Classification of motor commands using a modified self-organising feature map.

In this paper, a control system for an advanced prosthesis is proposed and has been investigated in two different biological systems: (1) the spinal withdrawal reflex system of a rat and (2) voluntary movements in two human males: one normal subject and one subject with a traumatic hand amputation. The small-animal system was used as a model system to test different processing methods for the prosthetic control system. The best methods were then validated in the human set-up. The recorded EMGs were classified using different ANN algorithms, and it was found that a modified self-organising feature map (SOFM) composed of a combination of a Kohonen network and the conscience mechanism algorithm (KNC) was superior in performance to the reference networks (e.g. multi-layer perceptrons) as regards training time, low memory consumption, and simplicity in finding optimal training parameters and architecture. The KNC network classified both experimental set-ups with high accuracy, including five movements for the animal set-up and seven for the human set-up.

Changes in expression of PACAP in rat sensory neurons in response to sciatic nerve compression.

In the present study, expression of pituitary adenylate cyclase-activating polypeptide (PACAP) in rat dorsal root ganglion (DRG) neurons and sciatic nerve following experimental sciatic nerve compression was studied with the use of quantitative immunohistochemistry and in situ hybridization. Previously, we have investigated changes in PACAP expression after nerve transection and, here, the far more frequently encountered condition of nerve compression injury is examined. Nerve compression was performed unilaterally on the rat sciatic nerve, at mid-thigh level, by application of a narrow silicone tube around the nerve for 3, 7, 14 or 28 days, respectively. We detect a statistically significant upregulation in the number and density of PACAP mRNA expression in both small and large DRG neurons in response to nerve compression. An increased number of PACAP-immunoreactive neurons is also found in the ipsilateral DRG. In addition, PACAP immunoreactivity is observed in the compressed sciatic nerve segment and adjacent nerve tissue after nerve compression. The present findings can be compared with previous studies where we have shown that PACAP expression is upregulated in DRG; in response to peripheral inflammation (primarily in small-medium neurons), and after axotomy (dramatic upregulation in medium-large neurons). In view of the recent findings of an increased PACAP expression in DRG after nerve compression, as well as the previous findings of a modulation of PACAP expression in response to axotomy and inflammation, it is likely that PACAP is also involved in the modulation of the response to peripheral nerve compression.

Effect of anti-nerve growth factor treatment on pituitary adenylate cyclase activating polypeptide expression in adult sensory neurons exposed to adjuvant induced inflammation.

Expression of pituitary adenylate cyclase activating polypeptide (PACAP) is increased in sensory neurons exposed to adjuvant induced peripheral inflammation. Local elevation in expression of the neurotrophin nerve growth factor (NGF) is a main factor contributing to the neuronal response to inflammation. This study examines the role of endogenous NGF in inflammation-associated increases in PACAP expression using the adjuvant-induced peripheral inflammation model with or without systemic administration of antibodies against NGF. Quantitative in situ hybridization was used to detect changes in neuronal PACAP mRNA expression and to correlate this expression with neuronal mRNA expression of the NGF receptor tyrosine kinase (trk) A. The results from this study show that inflammation triggered increases in PACAP expression occurs in small- to medium-sized dorsal root ganglion (DRG) neurons that also express trkA, and that this elevation in PACAP expression is prevented by systemic injection of anti-NGF. This supports a role for NGF as a positive regulator of PACAP expression during inflammation.

Exogenous NT-3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation.

Expression of pituitary adenylate cyclase-activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT-3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4-L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT-3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT-3 infusion, suggesting a role for NT-3 in returning the 'injured phenotype' back towards an 'intact phenotype'. NGF dramatically up-regulated PACAP expression in trkA-positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT-3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT-3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small-medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.

Migration of cells into and out of peripheral nerve isografts in the peripheral and central nervous systems of the adult mouse.

Peripheral nerve (PN) isografts provide a favourable environment for axon regeneration after peripheral and central nervous system (CNS) injury, but definitive information on the extent of cellular intermixing between donor and host tissues is lacking. We wished to compare migration patterns in fresh and predegenerate PN grafts, and also compare the extent of cell migration after transplantation to peripheral nervous system (PNS) versus CNS. To discern how host and donor cells interact after PN transplantation, sciatic nerve segments were transplanted from inbred adult mice into PN defects (PN-PN grafts) or into lesioned cerebral cortex of opposite gender siblings. Migrating male cells were identified using a Y-chromosome-specific probe and in situ hybridization methods, and characterized immunohistochemically. The extent of donor and host cellular intermixing was similar in fresh and predegenerate PN-PN isografts. There was substantial intermixing of donor and host cells by 8 days. Many host cells migrating into epineurial regions of grafts were immunopositive for F4/80 (macrophages). The endoneurium of grafted PN was also colonized by host cells; some were F4/80+ but many were immunostained with S-100 (Schwann cell marker). Donor S-100+ Schwann cells rapidly migrated out into proximal and distal host PN and by 12 weeks were found at least 2 mm from the grafts. Endoneurial microvessels in grafts were mostly donor-derived. By comparison, in male PN grafts to female CNS, even after 6 weeks few donor cells had migrated out into surrounding host cortex, despite the observation that almost all grafts contained regenerating axons and were thus attached to host CNS tissue.

