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Inflammatory cells and mediators in the silicone chamber model for regeneration
Abstract
In the present study the inflammatory response was quantitatively evaluated during peripheral nerve regeneration. The fluid from silicone nerve regeneration chambers, inserted in rats, was collected during the early period of regeneration of transected sciatic nerves (6 h-7 d) and analysed with respect to inflammatory cells and mediators (leukotriene B4, LTB4, and interleukin-1 alpha, IL-1 alpha). Leucocytes were detected during the entire period (up to 7 d after implantation). The highest concentration was detected after 24 h. PMNG (polymorphonuclear granulocyte) was the predominant cell type in the chamber fluid during the initial 5d of regeneration. Analysis of the concentration of LTB4 demonstrated two peaks (at 24 h and 5 d). The IL-1 alpha concentration displayed an early and relatively smaller peak after 24 h and a second and much larger peak after 7 d, concomitant with an increase of the number of mononuclear cells.
... Peripheral nerve transection disrupts contact and communication between the neuron and its target, but the gap between the stumps can be bridged by implanting a tubular prosthesis thereby allowing axonal regeneration and target reinnervation [6]. If the tube is left empty, the gap is eventually filled with a plasma like fluid that polymerizes to form a fibrin bridge connecting the stumps [5]. Non-neuronal cells migrate from proximal and distal stumps and precede axonal sprouting and regeneration [5,11,12]. ...
... If the tube is left empty, the gap is eventually filled with a plasma like fluid that polymerizes to form a fibrin bridge connecting the stumps [5]. Non-neuronal cells migrate from proximal and distal stumps and precede axonal sprouting and regeneration [5,11,12]. However, if the gap between the stumps is filled with a substance such as collagen or laminin, the initial steps of regeneration can be accelerated, resulting in an increased number of regenerated axons [4,10,19]. ...
After axotomy, regeneration can be enhanced by bridging the transected nerve with a biocompatible tube, and the effect of trophic substances or molecules from the extracellular matrix can be investigated by filling the prosthesis. In this study, we assessed the importance of the molecular organization and aggregational state of collagen type I in axonal regeneration and guidance. Two types of collagen were used, namely, a collagen gel derived from bovine tendon that displays supraorganization after extrusion, and collagen from rat tail which does not self-organize under such conditions. Adult male Wistar rats were divided into four groups. In the first group (n=3), the polyethylene tube was filled with bovine collagen, while in the second (n=3), the prosthesis was filled with rat-derived collagen. In the third group (n=3), the tube was left empty, and the fourth group (n=3), consisted of unoperated rats. Six weeks after tubulization, the number of axons was significantly higher with bovine collagen than with rat collagen (7,661 ± 1,018 versus 4,110 ± 1,027, p
... Nerve guidance channels have been synthesized from a wide assortment of natural and synthetic polymers ( Table 1). The channel serves to prevent the ingrowth of fibrous scar tissue, to concentrate neurotrophic molecules released from the injured nerve stumps, and to direct growth from the proximal to the distal nerve stump (Danielsen et al., 1993;Longo et al., 1983;Lundborg et al., 1982;Williams et al., 1983). The dimensions, material of construction, and luminal components all affect the regenerative capacity of a given nerve guidance channel design; see Figure 1 for a depiction of critical design parameters integral for nerve guidance channel function. ...
The highly debilitating nature of spinal cord injuries has provided much inspiration for the design of novel biomaterials that can stimulate cellular regeneration and functional recovery. Many experts agree that the greatest hope for treatment of spinal cord injuries will involve a combinatorial approach that integrates biomaterial scaffolds, cell transplantation, and molecule delivery. This manuscript presents a comprehensive review of biomaterial-scaffold design strategies currently being applied to the development of nerve guidance channels and hydrogels that more effectively stimulate spinal cord tissue regeneration. To enhance the regenerative capacity of these two scaffold types, researchers are focusing on optimizing the mechanical properties, cell-adhesivity, biodegradability, electrical activity, and topography of synthetic and natural materials, and are developing mechanisms to use these scaffolds to deliver cells and biomolecules. Developing scaffolds that address several of these key design parameters will lead to more successful therapies for the regeneration of spinal cord tissue.
... Most of the experimental and clinical data support the view that an autologous nerve with blood vessels is the best material to bridge the gaps (Millesi et al., 1972), but the length of available nerve grafts is limited and a transplanting nerve donor is required. Another possibility to bridge nerve gaps is the use of tubes made with nonhomotopic biological material such as decalcified bone, or artificial materials such as nylon fiber, silicone or polyurethane (Lundborg et al., 1982;Danielsen et al., 1993). Despite the fact that they can provide tubular support for nerve regeneration, they cannot be degraded or absorbed in vivo, requiring a second surgical procedure to withdraw them. ...
We evaluated peripheral nerve regeneration using a tubular nerve guide of resorbable collagen filled with either bone marrow-derived cells (BMDCs) in Dulbecco's cell culture medium (DMEM) or with DMEM alone (control). The control group received just the culture medium (vehicle). The left sciatic nerves of ten isogenic mice were transected and the tubular nerve guides were sutured to the end of the proximal and distal nerve stumps. Motor function was tested at 2, 4 and 6 weeks after surgery using the walking track test. The pawprints were analyzed and the print lengths (PL) were measured to evaluate functional recovery. After 6 weeks, mice were anesthetized, perfused transcardially with fixative containing aldehydes, and the sciatic nerves and tubes were dissected and processed for scanning and transmission electron microscopy. Scanning electron microscopy of the collagen tube revealed that the tube wall became progressively thinner after surgery, proving that the tube can be resorbed in vivo. Quantitative analysis of the regenerating nerves showed that the number of myelinated fibers and the myelin area were significantly increased in the experimental group. Also, motor function recovery was faster in animals that received the cell grafts. These results indicate that the collagen tube filled with BMDCs provided an adequate and favorable environment for the growth and myelination of regenerating axons compared to the collagen tube alone.
The central nervous system, in which the spinal cord is included, has a limited capacity of self-regeneration. Therefore, diseases affecting the spinal cord normally have a poor prognosis. This highlights the necessity of developing innovative therapies targeting spinal cord regeneration. Accordingly, tissue engineering has been on the forefront of promoting regeneration in several tissues and organs through the development of scaffolds enabling the growth and proliferation of transplanted and/or endogenous cells. In this chapter, it is intended to provide an overview of the most recent and auspicious scaffold applications to enhance spinal cord regeneration. Focus will be given to natural (extracellular matrix-derived or not), synthetic, and other relevant scaffold compositions. The advantages and disadvantages of each type of formulation together with an insight about the future perspectives of the field will also be discussed.
Pain that arises from disease in which damage to, or disease involving, peripheral sensory nerves remains one of the most difficult clinical problems faced by physicians. Despite advances in our understanding of what might cause this pain, fully effective treatment evades us. Much of this difficulty is due both to a lack of understanding of the diverse origins of this type of pain and the specific mechanisms that apply in each instance. Although the development of clinically relevant models of nerve injury pain has improved our understanding of the possible causes of this pain, additional research will be needed to establish the importance of these findings for humans.
Injuries that result in the loss of limb functionality may be caused by the severing of the peripheral nerves within the affected limb. Several bioengineered peripheral nerve scaffolds have been developed in order to provide the physical support and topographical guidance necessary for the naturally disorganized axon outgrowth to reattach to distal nerve stumps as an alternative to other procedures, like nerve grafting. PDMS has been chosen for the base material of the scaffolds due to its biocompatibility, flexibility, transparency, and well-developed fabrication techniques. The process of observing the axon outgrowth across the nerve gaps with PDMS scaffolds has been challenging due to the limited number and fineness of longitudinal sections that can be extracted from harvested nerve tissue samples after implantation. To address this, multilayer microchannel scaffolds were developed with the object of providing more refined longitudinal observation of axon outgrowth by longitudinally ‘sectioning’ the device during fabrication, removing the need for much of the sample preparation process. This device was then implanted into the sciatic nerves of Lewis rats, and then harvested after two and four weeks to analyze the difference in nerve regeneration between two different time periods. The present layer by layer structure, which is separable after nerve regeneration and is treated as an individual layer during the histology process, provides the details of biological events during axonal regeneration. Confocal microscopic imaging showed the details of peripheral nerve regeneration including nerve branches and growth cones observable from within the microchannels of the multilayer PDMS microchannel scaffolds.
