerve, becomes invaded by regenerating axons. The origin of these fibres is controversial: it is debated whether or not they represent collateral sprouts from intact axons. Here we demonstrate by double retrograde tracing, using one tracer for the ETS attached segment and another for the sciatic nerve proper double-labelled sensory neurons in 67% of the rats receiving an ETS segments. Double-labelled motor neurons were observed in 11% of the rats. The results show that a nerve segment attached ETS to an intact nerve can induce collateral sprouting of both sensory and motor axons although the extent of such branching may vary with the experimental conditions.

Sutures alone are sufficient to support regeneration across a gap in the continuity of the sciatic nerve in rats.

We have proposed that it is sufficient to provide a simple substratum on which regenerating axons may traverse a gap in a peripheral nerve. To test this hypothesis we set up a new experimental model in which sutures were used to bridge a 10 mm long defect in a peripheral nerve. A defect was created bilaterally in 25 rat sciatic nerves. The cut ends of the nerve were joined by three laps of a continuous suture, on one side with 8/0 polyamide (non-absorbable) and on the other with 8/0 polyglactin (absorbable), leaving a 7 mm gap. At two weeks a matrix that contained capillaries, fibroblast-like cells, and mononuclear cells had formed between the nerve endings, and the sutures were surrounded by foreign-body-like tissue reactions. At four weeks axons had grown into the distal nerve segment on both sides in 65%-90% of the cases as indicated by a response to the pinch reflex test and immunocytochemistry for presence of neurofilament protein. Axons were organised in minifascicles and these tended to grow larger as the demarcation of the entire regenerated segment by a perineurial-like structure improved with time. At 12 weeks axonal counts of cross-sections of the distal tibial trunk showed many myelinated nerve fibres but no significant difference in axonal counts or degree of myelination between the polyamide and polyglactin sides. The results show that conventional sutures alone are sufficient to support regeneration across a short gap in a peripheral nerve, a method that may be of potential clinical value.

Heme oxygenase-1, heme oxygenase-2 and biliverdin reductase in peripheral ganglia from rat, expression and plasticity.

The expression of inducible and constitutive heme oxygenase and biliverdin reductase was studied in normal and cultured peripheral ganglia from adult rats, using immunocytochemistry and in situ hybridization. Dramatic changes were induced by one to two days' culturing of dorsal root ganglia, nodose ganglia, otic ganglia, sphenopalatine ganglia and superior cervical ganglia. An up-regulation of inducible heme oxygenase was found in satellite cells of the cultured nodose ganglia, dorsal root ganglia, sphenopalatine ganglia and otic ganglia, whereas only a few satellite cells in the superior cervical ganglia responded with an increase in inducible heme oxygenase immunoreactivity. In the superior cervical ganglia inducible heme oxygenase also appeared in a subpopulation of macrophages. During culturing, expression of inducible heme oxygenase immunoreactivity also increased in axons and in nerve cell bodies. In situ hybridization corroborated the immunocytochemical findings, revealing a strong up-regulation of inducible heme oxygenase messenger RNA in satellite cells, and less pronounced up-regulation in nerve cell bodies. Constitutive heme oxygenase immunoreactivity was found in most neurons in all of the ganglia studied. No significant changes in constitutive heme oxygenase immunoreactivity could be observed in cultured ganglia. Biliverdin reductase immunoreactivity was barely detectable in any of the normal ganglia; however, after culturing it appeared in axons, single nerve cell bodies and nerve cell nuclei. The results show that inducible heme oxygenase is up-regulated in peripheral ganglia after axonal injury, and suggest a role for carbon monoxide in cellular signaling and a requirement for the antioxidant (bilirubin) during the regeneration process.

Alteration of PACAP distribution and PACAP receptor binding in the rat sensory nervous system following sciatic nerve transection.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely expressed neuropeptide that has been involved in nerve regeneration, neurone survival and nociception. In this study, the distribution of PACAP and PACAP-receptors were investigated in rat dorsal root ganglia (DRG), spinal cord and medulla oblongata at 3, 7 or 14 days following unilateral sciatic nerve transection using immunohistochemistry, 125I-PACAP-binding and in situ hybridisation. In control (contralateral side) DRG, about 30% of the nerve cell bodies (92% being small) were PACAP-immunoreactive (PACAP-IR). In the spinal cord, PACAP-IR fibres were seen in laminae I-II but not in the gracile nuclei. Following sciatic nerve transection, PACAP-IR fibres appeared in the gracile nuclei and occasionally in the deeper laminae of the dorsal horn consistent with the relative increase in larger PACAP-IR DRG neurones. However, the relative number of small PACAR-IR neurones was significantly lower on the transected side as compared to the control side suggesting a dual reaction for PACAP in the DRG following nerve injury. 125I-PACAP-binding was found in laminae I-II, around the central canal and in the gracile nuclei but not in the DRG. At 14 days after transection, 125I-PACAP-binding density was significantly reduced in the ipsilateral dorsal horn. PACAP-receptor (PAC(1)) mRNA was detected in neurones of the dorsal and ventral horn and in the gracile nuclei with no overt changes observed after transection. Very few DRG nerve cell bodies contained PAC(1) mRNA. The findings are consistent with a role for PACAP both in nociception and regeneration.