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.

The geometric design of micromachined silicon sieve electrodes influences functional nerve regeneration.

A neural interface could be used to control a limb prosthesis. Such an interface can be created by facilitating axonal regeneration through a sieve electrode and then register nerve signals intended to control the prosthesis. A key question is how to design the electrodes to ensure the best possible regeneration. Our previous studies have indicated that regeneration can be achieved using electrodes with square-shaped, 100 x 100 microm, via holes (holes that axons will regenerate through). Other reports have indicated a suitable range of these holes between 40 and 65 microm. In the present study we used silicon sieve electrodes with via holes of either 30 or 90 microm. The transparency, i.e. the percentage of the total via hole area, of these electrodes was either 20 or 30%. The electrodes were inserted into a silicone chamber which was used to bridge a gap in a rat sciatic nerve. After 12 weeks of nerve regeneration electrodes with a hole size of 30 microm and a 30% transparency had the most favourable result as judged by the regained gastrocnemius muscle force and the formation of reactive tissue inside the chamber. The sieve electrode transparency is crucial for ensuring regeneration.

Effects of gradual bone lengthening on the rabbit tibial nerve.

Little is known about the effect of gradual bone lengthening on peripheral nerves. In the present study, an external fixation device was applied to the rabbit tibia, which was then divided. After seven days, the tibia was subjected to 0.7 mm/day callus distraction for periods of up to one month. The tibial nerve was fixed in glutaraldehyde and plastic sections were cut in longitudinal and transverse planes for light and electron microscopy. Light microscopy showed a 64% increase in the gap length at the node of Ranvier in myelinated axons from the experimental side compared with the control side. The cross-sectional area of the non-myelinated axons was not altered significantly. We conclude that gradual stretching of the nerve elongates the nerve fibres at least at the region of the nodes, perhaps a point of least resistance. Diameters of fibres seem to be held more constant during the lengthening procedure.

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.

Hydroxyapatite granule/carrier composites promote new bone formation in cortical defects.

BACKGROUND: A great deal of interest has been focused on finding substitutes for autogenous bone grafts. Among the most interesting materials are different calcium phosphate compositions (e.g., hydroxyapatite [HA]), due to their biocompatible properties in hard and soft tissue. PURPOSE: The bone response to porous ceramic HA granules in combination with two lipid and one polysaccharide carrier was evaluated in an experimental bone defect model in rabbits. MATERIALS AND METHODS: Circular defects (Ø 4 mm) were made in both tibias of 32 rabbits. The 64 defects were divided into four groups. Group A was augmented with a composite of HA granules and a phospholipid-diacetyl-glycerol carrier, group B with HA granules and a phospholipid carrier, group C received HA granules and a sodium hyaluronan carrier, and group D served as control. The animals were killed after 6 weeks and ground sections were evaluated using light microscopic morphometry. X-ray microfluorescence (XRF) was applied in order to evaluate the suitability of this method to examine bone-biomaterial interfaces. Calcium distribution was studied using x-ray fluorescence line scans at selected interface regions of two sections in group B. RESULTS: The HA/phospholipid composites were easier to shape and handle than the HA/hyaluronan composite. Group A had 36% newly formed bone area within the defect. Groups B and C showed significantly more newly formed bone within the defect (47% and 49%, respectively) compared to the control group (31%). The XRF analysis revealed that the amount of calcium in the newly formed bone was similar to that observed for the HA granules and slightly lower when compared to the mature, lamellar bone. CONCLUSIONS: Synchrotron radiation may be a new, suitable technique to study the interface between bone and biomaterials with regard to mineral content. The results suggest that HA granule/lipid and HA granule/hyaluronan composites have interesting properties as bone-substitute materials.

Perforated silicon nerve chips with doped registration electrodes: in vitro performance and in vivo operation.

An in vitro model was developed for the study of signal transduction between a Cu-wire, miming a neural signal source, and recording electrodes on perforated silicon chips. Phosphorous doped electrodes were used to achieve an all silicon device. The model was used to study signal amplitude as a function of the spatial position, and distance to the signal source. Recordings of the signal crosstalk to neighboring electrodes on the chips were made. It was found that the amplitude decreased by a factor of two at a distance of 50 microns between the electrode surface and the signal source. The chip electrode signal crosstalk was found to be 6 dB using an external reference electrode. Improvements were accomplished with an on chip reference electrode giving a crosstalk suppression of 20 dB. Impedance analysis showed that doped silicon electrodes displayed similar characteristics as Cu-electrodes at frequencies above 3 kHz. Sieve electrodes were implanted in the rat sciatic nerve and following a 10-week nerve regeneration period the dorsal and ventral (L5) roots in the spinal cord were stimulated. Compound action potentials were recorded via the chip. Stimulating the regenerated sciatic nerve via the sieve electrode also induced lower leg muscle contraction activity.