The use of tubes as an alternative to primary nerve suture in fresh nerve transections has been introduced as a biologic approach to nerve injuries, creating optimal conditions for axonal regeneration over a short empty space intentionally created between the proximal and distal nerve ends. The idea may seem controversial and has been criticized using the arguments that silicone in itself may create problems like inflammation and the tube may compress the nerve ends. With the use of appropriately sized tubes for bridging a maximum 5-mm gap in human median and ulnar nerves, the authors have found the technique to be useful and persistent at follow-up examinations for up to 4 to 5 years. In addition, from the intellectual point of view, the principle illustrates the concept by which emphasis is placed on the intrinsic healing capacities of the nerve rather than on the technical skill of the surgeon. The thin mesothelial lining found around the silicone tube lacks primary inflammatory signs at follow-up after 1 year, and no signs of compression are seen. It may be an advantage because it allows sliding of the repair site against the surrounding tissues. Tubes made of bioresorbable material may seem ideal, but they may introduce new problems associated with the resorption process in terms of a substantial unrestricted macrophage invasion, fibrosis, and disorganized axonal growth. For an extended nerve defect, the use of autologous nerve grafts is still the gold standard, because no tubular conduit or other conduit has so far proved equal to autologous nerve grafts, at least not for reconstruction of human median and ulnar nerve trunks. Alternatives other than tubes are currently being developed and investigated. For the future, the use of tubes for repair and reconstruction of nerves may have interesting potentials, because such a structure allows several types of tissue engineering. Various matrices containing, for instance, appropriate cells, factors, or other stimulating agents can be introduced in the tube lumen and can also be incorporated in a slow-release form in the walls of the tube and manipulated. Cultured Schwann cells or other cellular components, with or without manipulated production machinery, are probably the cells of choice for introduction in the tubes. Tubes may thus prove to be interesting alternatives to conventional repair techniques for primary repair of nerves and for reconstruction of segmental defects and for neuroma treatment in the future.
Restoration of function following complete nerve injuries with subsequent nerve repair is still not satisfactory and in many cases poor, especially when a gap has to be bridged by a graft. In such situations, there may be insufficient access to autologous graft material. Alternatives have to be developed and a close collaboration between basic scientists and clinicians is required. In the present article, current studies on experimental nerve grafts are discussed and some new alternatives to autologous nerve grafts are reviewed.
A peripheral nerve injury has a profound impact on the patient’s life and such an injury causes high costs for society. A peripheral nerve injury is a very complex injury because it involves both peripheral and central components of the nervous system all the way from targets at muscle and skin receptors up to the brain. In the neuron and its supporting cells—mainly Schwann cells—there are numerous steps in intracellular signaling, signal transduction, that are initiated by the injury with the aim of putting the neuron and the Schwann cells into a regenerative and proliferative condition, respectively. The present article focuses on the basic reactions of the neuron and the Schwann cells during injury and regeneration from a neurobiologic viewpoint. Greater future understanding about the signal transduction mechanisms in neurons and Schwann cells will help to develop pharmacologic strategies in addition to the current surgical treatment options.
Changes in the distribution of the magnetic resonance (MR)-observable brain metabolites N-acetyl aspartate (NAA), total choline (Cho), and total creatine (Cre), following mild-to-moderate closed-head traumatic brain injury (mTBI) were evaluated using volumetric proton MR spectroscopic imaging (MRSI). Studies were carried out during the subacute time period following injury, and associations of metabolite indices with neuropsychological test (NPT) results were evaluated. Twenty-nine subjects with mTBI and Glasgow Coma Scale (GCS) scores of 10-15 were included. Differences in individual metabolite and metabolite ratio distributions relative to those of age-matched control subjects were evaluated, as well as analyses by hemispheric lobes and tissue types. Primary findings included a widespread decrease of NAA and NAA/Cre, and increases of Cho and Cho/NAA, within all lobes of the TBI subject group, and with the largest differences seen in white matter. Examination of the association between all of the metabolite measures and the NPT scores found the strongest negative correlations to occur in the frontal lobe and for Cho/NAA. No significant correlations were found between any of the MRSI or NPT measures and the GCS. These results demonstrate that significant and widespread alterations of brain metabolites occur as a result of mild-to-moderate TBI, and that these measures correlate with measures of cognitive performance.
A low-hemorrhage-risk surgical approach to expose the optic nerve (ON) in rabbits through the orbital process of the frontal bone without removal of the bony orbit and resection of the rectus muscle was explored and assessed in this study. This approach will be used to investigate a new visual prosthesis that requires intraorbital ON stimulation with penetrating electrodes. Animals Chinese Albino rabbits (n = 10).
Rabbits were classified into a surgery and a control group (five in each). In the surgery group, the ON exposure was explored by the newly proposed surgical approach. Surgical time, blood loss, visually evoked potentials (VEP) at four different scheduled time points, and H&E-stained histology of the ON at one month after surgery were recorded and analyzed to assess the ease and safety of the approach.
The average surgical time for the ON exposure was 16.40 +/- 1.14 min with average blood loss of 0.52 +/- 0.08 mL. Within the one-month follow-up, the ON exhibited a naturally reversible conduction change in terms of VEP amplitude. Histological examination of the ON was unremarkable. A postoperative mild ptosis of the surgical eye resolved within one month after surgery.
The ease and safety of this new surgical approach allowed it to be easily used by non-expert operators and widely applied in rabbit experiments for various research purposes requiring exposure of the ON.
In the present study, we determined the regeneration rate and the initial delay in rat sciatic nerve grafts first made hypercellular by predegeneration then acellular by freeze-thawing. 7-day predegenerated nerve pieces from the distal nerve stump on the right side were made acellular by repeated freeze-thawing and inserted as grafts into a 10-mm long freshly created defect on the left contralateral side. Freshly made (no predegeneration period) acellular nerve grafts were used as controls. Both types of grafts supported outgrowth of regenerating axons as demonstrated by the sensory pinch test. However, the predegenerated acellular nerve grafts had a significantly shorter initial delay period (2.7 days) as compared with freshly made acellular nerve grafts (9.5 days). The initial delay period for predegenerated acellular nerve grafts was similar to that for fresh cellular nerve grafts but significantly longer than that for predegenerated cellular nerve grafts [24]. The rate of regeneration appeared independent of the type of grafts used. We suggest that modifications of the basal lamina and/or factors produced during the predegeneration period by non-neuronal cells survive the freeze-thawing cycle and account for the decrease in the initial delay period.
The importance of cell invasion for regeneration in nerve segments was investigated in rats. The regeneration distance of axons in predegenerated nerve segments was compared to the outgrowth in nerve segments where cell invasion had been prevented. A 10 mm long nerve segment, which was predegenerated (preserved or impaired blood circulation) or kept in a Millipore chamber (pore size 0.22 microns), was sutured as a nerve graft at the contralateral side three days or two weeks after the initial procedure. At two weeks immunocytochemical staining and routine histologic analysis revealed pronounced myelin breakdown and presence of ED1 and ED2 positive macrophages in the predegenerated nerve segment. Nerve segments, which were kept in the Millipore chamber, showed no invasion of macrophages and the myelin sheaths were preserved. The regeneration distances of axons in the nerve segments, evaluated with the pinch reflex test, were significantly longer in the predegenerated nerve segments compared to the nerve segments kept in Millipore chambers. Nerve grafts, which were taken from predegenerated nerves with intact blood circulation, showed the longest regeneration distances. It is suggested that the regeneration process can be impaired in nerve segments where cell and macrophage invasion as well as myelin breakdown are prevented and that preservation of the blood circulation during the degeneration process is important.
In the present study we tested how nerve grafts with different pre-degeneration periods (1-28 days) influenced the early regenerative response in the rat sciatic nerve. The sciatic nerve on the right side was crushed and after 1-28 days of pre-degeneration, a 10 mm segment was used as an autologous nerve graft and transposed to a freshly made 10 mm long nerve defect on the left side. The regeneration distance was measured by the sensory pinch test 2-10 days after nerve repair. A newly developed mathematical model was used to calculate regeneration rates and initial delay periods from the measured regeneration distances. Pre-degenerated nerve grafts improved nerve regeneration by decreasing the initial delay period as compared to fresh nerve grafts without affecting the regeneration rate. Only one day of pre-degeneration was sufficient to reduce the initial delay period from 3.6 days to 1.7 days. The maximal effect on the initial delay period was achieved after 3 days of pre-degeneration. The initial delay period at later pre-degeneration intervals (7-14 days) was about 1 day. The effect persisted for at least 28 days of pre-degeneration. The regeneration rate was 1.5 mm/day for fresh nerve grafts and between 1.8-2.1 mm/day for pre-degenerated grafts. The results suggest that the effects of pre-degeneration are not only due to the increased cell proliferation in the graft, but that also trophic and/or inflammatory mechanisms may be of importance. Grafts pre-degenerated by crush may have clinical implications since they are easy to perform if an elective nerve grafting procedure is planned.