Vascular endothelial growth factor stimulates Schwann cell invasion and neovascularization of acellular nerve grafts.

The aim of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on regeneration of the rat sciatic nerve in vivo. To that end we used 10-mm long cell-free nerve grafts to bridge a gap in the sciatic nerve. The grafts were pretreated with either VEGF (50, 100 or 250 ng/ml), nerve growth factor (NGF, 100 ng/ml) or laminin (100 ng/ml) before implantation. Outgrowth of axons, Schwann cells, blood vessels and macrophages were studied 10 days post-implantation by the use of immunocytochemistry and histochemistry. Grafts pretreated with VEGF stimulated the outgrowth of Schwann cells and blood vessels but not axons. In such grafts, the Schwann cells also exhibited a dramatic change in morphology and became filled with large lipid-containing vacuoles. These cells also showed an intense immunoreactivity for the VEGF receptor flk-1. Neither pretreatment with laminin nor NGF affected the outgrowth of Schwann cells. However, NGF treatment increased the number of axons in the graft but was not able to counteract injury-induced downregulation of substance P in the dorsal root ganglia. The results show that local application of VEGF promotes at least two events, invasion of Schwann cells and neovascularization, which are important during nerve regeneration. The findings suggest that the effects of the pretreatment by the growth factors is local and limited to the graft, whereas central events like neuropeptide synthesis is not affected.

Peripheral but not central axotomy promotes axonal outgrowth and induces alterations in neuropeptide synthesis in the nodose ganglion of the rat.

We investigated the effects of central and peripheral axotomy of the sensory neurons in the nodose ganglion on neurite outgrowth and neuropeptide expression. Axonal outgrowth was studied in ganglia subjected to a conditioning lesion of the vagus nerve 6 days prior to in vitro explantation. In such cultures, a conditioning effect, i. e. a shorter initial delay and faster axonal outgrowth, was observed after peripheral axotomy, while central axotomy had no effect. Neuropeptide expression was measured by immunocytochemistry 3 days after axotomy. Peripheral axotomy induced an increase in the number of neurons expressing the C-terminal flanking peptide of neuropeptide Y (C-PON), galanin (GAL) and vasoactive intestinal peptide (VIP). In contrast, central axotomy did not affect neuropeptide expression. These results suggest that both axonal outgrowth and expression of neuropeptides in the sensory neurons of the nodose ganglion could be regulated by the contact of the cells with their peripheral, but not their central targets.

Estrogen and progesterone stimulate Schwann cell proliferation in a sex- and age-dependent manner.

The effects of estrogen and progesterone on Schwann cell proliferation were studied in cultured segments of the rat sciatic nerve from adult male, female, and newborn rats, by measurement of [3H thymidine incorporation or bromo-deoxy-uridine- (BrdU)-labelling and immunocytochemistry. Estrogen (100 nM-500 nM) enhanced [3H] thymidine incorporation in segments from male and newborn rats, while it had no effect on segments from female rats. Progesterone stimulated thymidine incorporation in segments from female and newborn rats (100 nM-500 nM), but caused only a small proliferative response in Schwann cells from male rats at high concentrations. The proliferative effects of estrogen and progesterone were blocked when the segments were cultured in the presence of inhibitors of their respective receptors, ICI 128 780 and zk 112994. The data suggest that Schwann cells possess distinct receptors for estrogen and progesterone and that these receptors may be involved in the control of Schwann cell proliferation. It also shows that the response of Schwann cells to sex hormones varies with sex and perhaps also with age.

Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system.

Vascular endothelial growth factor (VEGF) is a mitogen for endothelial cells, and it promotes angiogenesis in vivo. Here we report that VEGF(165) has neurotrophic actions on cultured adult mouse superior cervical ganglia (SCG) and dorsal root ganglia (DRG), measured as axonal outgrowth. Maximal effect was observed at 10-50 ng/ml for SCG and 100 ng/ml for DRG. VEGF-induced axonal outgrowth was inhibited by the mitogen-activated protein kinase kinase inhibitor PD 98059 but not by the protein kinase inhibitor K252a. VEGF also increased survival of both neurons and satellite cells and the number of proliferating Schwann cells. Immunocytochemistry and immunoblotting revealed that VEGF was expressed in virtually all nerve cells in the SCG but only in a population of small-diameter (<35 micrometers) neurons representing approximately 30% of the neurons in DRG. Immunostaining showed that the VEGF receptor fetal liver kinase receptor (flk-1) was found on nerve cell bodies in DRG and to a lesser extent on neurons in SCG. Growth cones of regenerating axons from both types of ganglia exhibited flk-1 immunoreactivity, as did Schwann cells. We conclude that VEGF has both neurotrophic and mitogenic activity on cells in the peripheral nervous system.