Pituitary adenylate cyclase-activating polypeptide and islet amyloid polypeptide in primary sensory neurons: functional implications from plasticity in expression on nerve injury and inflammation.

Primary sensory neurons serve a dual role as afferent neurons, conveying sensory information from the periphery to the central nervous system, and as efferent effectors mediating, e.g., neurogenic inflammation. Neuropeptides are crucial for both these mechanisms in primary sensory neurons. In afferent functions, they act as messengers and modulators in addition to a principal transmitter; by release from peripheral terminals, they induce an efferent response, "neurogenic inflammation," which comprises vasodilatation, plasma extravasation, and recruitment of immune cells. In this article, we introduce two novel members of the sensory neuropeptide family: pituitary adenylate cyclase-activating polypeptide (PACAP) and islet amyloid polypeptide (IAPP). Whereas PACAP, a vasoactive intestinal polypeptide-resembling peptide, predominantly occurs in neuronal elements, IAPP, which is structurally related to calcitonin gene-related peptide, is most widely known as a pancreatic beta-cell peptide; as such, it has been recognized as a constituent of amyloid deposits in type 2 diabetes. In primary sensory neurons, under normal conditions, both peptides are predominantly expressed in small-sized nerve cell bodies, suggesting a role in nociception. On axotomy, the expression of PACAP is rapidly induced, whereas that of IAPP is reduced. Such a regulation of PACAP suggests that it serves a protective role during nerve injury, but that of IAPP may indicate that it is an excitatory messenger under normal conditions. In contrast, in localized adjuvant-induced inflammation, expression of both peptides is rapidly induced. For IAPP, studies in IAPP-deficient mice support the notion that IAPP is a pronociceptive peptide, because these mutant mice display a reduced nociceptive response when challenged with formalin.

Reactive capsule formation around soft-tissue implants is related to cell necrosis.

Low-density polethylene disks with smooth or course surfaces were implanted in the abdominal wall of rats, and the tissue response was evaluated after 1, 6, or 12 weeks. Cell damage was detected by two different methods. Cells with increased membrane permeability could be identified using fluorescence microscopy by injection of propidium iodide prior to the killing of the rats. Second, cell death was verified by detection of DNA fragmentation. At 1 week a considerable number of the interfacial cells was stained with propidium iodide. Propidium-iodide-positive cells also were enriched at the edges of the disks irrespective of surface texture. The numbers of positive interfacial cells decreased markedly over time. Cells with DNA fragmentation initially displayed a scattered distribution; at later time points they appeared mainly in the outer portion of the enveloping capsule. The reactive capsule was thicker for the smooth surface, and there was a positive correlation between capsule thickness and propidium-iodide-positive cells at earlier implantation periods. The results suggest that the thickness of the reactive capsule is related to the extent of cell necrosis. It is suggested that the major initiator for this cell necrosis is mechanical shear since cell necrosis was found mainly in areas where mechanical shear could be expected.

Tissue reactions to polyethylene implants with different surface topography.

This study investigates the importance of implant surface topography on soft tissue response. The tissue response in the rat abdominal wall to discs of low density polyethylene with smooth to coarse surfaces was evaluated after one, six or 12 weeks. Capsule thickness and immunohistochemical quantification of monocytes-macrophages were used as measures. The macrophage specific antibody ED1 was used for identification of newly recruited macrophages and the ED2 antibody for the mature tissue macrophages. The smoother surfaces gave a thicker capsule than the rougher surfaces, and at one week also larger total numbers of cells and ED1 positive macrophages at interface. The capsule thickness increased over time for the smooth and intermediate surface topographies. In contrast, the cell numbers generally decreased over time. In conclusion, a coarse surface elicited lesser tissue reaction compared with a smooth surface.

Pituitary adenylate cyclase-activating peptide is upregulated in sensory neurons by inflammation.

The neuropeptide pituitary adenylate cyclase-activating peptide (PACAP) is expressed in sensory neurons. Expression of several neuropeptides is up-regulated in sensory neurons following inflammation. To examine whether also PACAP expression is regulated by inflammation, PACAP expression in L5 dorsal root ganglion (DRG) was determined, using in situ hybridization, after unilateral adjuvant-induced inflammation in the rat paw. At 12 h and day 3, but not day 21, the percentage of neurons expressing PACAP mRNA was greater in the innervating L5 DRG. Similarly, PACAP mRNA expression in individual neurons was higher in the innervating L5 DRG at 12 h and day 3, but not day 21. Up-regulated PACAP expression following adjuvant injection suggests a role for PACAP in inflammation.