It is important to develop methods which increase nerve regeneration since restoration of function following injury to peripheral nerves often requires outgrowth of the injured axons over long distances. In this study, axonal outgrowth after bilateral crush injury to the sciatic nerve of the rat was measured. One group with large-diameter nonpermeable silicone tubes and one group with large diameter permeable silicone tubes applied around the crush site on one side had regeneration following nerve injury compared to controls on the other side. The length of regeneration of the regenerating axons were then measured 4, 5, and 6 days following the crush injury using the "pinch reflex test." The presence of axons at the pinch level was confirmed by immunocytochemical staining for neuro-filaments. The length of regeneration for rats with nonpermeable tubes was significantly greater than that of the contralateral control side and was so at all times (p < 0.05). The effect was present but not that pronounced where permeable tubes were used. We conclude that the outgrowth of regenerating sensory axons after sciatic nerve crush injury in the rat can be increased by enclosing the regeneration site in a silicone tube. The observed effect may be due to local mechanisms such as macrophage invasion or prevention of rapid wash-out of fluid from the crush zone.
Forty-four rat sciatic nerves with partial defects were repaired with a silicone chamber. Each partial defect was created by resecting a 10 mm segment from the tibial fascicle leaving the peroneal fascicle intact. The proximal and the distal stumps of the tibial fascicle together with the intact peroneal fascicle were encased in a single silicone chamber. After seven days a fibrin matrix had surrounded the peroneal fascicle and spanned the defect between the tibial stumps. This matrix was later invaded by non-neuronal cells and regenerating axons. Non-myelinated nerve fibres had almost regenerated across the defect by 16 days. The tetanic force of the gastrocnemius muscle 120 days after repair showed 80% recovery, which was no different from that of partial defects repaired with conventional nerve grafts. The results suggest that the silicone chamber technique could be applicable to the treatment of partially transected nerve trunks.
In the rat sciatic nerve, a gap of around 10 mm in nerve continuity seems to be the maximal distance which can be successfully repaired by silicone tubes. In this study we tested if a new artificial nerve graft, composed of eight polyamide filaments (diameter 250 microns) placed inside silicone tubes (1.8 mm inner diameter), could be used to bridge an extended gap (15 mm) in rat sciatic nerve. Silicone tubes containing eight polyamide sutures were found to support regeneration across such a gap. After 4 weeks sensory fibres had bridged the gap and grown into the distal nerve segment as revealed by a positive pinch reflex test as well as positive staining for neurofilaments in the distal nerve segment. Myelinated axons could be observed in the tissue matrix formed in between and peripheral to the synthetic filaments along the whole length of the tube. In contrast, when silicone tubes without filaments were used to bridge the 15 mm gap, the tubes contained only fluid or in two cases a thin tissue strand. No positive pinch reflex response was elicited in the nerve segment distal to such a tube. We conclude that the new artificial nerve graft can be used to support regeneration across extended gaps in nerves.
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.
Nerve injury pain remains a complex clinical challenge. Although the development of animal models of nerve injury pain has aided our understanding of potential pathophysiologic mechanisms for this condition, effective treatment still remains beyond our reach. Several classes of agents appear to block pain behavior in these animal models and humans, but they are often limited in their use by low efficacy, or undesirable side-effects. A prerequisite for the improvement of nerve injury pain includes the development of clinically-relevant animal models in which therapeutic targets can be identified.
To test if fabricated gelatin conduits can be used to bridge nerve inter-stump gaps and support regeneration.
Experimental laboratory study.
Department of Anatomy and Neurobiology, Graduate School of Medicine, Kyoto University, Japan.
Twenty-four adult mice.
Mouse's sciatic nerve was resected and both proximal and distal nerve stumps sutured into each end of a gelatin conduit, to bridge a 7-mm gap.
Nerve regeneration.
At one week post-implantation, a scaffolding fibrin matrix containing few mononuclear cells formed inside the conduit. At three weeks, a well regenerated nerve composed of myelinated and unmyelinated axons, associated Schwann cells and surrounding perineurial sheath bridged the gap.
Biodegradable gelatin conduits direct and support nerve regeneration and are therefore promising tools for use in entubulization repair of nerve defects.
In spite of an enormous amount of new experimental laboratory data based on evolving neuroscientific concepts during the last 25 years peripheral nerve injuries still belong to the most challenging and difficult surgical reconstructive problems. Our understanding of biological mechanisms regulating posttraumatic nerve regeneration has increased substantially with respect to the role of neurotrophic and neurite-outgrowth promoting substances, but new molecular biological knowledge has so far gained very limited clinical applications. Techniques for clinical approximation of severed nerve ends have reached an optimal technical refinement and new concepts are needed to further increase the results from nerve repair. For bridging gaps in nerve continuity little has changed during the last 25 years. However, evolving principles for immunosuppression may open new perspectives regarding the use of nerve allografts, and various types of tissue engineering combined by bioartificial conduits may also be important. Posttraumatic functional reorganizations occurring in brain cortex are key phenomena explaining much of the inferior functional outcome following nerve repair, and increased knowledge regarding factors involved in brain plasticity may help to further improve the results. Implantation of microchips in the nervous system may provide a new interface between biology and technology and developing gene technology may introduce new possibilities in the manipulation of nerve degeneration and regeneration.
Silicone tubes of appropriate sizes were used to enclose the injured zone of transsected ulnar and median nerves in the human forearm as an alternative to conventional microsurgical repair of the nerve trunk. A gap measuring 3-5 mm was left intentionally between the nerve ends inside the tube. The clinical early results from a prospective randomised study that compared these two principles have recently been presented. Seven patients (five men and two women), aged 15-49 years (median 20) were reexplored 12-44 months (median 22) after the initial procedure because of local discomfort from the tube in four patients. There was a new nerve structure bridging the former gap and in most cases it was impossible to distinguish the site of the injury. In all cases there was a thin capsule around the silicone tube that microscopically consisted of connective tissue with thin walls and no signs of inflammation, granuloma or macrophages (n = 4), while in two cases a mild foreign body reaction was seen at a single site (n = 1) or at patchy areas (n = 1). These results indicate that after more than one year there is a limited tissue reaction around silicone tubes used to repair median and ulnar nerves in humans.
The functional outcome of microsurgical repair of divided nerves is disappointing since many regenerating axons fail to reach appropriate targets. Sorting of regenerating axons according to target tissue might be used to improve functional regeneration. The aim of the present study is to see if regenerating axons can be sorted into functionally different bundles with target-derived molecules. The proximal stump of the adult rat sciatic nerve was sutured into the inlet of a silicon Y-tube. The two branches of the Y-tube were filled with agarose primed with filtrates prepared from skin and muscle homogenates from the operated rat. The tibial and sural nerves were inserted in the two branches of the Y-tube. Six weeks later the sciatic nerve axons showed vigorous regeneration into both branches. Electron microscopic examination of regenerated nerve segments showed numerous myelinated and unmyelinated axons. The proportion of myelinated axons was significantly larger in the muscle-gel branch than in the skin-gel branch. Retrograde tracing from the nerve regenerates with Fast Blue and Fluoro-Ruby showed that ventral horn neurons at L4-L5 segmental levels were preferentially labeled from the muscle-gel branch. Neurons in corresponding dorsal root ganglia were labeled from both Y-tube branches (no significant numerical difference). A few neurons of both types contained both tracers. Measurements revealed that sensory neurons labeled from the muscle-gel branch were significantly larger (mean perikaryal area 870 microm(2)) than neurons labeled from the skin-gel branch (mean area 580 microm(2)). We conclude that regenerating motor and sensory axons can be sorted with target-derived molecules.
Vasopressin (antidiuretic hormone) is emerging as a potentially major advance in the treatment of a variety of shock states. Increasing interest in the clinical use of vasopressin has resulted from the recognition of its importance in the endogenous response to shock and from advances in understanding of its mechanism of action. From animal models of shock, vasopressin has been shown to produce greater blood flow diversion from non-vital to vital organ beds (particularly the brain) than does adrenaline. Although vasopressin has similar direct actions to the catecholamines, it may uniquely also inhibit some of the pathologic vasodilator processes that occur in shock states.
There is current interest in the use of vasopressin in the treatment of shock due to ventricular fibrillation, hypovolaemia, sepsis and cardiopulmonary bypass. This article reviews the physiology and pharmacology of vasopressin and all of the relevant animal and human clinical literature on its use in the treatment of shock following a MEDLINE (1966–2000) search.
After injury, axonal regeneration occurs across short gaps in the peripheral nervous system, but regeneration across larger gaps remains a challenge. To improve regeneration across extended nerve defects, we have fabricated novel microfilaments with the capability for drug release to support cellular migration and guide axonal growth across a lesion. In this study, we examine the nerve repair parameters of non-loaded filaments. To examine the influence of packing density on nerve repair, wet-spun poly(L-Lactide) (PLLA) microfilaments were bundled at densities of 3.75, 7.5, 15, and 30% to bridge a 1.0-cm gap lesion in the rat sciatic nerve. After 10 weeks, nerve cable formation increased significantly in the filament bundled groups when compared to empty-tube controls. At lower packing densities, the number of myelinated axons was more than twice that of controls or the highest packing density. In a consecutive experiment, PLLA bundles with lower filament-packing density were examined for nerve repair across 1.4- and 1.8-cm gaps. After 10 weeks, the number of successful regenerated nerves receiving filaments was more than twice that of controls. In addition, nerve cable areas for control groups were significantly less than those observed for filament groups. Axonal growth across 1.4- and 1.8-cm gaps was more consistent for the filament groups than for controls. These initial results demonstrate that PLLA microfilaments enhance nerve repair and regeneration across large nerve defects, even in the absence of drug release. Ongoing studies are examining nerve regeneration using microfilaments designed to release neurotrophins or cyclic AMP.
The use of autologous nerve grafts for bridging defects in nerve continuity requires the sacrifice of healthy nerves. Development of alternatives to autologous nerve grafts is therefore well motivated. Such "bioartificial nerve grafts" can be tissue-engineered on the basis of the use of a stroma/matrix/scaffold acting in concert with cells and neurotrophic factors. Such a scaffold can be of synthetic or biological nature and can be resorbable or non-resorbable. It should act together with Schwann cells which can be pre-cultured or acutely dissociated. Neurotrophic factors, stimulating axonal growth, can be incorporated in the scaffold and can also be supplied by cells which are seeded into the stroma. So far, tissue-engineered nerve conduits have been used mainly for experimental purposes in experimental animals. However, an increasing amount of experience from the clinical use of alternatives to nerve grafts now exists, mainly tubes made of silicone or poly-glycolic acid (PGA), veins, collagen and muscle basal laminae.
The long-term outcome from silicone tube nerve repair was compared with the outcome from routine microsurgical repair in a clinical randomized prospective study, comprising 30 patients with median or ulnar nerve injuries in the distal forearm. Postoperatively, the patients underwent neurophysiological and clinical assessments of sensory and motor function regularly over a 5-year period. After 5 years there was no significant difference in outcome between the two techniques except that cold intolerance was significantly less severe with the tubular technique. In the total group there was ongoing improvement of functional sensibility throughout the 5 years after repair. It is concluded that tubular repair of the median and ulnar nerves is at least as good as routine microsurgical repair, and results in less cold intolerance.
The inflammatory response to nickel chromium (NiCr), cobalt chromium (CoCr), and titanium (Ti) implants at 7 and 28 days was investigated using real-time PCR analysis along with histological and immunohistochemical staining. Contrasting inflammatory profiles were found in response to the different metal compositions. The inflammatory profile induced by CoCr remained consistent and elevated during the 28-day period with high cell counts associated with the implants and a progressive recruitment of T lymphocytes. The response to NiCr was also elevated, but with an initially low T-lymphocyte infiltration that increased by the later time period. Ti indicated an early increased inflammatory response that had reduced by 28 days. Changes in gene expression demonstrated that Ti induced very low levels of expression of the three inflammatory cytokine genes. NiCr initiated a significant upregulation in gene expression for IL-6 and TNF-alpha. CoCr resulted in the highest upregulation of IL-2 indicative of T-lymphocyte activation to this material.
The efficiency of denatured muscle grafting in nerve repair has been confirmed in experimental models and animals. The first clinical trials to repair digital nerves and mixed sensory-motor nerves were encouraging regarding sensory recovery but motor recovery was poor, probably because of delayed repair. We present the functional outcome of repair of motor nerves using denatured muscle graft and compare the results with those using standard nerve graft techniques.
This prospective study included 9 radial nerve defects repaired with denatured muscle grafts and 23 radial nerve defects repaired using nerve grafts. Missile induced nerve injury, mid-arm level of lesion, a nerve gap smaller than 6 cm, and a preoperative interval of less than 5 months were characteristics shared by all patients. None of the patients had concomitant vascular injury, severe scarring, or significant soft tissue damage in the region of nerve repair. Motor recovery was estimated with 0-5 points, at least 4.7 years after surgery, according to the BMRC scale.
A successful outcome (>or=M3) was achieved in 7 out of the 9 patients treated using a muscle graft and in 21 out of the 23 patients treated using nerve grafts (P > 0.05). Excellent recovery and the clinically significant re-establishment of thumb extension (M5 grade) were never achieved in the patients treated using muscle grafts. The average motor score was significantly better in patients treated with nerve grafts than in those who received muscle grafts (3.8 +/- 0.9 and 3.2 +/- 0.8; P = 0.035). With the patients who received muscle grafts, an inverse correlation existed between motor recovery and the length of the nerve gap (P = 0.017).
Denatured muscle grafts can be useful for bridging short radial nerve defects, but the quality of recovery is significantly worse than after nerve graft repair. Even if relatively short nerve defects are bridged with denatured muscle grafts, the outcomes correlate inversely with the length of the gap.
There has been no comprehensive study that maps the production of the range of inflammatory cytokines following implantation of a material. There is an urgent requirement for specific data on the real time production of biological markers in order to study their effects in vitro and more accurately predict the in vivo response. This study determined the production of IL-1beta, IL-2, IL-4, IL-6, IL-10, IFNgamma and TNF-alpha in response to a synthetic material implanted in a rat soft tissue model for up to 90 days. IL-1 beta and TNF-alpha were elevated over the total experimental time course with values in the order of 500 pg/ml for IL-1beta and 40 pg/ml for TNF-alpha. The cytokines IL-2, IL-4, IL-6 and IL-10 were also detected and their production reduced with increasing time.
Although crushed axons in a dorsal spinal root normally regenerate more slowly than peripheral axons, their regeneration can be accelerated by a conditioning lesion to the corresponding peripheral nerve. These and other observations indicate that injury to peripheral sensory axons triggers changes in their nerve cell bodies that contribute to axonal regeneration. To investigate mechanisms of activating nerve cell bodies, an inflammatory reaction was provoked in rat dorsal root ganglia (DRG) through injection of Corynebacterium parvum. This inflammation enhanced regeneration in the associated dorsal root, increasing 4-fold the number of regenerating fibers 17 d after crushing; peripheral nerve regeneration was not accelerated. A milder stimulation of dorsal root regeneration was detected after direct injection of isogenous macrophages into the ganglion. It is concluded that changes favorable to axonal regeneration can be induced by products of inflammatory cells acting in the vicinity of the nerve cell body. Satellite glial cells and other unidentified cells in lumbar DRG were shown by thymidine radioautography to proliferate after sciatic nerve transection or injection of C. parvum into the ganglia. Intrathecal infusion of mitomycin C suppressed axotomy-induced mitosis of satellite glial cells but did not impede axonal regeneration in the dorsal root or the peripheral nerve. Nevertheless, the similarity in reactions of satellite glial cells during 2 processes that activate neurons adds indirect support to the idea that non-neuronal cells in the DRG might influence regenerative responses of primary sensory neurons.
The Schwann cells and fibroblast-like cells of the intact sciatic nerve of adult rats synthesize very little nerve growth factor (NGF). After lesion, however, there is a dramatic increase in the amounts of both NGF-mRNA and NGF protein synthesized by the sciatic non-neuronal cells. This local increase in NGF synthesis partially replaces the interrupted NGF supply from the periphery to the NGF-responsive sensory and sympathetic neurons, whose axons run within the sciatic nerve. Macrophages, known to invade the site of nerve lesion during wallerian degeneration, are important in the regulation of NGF synthesis. Here we demonstrate that the effect of macrophages on NGF-mRNA levels in cultured explants of sciatic nerve can be mimicked by conditioned media of activated macrophages, and that interleukin-1 is the responsible agent.
Interleukin 1 (IL-1) is a protein with several biological activities regulating host defense and immune responses. We report here the isolation of human IL-1 cDNA. It encodes a precursor polypeptide of 269 amino acids (30,747 Mr). mRNA isolated by hybridization to this cDNA was translated in a reticulocyte cell-free system, yielding immunoprecipitable IL-1. Furthermore, this hybrid-selected mRNA was injected into Xenopus laevis oocytes, which subsequently secreted biologically active IL-1. The cDNA nucleotide sequence suggests that IL-1 is initially translated as a precursor molecule that is subsequently processed into the 15,000-20,000 Mr protein usually associated with IL-1 activity.
We studied the effects of leukotrienes on in vitro functions of neutrophil polymorphonuclear (PMN) granulocytes. Leukotriene B4 (LTB4) evoked a stimulated and directed migration of neutrophils under agarose with an optimum concentration of 10(-6)M, whereas two nonenzymatically formed isomers (compounds I and II) induced this response at 10(-5)M. Leukotriene C4 (LTC4) and 5-hydroxyeicosate-traenoic acid (5-HETE) did not affect this PMN migration. At the same optimum concentrations, LTB4 and compounds I and II augmented PMN adherence to nylon fibers. The chemotactic and adherence responses were of the same magnitude as with formal-Met-Leu-Phe (fMLP) at 10(-7)M. None of the leukotrienes influenced the spontaneous or phagocytosis-associated chemiluminescence or the ability to kill Staphylococcus aures. The cyclooxygenase inhibitor, indomethacin, inhibited only partly the fMLP-induced migration at high concentrations and stimulated migration at 2.5 x 10(-7)M, suggesting that arachidonic acid was then mainly metabolized by the lipoxygenase pathways. The lipoxygenase and cyclooxygenase inhibitor, eicosatetraynoic acid, inhibited both spontaneous and stimulated migration at greater or equal to 2.5 x 10(-5)M, but not at lower concentrations. Thus, since LTB4, and to a lesser degree compounds I and II, stimulated migration and adhesion, it is suggested that these mediators could be of importance for the emigration of neutrophils from blood vessels to areas of inflammation.
The production and mRNA expression of IL-1α and IL-1β by human monocytes was examined after two different stimuli, a protein kinase C (PKC) activator phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). LPS induced production of high levels of both IL-la and IL-1β protein (quantitated with type-specific ELISA assays), while after PMA stimulation only IL-1/β protein could be detected. The IL-1α and IL-1/β mRNA levels quantitated by Northern blotting were in line with the respective protein levels and nuclear run off analysis revealed that PMA did not activate the IL-la transcription. The production of the IL-1α and IL-1/β protein as well as the mRNA expression could be inhibited with protein kinase inhibitor H7, but not with HA 1004, indicating that PKC activation is essential for the activation of these genes. Thus these data indicate that PKC activation alone is sufficient for the induction of the IL-1/β gene, but some additional signals (provided by LPS) are required for the activation of the IL-1α gene.
The spatial-temporal progress of nerve regeneration was examined in silicone chambers of three different volume capacities: 11, 25, and 75 μl. In all chambers, the stumps of a transected rat sciatic nerve were sutured into the ends of the chamber leaving a 10 mm gap between the stumps. Chambers were implanted empty (E chambers) or prefilled with saline (PF chambers). A coaxial and continuous fibrin matrix had formed in all chambers by 1 week. In E chambers, the matrices had a proximal-distal taper that was more pronounced in E25 and E75 chambers due to significantly larger matrix diameters in the proximal region. At 3 weeks, vascular and Schwann cell migration and axonal regeneration were less advanced in the E25 and E75 than in the control E11 chambers. The retardation correlated with the presence of an avascular organization of circumferential cells. Saline prefill-ing affected the caliber and density of fibrin fibers in the 1 week matrices of PF25 and PF75 chambers. The matrices did not have a prominent taper and diameters were progressively larger with increasing chamber volume. Saline prefilling did not affect regeneration progress in 3 week PF11 chambers but did enhance regeneration in the PF25 chambers; a 1.5-fold larger diameter nerve formed at 3 weeks that contained 2,6-fold more axons. Progress in the PF75 chamber was retarded. We conclude that the volume, timing, and nature of the fluid filling a silicone chamber have significant influence on the formation of fibrin matrices. Alterations in matrix formation correlate with substantial changes in the subsequent progress of intrachamber regeneration events.
In 1972, Gery and co-workers1 detected a factor that promotes murine thymocyte proliferation in culture supernatants of human peripheral blood adherent leukocytes. This factor is active across species lines, does not support the growth of interleukin 2 (IL-2)-dependent lymphocyte lines, is produced by monocytic rather than lymphocytic leukocytes, and has subsequently been termed interleukin 1 (IL- 1)2. More recently, it has become evident that IL-1 activities can be produced by virtually every nucleated cell type and, in addition, IL-1 has been reported to have stimulatory effects on the growth and differentiation of numerous cell types. In this review, Joost Oppenheim and his colleagues discuss the biochemical characteristics, gene cloning, cell sources, biological properties and actions of IL-1, and give reasons why this pleitotropic, nonspecific hormone-like cytokine is of considerable concern to immunologists.
The presence of neuronotrophic factors (NTFs) in noninjured sciatic nerve extract and the course of their accumulation from 3 h to 30 days after nerve transection was examined. Rat sciatic nerves were transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers leaving a 10-mm interstump gap. Previous studies had shown that regeneration occurs in chambers containing both stumps but is absent in chambers lacking the distal stump. Chambers became completely filled with fluid 10 to 12 h after implantation. Fluid from chambers without nerve stumps (open-ended) implanted adjacent to nerve-containing chambers had markedly lower trophic activities than those containing one or both stumps. In fluid collected from chambers containing both proximal and distal nerve stumps, the highest titers of NTFs directed to sensory neurons were measured at 3 h posttransection whereas the highest titers of NTFs directed to sympathetic and spinal cord neurons were detected at 1 and 3 days, respectively. Chambers containing only the proximal or only the distal stumps showed similar temporal dynamics for sensory and sympathetic NTFs. Sensory and sympathetic neuronotrophic activity in extracts of proximal and distal stumps followed a similar temporal course to those in chamber fluid. Extracts of nonlesion nerve segments 5 mm from the transection site contained higher sensory and lower sympathetic trophic activity than extracts including the transection site. Spinal cord activity was undetectable in all extracts. Antiserum to nerve growth factor had no effect on fluid or extracts containing high sensory or sympathetic activities. These observations suggested that (i) some NTFs may be present in normal nerves and others may be synthesized or accumulated in response to nerve injury, (ii) sensory, sympathetic, and spinal cord NTFs are separate agents and immunochemically distinct from nerve growth factor, (iii) NTFs predominantly originate from nerve stumps rather than from surrounding fluid, and (iv) proximal and distal nerve stumps accumulate and release NTFs at similar rates.
Rat sciatic nerves can be transected and their proximal and distal stumps sutured into the openings of cylindrical silicone chambers. Anatomical regeneration has been demonstrated across 10 mm long chambers containing both stumps, although little or no axonal outgrowth occurs in chambers omitting the distal stump or exceeding the 10 mm length. We have previously shown that chambers containing both proximal and distal stumps accumulate within days of implantation a clear fluid containing neuronotrophic factors (NTFs) directed to neurons from neonatal mouse dorsal root ganglia. We report here that these chamber fluids also have considerable neuronotrophic activity for chick embryo neurons from embryologic day 4 (E4) lumbar spinal cord, E12 sympathetic ganglia, E12 (but not E8) dorsal root ganglia and E8 ciliary ganglia. Thus, the neuronal types supported by trophic factors of these fluids include all those which contribute axons to the sciatic nerve, namely sensory, spinal motor, and sympathetic. In fluid collected 1 week after implantation, NTF levels directed to different neurons varied independently from one another in chambers with different nerve insertions, suggesting that these activities reside in separate factors. Fluid collected from chamber arrangements allowing little proximal fiber regrowth did not always contain correspondingly lower titers of NTFs. However, generally higher titers of all NTFs were found in chambers containing either or both nerve stumps that in nerve-free chambers. Fluids collected from nerve-containing chambers were subjected to heat, dialysis or trypsin treatments. The behavior of their neuronotrophic activities suggests their association with proteins.
The biocompatibility of biomaterials at implant sites is controlled by the tissue/material interaction. A major cell in the tissue reaction is the macrophage. A summary is presented on macrophage mediation of cellular and humoral regulatory pathways in inflammatory and immune responses.
Murine peritoneal exudate cells (PEC), analyzed immediately after isolation, did not express detectable IL 1 activity or IL 1-specific mRNA. Stimulation of these cells by adherence induced the expression of intracellular, membrane, and extracellular IL 1 activities within 4 hr. Analysis of mRNA from these cells showed a concurrent induction of both IL 1 alpha and IL 1 beta mRNA within 1 hr. However, this stimulation of IL 1 expression was transient, since PEC cultured for 5 days no longer expressed IL 1 bioactivity or specific mRNA. Stimulation of these quiescent cells with bacterial lipopolysaccharide induced the re-expression of intracellular, membrane, and extracellular IL 1 activities as well as IL 1 alpha and IL 1 beta mRNA. We found no qualitative difference in the degree or rate of induction of IL 1 alpha compared with IL 1 beta mRNA. These results indicate that resting macrophages are IL 1 negative, and that the IL 1 inducing stimuli used in this study act transiently to increase the levels of IL 1 alpha and IL 1 beta mRNA.
Matrix formation within silicone tubes of different geometries implanted between the stumps of the transected rat sciatic nerve was studied. A matrix, composed of longitudinally oriented fibrin strands containing fibronectin, was formed within one day. The matrix then increased in size. The matrix contained macrophages and other inflammatory cells. Matrix size could be increased if the diameter of the tube was increased or if holes were made in the implanted tube. In contrast, matrix diameter decreased if the tube length was increased or if circulation was compromised in the inserts. The results suggest that the size, orientation and cellular components of the matrix have profound effects on the regenerative response of the transected nerve.
The silicone nerve regeneration chamber is a useful model to investigate the cellular and molecular events underlying successful regeneration in the peripheral nervous system. In this model a transected rat sciatic nerve with a 10-mm interstump gap, is repaired with a silicone chamber. The spatial-temporal sequence of regeneration in the silicone chamber has been examined in detail. The chamber rapidly becomes filled with fluid which contains neurotrophic activity for neurons in vitro. The second event to occur is the formation of a fibrin matrix connecting the two nerve stumps. This matrix is then invaded by cellular elements in the following order: perineurial-like cells, vasculature, Schwann cells, and axons. The silicone chamber model also allows manipulation of the regeneration process. Prefilling the chamber at the time of implantation with phosphate-buffered saline or dialyzed plasma stimulates nerve regeneration. Multiple injections into the chamber of a mixture containing laminin, testosterone, and ganglioside GM1 increase the size and the vascularization of the regenerate. Specially designed chambers divided into two compartments by a longitudinally inserted nitrocellulose strip have been used to examine the effects of substrate-bound trophic factors on nerve regeneration. Fibroblast growth factor containing chambers have an improved regeneration and vascularization as compared to controls.
The effects of application of a chromic catgut suture (conditioning lesion) placed close to the sciatic or tibial nerves on regeneration of the sciatic nerve after a crush lesion (test lesion), that had been induced after an appropriate conditioning interval (two or four weeks) were assessed. The catgut suture induced an inflammatory reaction around the nerve during the four weeks after application of the catgut suture (conditioning interval) but no signs of degeneration of nerve fibres were seen. There was a significant increase in length of outgrowth of sensory nerve fibres as measured by the pinch reflex test when the test lesion was applied after two and four weeks' exposure to the catgut suture. The rate of regeneration was increased by about 11% when the catgut suture had been applied for four weeks before the test lesion was made. The findings indicate that an inflammatory reaction around a peripheral nerve may act as a conditioning lesion, thereby stimulating regeneration of the nerve.
Plasma levels of interleukins 1, 2, 4 and 6 and tumor necrosis factor (TNF) were measured from 0 to 30 days in rats after a unilateral crush of the sciatic nerve at the level of the sciatic notch and after sham operations without nerve crush. Interleukin-6 was observed to peak and return to baseline levels within 24 h and remained at baseline for the duration of the experiment. An initial sharp rise in interleukin-1 and TNF was observed in all animals 1-2 days after the operation. A transient increase in interleukin-1 and TNF was also observed only in nerve-injured animals between 10 and 14 days after injury. A large increase in interleukin-2 appeared only in nerve-injured animals beginning at 11 days after injury and remained elevated for the remaining study period. No alterations in plasma interleukin-4 were observed at any time point. The experiments provide preliminary evidence for significant trauma-induced alterations in plasma cytokines which could provide a basis for some of the diffuse responses of peripheral neurons to trauma. The biphasic nature changes in plasma cytokines suggest that the immune system may participate in tissue reactions involved in the recovery from nerve injury.
The inflammatory reaction elicited after implantation of non-biologic materials was studied by analysis of the exudate in pure titanium or polytetrafluorethylene chambers 1-9 d after insertion in the abdominal wall of the rat. In chambers made of pure titanium, the number of leucocytes increased about twofold from 1.8 x 10(6)/ml to 4.1 x 10(6)/ml between 24 h and 6 d, whereas a larger increase from 2.1 x 10(6)/ml to 8.3 x 10(6)/ml was observed in the polytetrafluorethylene chambers. Irrespective of implant material, polymorphonuclear granulocytes constituted the vast majority of leucocytes (greater than 81%). After 24 h the leukotriene B4 content was slightly higher in polytetrafluorethylene chambers than in titanium chambers. In contrast to the titanium chambers, a marked increase in leukotriene B4 levels was detected in polytetrafluorethylene chambers 6 d after insertion, whereas the greatest amount of leukotriene B4 in titanium chambers was measured at 9 d. Interleukin 1 was only detected in both types of chambers after 6 d. The present study, together with previous findings, shows that the materials used in this study elicit different degrees of inflammatory reactions in the chamber exudate. Since the number of leucocytes in the exudate retrieved from the chambers correlated well with the leukotriene B4 levels, it is suggested that leukotriene B4, but not interleukin 1, may be an important mediator for polymorphonuclear granulocyte migration into the implant-tissue interface.(ABSTRACT TRUNCATED AT 250 WORDS)
LPS is known to be a potent activator of macrophages and induces the production of TNF-alpha and IL-1. However, the signaling events and regulatory mechanisms required for the activation of macrophages by LPS have not been resolved precisely. We show that LPS modulates its own response in macrophages. Proteose peptone-induced murine peritoneal macrophages (P-PEM) produce significant amount of TNF-alpha and IL-1 after stimulation with LPS. However, preexposure of macrophages to low doses (less than 1 ng/ml) of LPS renders them refractory to stimulation by a second round of LPS, as evaluated by production of TNF-alpha. The loss of sensitivity to a second round of LPS was selective for TNF-alpha production as the LPS-primed macrophages retained the ability to produce IL-1. Northern blot analysis was performed with total RNA obtained from control and LPS- (1 ng/ml) primed P-PEM after 3-h stimulation with a second round of LPS. The expression of TNF-alpha mRNA was inhibited in LPS-primed P-PEM, whereas the expression of IL-1 beta mRNA was the same in control and LPS-primed P-PEM, consistent with the data of biologic activities of these two cytokines. Zymosan-induced TNF-alpha production was the same in control and LPS-primed macrophages, indicating that not all of the pathways required for TNF-alpha production were affected by LPS priming. Monokines such as human (h) rIL-1 alpha, hrTNF-alpha, hrIL-6, and murine rIFN-beta could not substitute for the action of low doses of LPS, and addition of indomethacin could not restore TNF-alpha production. These results suggest that exposure of macrophages to low doses of LPS suppresses the production of TNF-alpha, but not of IL-1, by inhibiting the expression of mRNA through a noncyclooxygenase-dependent mechanism. Thus, LPS-induced production of TNF-alpha and IL-1 in macrophages are differently regulated.
Controversy has existed as to the ability of leukotriene B4 (LTB4) to enhance adhesive properties of human neutrophils (PMN) and endothelial cells. We found that LTB4 induced a rapid but transient adhesion of PMN to an albumin-coated plastic surface and to cultured human umbilical vein endothelial cells (HUVEC). Although the adhesive response of PMN to the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was longer lasting, peak hyperadherence was of similar magnitude as to LTB4 and was less susceptible to assay conditions. Adherence induced by either LTB4 or fMLP could be abrogated by the monoclonal antibody 60.3, indicating similar dependence on the leucocyte adhesion protein CD18. Lipoxin A did not induce PMN hyperadherence. Treating HUVEC with LTB4, but not with its omega-oxidized metabolites 20-OH- and 20-COOH-LTB4, lipoxin A, or with fMLP conferred a rapid, dose-related, enhanced adhesion of PMN. This effect was dependent on CD18 and on divalent cations. It disappeared with prolonged exposure to LTB4, required a metabolically active HUVEC, and was not due to passive binding of LTB4 to HUVEC. Thus, LTB4 induces a transient expression of hyperadhesiveness in HUVEC as well as in neutrophils, and both effects are dependent on expression of CD18.
The proximal and distal stumps of severed mouse sciatic nerves were inserted into opposite ends of polyethylene tubes. The tubes were implanted either empty or filled with collagen alone or in combination with interleukin-1 (IL1). Six weeks later, neurons in the L3-L5 dorsal root ganglia were back-filled with HRP. The number of HRP reactive sensory neurons detected in the IL1-treated animals was significantly greater than that seen in the other experimental groups. Thus, exogenous IL1 may partially mimic the effects obtained with in vivo administration of nerve growth factor in protecting sensory neurons from lesion-induced death.
A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.
Of the many factors determining host biocompatibility responses to implanted biomedical polymers, the cellular interactions at the tissue/material interface have been recognized to be some of the most important. The present study has combined results both from an in vitro cell culture system and from an in vivo animal model to examine this host response. In vitro results suggest that a variety of polymer materials can differentially activate human monocytes to produce a protein(s) having different biological activities. The polymers tested induce the production of the regulatory inflammatory protein interleukin 1 as well as a factor that enhances fibroblast proliferation and collagen synthesis. The observed activities of these factors appear to be related but not identical, and are dependent upon the specific polymer. Evaluation of exudate and tissue responses to these same polymer materials in an in vivo model are also presented. Both in vitro and in vivo results support the hypothesis that monocyte/macrophage activation with subsequent synthesis of regulatory factors such as interleukin 1 plays a significant role in determining the host response to biomedical polymer implants.
These studies involved the evaluation of human monocyte/macrophage activation by biomedical polymers coated with human blood proteins. The biomedical polymers were polyethylene, polydimethylsiloxane, woven Dacron fabric, expanded polytetrafluoroethylene, Biomer, and tissue culture treated polystyrene as the control. They were adsorbed with human blood proteins: albumin, fibrinogen, fibronectin, hemoglobin, and gamma globulin. The protein adsorbed polymers were evaluated for their potential to activate the monocyte/macrophage cellular population in vitro as assessed by the induction of the monocyte/macrophage inflammatory mediator, Interleukin 1 (IL1). Suppression of IL1 was observed when protein adsorbed polymers were compared to the appropriate protein adsorbed control. Protein adsorbed polymers, when compared to polymers without protein adsorption, stimulated IL1 production. The data presented in this manuscript show the level of induction and secretion of IL1 was dependent on the biomedical polymer and the protein adsorbed, as well as the requirement of lipopolysaccharide. These results show differential interactions occur between the proteins, monocytes/macrophages, and biomedical polymers which alter activation and induction of IL1.
The main left coronary artery of rats was ligated near its origin under light ether anaesthesia and the infarction observed for 48 h. The ischaemic area was determined after an intravenous injection of pontamine sky blue dye 1 h before induction of cardiac arrest with potassium chloride. The unstained area (true ischaemic area) decreased with time to 27.1% of the left ventricle at 48 h, whereas the intensely stained area between the normal and the true ischaemic areas increased with time, suggesting that blood was flowing to the border from the normal surrounding tissue. The infarcted area, identified by its lack of triphenyltetrazolium chloride staining, became evident after 3 h and stabilised at 12 h (42% of left ventricle). The polymorphonuclear leucocyte counts in the hearts, differentiated by staining of their chloroacetate esterase, increased gradually up to 5500 cells per section at 24 h. The leukotriene B4 concentration, determined by radioimmunoassay after freezing of the beating heart in liquid nitrogen, increased to eight times that of the sham operated hearts and peaked at 8 h (9.4(0.6) ng per heart, mean(SEM) n = 5) before the leucocyte counts reached their maximum. A single oral dose of a selective 5-lipoxygenase inhibitor (AA-861, 80 mg.kg-1, 1 h before ligation) lowered the leukotriene B4 concentration to that of the sham operated hearts and decreased the leucocyte count by 49.4% and 41.2% at 12 and 24 h respectively. The inhibitor also reduced the infarct size at 48 h by 34.4%. It was concluded that leukotriene B4, generated in ischaemic cardiac tissue, may increase infarct size through migration of polymorphonuclear leucocytes.
Two distinct but distantly related complementary DNAs encoding proteins sharing human interleukin-1 (IL-1) activity (termed IL-1 alpha and IL-1 beta), were isolated from a macrophage cDNA library. The primary translation products of the genes are 271 and 269 amino acids long, although expression in Escherichia coli of the carboxy-terminal 159 and 153 amino acids produces IL-1 biological activity.
The properties of specific human interleukin 1 (IL 1) receptors on human Epstein Barr virus-transformed B lymphocytes (EBV-B) were studied. Purified human IL 1-beta from a myelomonocytic cell line (THP-1) was labeled with 125I by the Bolton-Hunter method without detectable loss of biological activity. Among four EBV-B cell lines tested, a pre-B cell type (VDS-O) specifically bound the highest amount of 125I-IL 1-beta. Maximal binding was reached within 20 min at 4 degrees C. Scatchard plot analysis of the binding of 125I-IL 1-beta to VDS-O cells yielded a Kd (dissociation constant) of 2.4 to 5.9 X 10(-10) M with 110 to 220 binding (receptor) sites/cell. The binding of 125I-IL 1-beta to VDS-O cells was also inhibited by F(ab)'2 fragments of anti-human IL 1 and recombinant human IL 1-alpha, as well as by unlabeled human IL 1-beta but not by recombinant lymphotoxin, recombinant tumor necrosis factor, or phorbol myristic acid, suggesting that IL 1-alpha and IL 1-beta bind specifically to the same receptor. The m.w. of IL 1 receptor on human EBV-B cells was estimated to be 60,000 by both the chemical cross-linking method and high pressure liquid chromatography (HPLC) gel filtration analysis of receptor extracted from membrane enriched fraction by a non-ionic detergent (CHAPS). The isoelectric point of solubilized human IL 1 receptor was 7.3 on HPLC chromatofocusing. The evidence of existence of IL 1 receptor on human EBV-B cells additionally supports the hypothesis that IL 1 may be an autocrine signal for these cells.
We have developed a silicone nerve regeneration chamber that is partitioned into two compartments by a strip of nitrocellulose paper. The modified two-compartment chamber allows the investigation of the effects on rat sciatic nerve regeneration of trophic or growth factors that are initially bound to the nitrocellulose partition. In this study we compared the effects of untreated nitrocellulose, a siliconized nitrocellulose strip, and a strip that had been soaked in a basic fibroblast growth factor (FGF) solution. FGF is a known angiogenic factor and a mitogen for endothelial cells, fibroblasts, and Schwann cells. All of these cell types are present in the peripheral nerve. In vitro analyses, using 3T3 cells as test cells, showed that some of the bound FGF remained active on the nitrocellulose paper for at least 8-10 days. In vivo experiments, examined at 16 days post-implantation, revealed that spatial migration of all cellular elements (perineurial-like cells, vasculature, and Schwann cells) across the chamber gap was slower with untreated nitrocellulose strips than with siliconized strips but was most advanced with FGF-treated ones. Most striking was the well-developed vascular arborization of the regenerate within the FGF chambers. Histologic sections from the proximal one-half of the chamber revealed that the regenerate in untreated strip chambers consisted of fibrin matrix and erythrocytes, whereas a well-developed structure with all the cellular elements of a regenerating nerve was seen in several of the FGF strip chambers. We conclude that FGF stimulates peripheral nerve regeneration in this model.
Adherence is an important regulatory signal for several monokines and the proto-oncogenes c-fms and c-fos in human peripheral blood monocytes. Although there is little if any constitutive expression of the IL-1 beta, TNF-alpha and CSF-1 genes in freshly isolated monocytes, adherence is sufficient to induce high steady-state levels of mRNA for TNF and c-fos and more slowly that of CSF-1. Expression of mRNA for the CSF-1R gene, c-fms, was transiently down-regulated by 4 h. In contrast, the induction of high levels of IL-1 beta mRNA were achieved independent of culture conditions. Although all of these genes could be induced by adherence, actual secretion of the mediators required the exposure to a second signal derived from LPS. Thus adherence rapidly primes monocytes for a variety of inflammatory responses, the magnitude of which depends on the nature of a second "activating" signal. It is likely that some of these products act locally as paracrine or autocrine factors to further regulate the phenotype of the differentiating macrophage.
In the nerve regeneration silicone chamber model, the regenerate which forms across a 10-mm gap between proximal and distal nerve stumps is a monofascicular structure with an outer perineurial-like cell sheath. Recent work has provided indications that the geometry of the regenerate within a silicone chamber can be altered by experimental modifications of the chamber matrix. In the present study we modified the standard silicone chamber into a two-compartment chamber by inserting a 6- or 10-mm-long siliconized nitrocellulose strip in order to obtain two separate regenerates. Light microscopy 16 days after implantation revealed that two separate nerve structures had formed, one on each side of the nitrocellulose partition and adjacent to it, and each with its own perineurial-like cell sheath. In chambers with 6-mm-long strips a monofascicular regenerate started from the proximal stump and divided into two separate structures as it approached the proximal end of the strip: the two fascicles joined again into a monofascicular structure in the distal portion of the chambers. The new two-compartment silicone chamber model appears suitable for future examinations of experimental fasciculation. In addition, the nitrocellulose partition should allow one to study specific effects of growth factors on axonal regeneration in vivo, as growth factors bind strongly to untreated nitrocellulose while retaining their biological activity.
Interleukin-1 (IL-1), a peptide hormone produced by activated macrophages, possesses the ability to modulate the proliferation, maturation and functional activation of a broad spectrum of cell types and may play a major role in the initiation and amplification of immune and inflammatory responses through its action on these diverse cell populations. IL-1 exhibits microheterogeneity in terms of its relative molecular mass (Mr, 13,000-19,000) and charge properties, and although murine IL-1 has been purified and some of its basic structure-function relationships have been elucidated, it has proved difficult to prepare sufficient amounts of IL-1 for direct and detailed sequence and structural studies. Here we report the cloning, sequence analysis and expression of murine IL-1 cDNA in Escherichia coli. The IL-1 cDNA codes for a polypeptide precursor of 270 amino acids. Biologically active IL-1 was produced in E. coli by expressing the carboxy-terminal 156 amino acids of the IL-1 precursor.
A number of hydroperoxy (HPETE) and hydroxy (HETE) products of the lipoxygenase pathway of arachidonic acid metabolism are chemotactic and chemokinetic for human neutrophils. We have investigated the relative chemokinetic potency of some of these products on human, rat and rabbit neutrophils. The most potent lipoxygenase product studied was 5,12-dihydroxy-6,8,10-14-eicosatetraenoic acid (5,12-diHETE), which was maximally chemokinetic and chemotactic between 0.1 and 1.0ng/ml for the three species. The 5, 11 and 12-HPETEs and HETEs were chemokinetic, but less active by at least two orders of magnitude, for human and rabbit neutrophils at concentrations between 0.1 and 10micrograms/ml. 15-HPETE and 15-HETE were inactive on human leucoctes, and none of the monosubstituted products studied were chemokinetic for rat neutrophils. These results indicate that 5,12-diHETE may be an important mediator in the local accumulation of leucocytes in the inflammatory response.
Arachidonic acid plays a central role in a biological control system where such oxygenated derivatives as prostaglandins, thromboxanes, and leukotrienes are mediators. The leukotrienes are formed by transformation of arachidonic acid into an unstable epoxide intermediate, leukotriene A4, which can be converted enzymatically by hydration to leukotriene B4, and by addition of glutathione to leukotriene C4. This last compound is metabolized to leukotrienes D4 and E4 by successive elimination of a gamma-glutamyl residue and glycine. Slow-reacting substance of anaphylaxis consists of leukotrienes C4, D4, and E4. The cysteinyl-containing leukotrienes are potent bronchoconstrictors, increase vascular permeability in postcapillary venules, and stimulate mucus secretion. Leukotriene B4 causes adhesion and chemotactic movement of leukocytes and stimulates aggregation, enzyme release, and generation of superoxide in neutrophils. Leukotrienes C4, D4, and E4, which are released from the lung tissue of asthmatic subjects exposed to specific allergens, seem to play a pathophysiological role in immediate hypersensitivity reactions. These leukotrienes, as well as leukotriene B4, have pro-inflammatory effects.
The respective capacities of adherent human monocytes to metabolize endogenous arachidonic acid into leukotrienes C4 (LTC4) and B4 (LTB4) in response to activation with an ionophore, A23187, or to phagocytosis of unopsonized zymosan particles and IgG-sensitized sheep erythrocytes ( EsIgG ) were compared under optimal conditions for each stimulus. Resolution of the cellfree supernatant, after ionophore activation, by reverse-phase high performance liquid chromatography (RP-HPLC) identified only two products which eluted at the retention times of LTC4 and LTB4. There was correspondence between their quantitation by integrated optical density and radioimmunoassay, and the recoveries from the initial supernatant were 80% by radioimmunoassay. Activation of adherent monocytes from 12 donors by ionophore and by zymosan particles released 68.1 ng and 10.0 ng LTB4 and 29.5 ng and 2.1 ng LTC4, respectively. With trypsin pretreatment, the monocytes responded fully to ionophore activation but were inhibited in their response to zymosan particles as assessed by phagocytosis and leukotriene release, indicating that the zymosan stimulus acted through a trypsin-sensitive membrane receptor. When the response of adherent monocytes from nine donors to zymosan particles and to EsIgG were compared at identical particle concentrations and with similar numbers of ingesting monocytes, zymosan elicited LTB4 release (mean 6.7 ng) from all and LTC4 (mean 1.5 ng) from eight donors, while EsIgG caused low level release of LTB4 (mean 0.7 ng) from six and LTC4 from only one of the donors. Neither zymosan nor ionophore stimulation led to the metabolism of exogenously added [3H]LTB4 or [3H]LTC4 as assessed by RP-HPLC of the cellfree supernatants and by quantitation of the eluted labeled products. Thus, transmembrane activation of adherent monocytes by their receptor for particulate activators, in contrast to stimulation of their IgG-Fc receptor, reproducibly releases substantial quantities of LTB4 and LTC4, and may represent an important mechanism for regulating the microenvironment in the nonimmune host.
The outgrowth of neurites from cultured neurons can be induced by the extracellular matrix glycoproteins, fibronectin and laminin, and by polyornithine-binding neurite-promoting factors (NPFs) derived from culture media conditioned by Schwann, or other cultured cells. We have examined the occurrence of fibronectin, laminin and NPFs during peripheral nerve regeneration in vivo. A previously established model of peripheral nerve regeneration was used in which a transected rat sciatic nerve regenerates through a silicone chamber bridging a 10 mm interstump gap. The distribution of fibronectin and laminin during regeneration was assessed by indirect immunofluorescence. Seven days after nerve transection the regenerating structure within the chamber consisted primarily of a fibrous matrix which stained with anti-fibronectin but not anti-laminin. At 14 days, cellular outgrowths from the proximal and distal stumps (along which neurites grow) had entered the fibronectin-containing matrix, consistent with a role of fibronectin in promoting cell migration. Within these outgrowths non-vascular as well as vascular cells stained with anti-fibronectin and anti-laminin. Within the degenerated distal nerve segment, cell characteristic of Bungner bands (rows of Schwann cells along which regenerating neurites extend) stained with anti-fibronectin and laminin. The fluid surrounding the regenerating nerve was found to contain NPF activity for cultured ciliary ganglia neurons which markedly increased during the period of neurite growth into the chamber. In previous studies using this particular neurite-promoting assay, laminin but to a much lesser extent fibronectin also promoted neurite outgrowth.(ABSTRACT TRUNCATED AT 250 WORDS)
The spatial-temporal progress of peripheral nerve regeneration across a 10-mm gap within a silicone chamber was examined with the light and electron microscope at 2-mm intervals. A coaxial, fibrin matrix was observed at 1 week with a proximal-distal narrowing that extended beyond the midpoint of the chamber. At 2 weeks, Schwann cells, fibroblasts, and endothelial cells had migrated into the matrix from both nerve stumps. There was a delay of 7-14 days after nerve transection and chamber implantation before regenerating axons appeared in the chamber. At 2 weeks, nonmyelinated axons were seen only in the proximal 1-5 mm of the chamber in association with Schwann cells. Axons reached the distal stump by 3 weeks and a proximal-distal gradient of myelination was observed. These observations define the parameters of a morphologic assay for regeneration in this chamber model which can be used to investigate cellular and molecular mechanisms underlying the success of peripheral nerve regeneration.