Anti-inflammatory properties of titanium in the joint environment. An experimental study in rats.

Little is known about the tissue reactions to various implant materials which coincide with an inflammatory reaction. We used the avridine arthritis rat model to evaluate the tissue response in the synovial, interstitial and subcutaneous tissues after implant insertion. Quantitative immunohistochemistry showed that normal joint synovial tissue is dominated by ED2-positive resident macrophages. Polyethylene implants induced a much stronger foreign-body reaction than titanium implants, as measured by the number of interfacial ED1-positive macrophages. The tissue response to titanium and polyethylene was also vastly different in arthritic synovial tissue compared with control tissue. It is likely that these biomaterials interact differently with inflammatory cells or intermediary compounds. It may be that arthritic synovial tissue produces reactive oxygen intermediates (free radicals) with which titanium has a unique anti-inflammatory interaction in vitro.

Analysis of the inflammatory response to titanium and PTFE implants in soft tissue by macrophage phenotype quantification.

Implants of commercially pure titanium and polytetrafluoroethylene (PTFE) were inserted in the rat abdominal wall for 1, 6 or 12 wk. The foreign body reaction was evaluated by immunohistochemical quantification of monocytes/macrophages and by the thickness of the foreign-body capsule. At all time intervals, the majority of interfacial cells were ED1-positive while ED2-positive cells were localized deeper in the tissue. Neither titanium nor PTFE displayed a significant change in capsule thickness over time. The total cell numbers decreased overtime for both types of material. At 12 wk the PTFE implants, compared to titanium, were surrounded by a significantly thicker reactive capsule with larger total cell numbers. No significant differences were seen in the macrophage subset response between the two types of implants. Thus, the present study showed differences between titanium and PTFE at 12 wk but not at earlier time points.

Islet amyloid polypeptide and calcitonin gene-related peptide expression are upregulated in lumbar dorsal root ganglia after unilateral adjuvant-induced inflammation in the rat paw.

After unilateral adjuvant-induced inflammation, expression of neuropeptides believed to be involved in the inflammatory response, e.g. substance P and calcitonin gene-related peptide (CGRP), is upregulated in innervating sensory neurons. Islet amyloid polypeptide (IAPP) is structurally related to CGRP and constitutively expressed in sensory CGRP-containing neurons; the role of IAPP in sensory neurons is unknown. To examine whether IAPP could play a role in inflammation, IAPP expression in L5 dorsal root ganglion (DRG) and its distribution in the dorsal horn were investigated after unilateral adjuvant-induced inflammation in the rat paw and compared with CGRP, using in situ hybridization and immunocytochemistry. At 12 h and day 3, but not day 21, the percentage of nerve cell profiles expressing IAPP and CGRP mRNA was greater in the ipsilateral L5 DRG; these changes paralleled the occurrence of edema around the tarsotibial joint and a slight limp. IAPP expression in individual nerve cell profiles was higher in the ipsilateral L5 DRG at 12 h, but not at days 3 and 21; the corresponding CGRP mRNA level was higher at days 3 and 21. At day 3, the higher expression of IAPP and CGRP on the ipsilateral side was accompanied by increased numbers of immunoreactive DRG neurons and fibers in the spinal cord dorsal horn. Largely, expression of IAPP and CGRP seems to be co-ordinately regulated by localized inflammation, although the rapid, but transient, upregulation in DRG neurons of IAPP mRNA expression and the slower, but sustained, upregulation of CGRP mRNA expression may indicate dissociated regulation of the peptides. Thus, IAPP could play a role in the initial phase of localized inflammation.

Supersensitivity in rat micro-arteries after short-term denervation.

Contractile responses to phenylephrine and high-K+ were investigated in vitro in microvascular preparations from the rat medial plantar artery, a branch from the saphenous artery, obtained after short-term denervation in vivo. Two groups of animals were studied: (1) animals undergoing surgical resection of the saphenous nerve, and (2) animals undergoing surgical resection of both the sciatic and saphenous nerves. The animals were operated on one side only. Microvascular preparations (diameter about 325 microns) were obtained 10 days after surgery. Vessels from the non-operated side served as controls. Immunocytochemistry showed a decreased number of both neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibres in vessels after resection of the saphenous nerve only. Resection of both the saphenous and the sciatic nerve caused a complete loss of immunoreactive nerve fibres. Mechanical measurements were performed using a wire myograph. In vessels subjected to resection of the saphenous nerve the sensitivity to phenylephrine was similar to controls. Vessels denervated by resection of both the saphenous and sciatic nerves showed significant increases in phenylephrine and potassium sensitivity. When depolarized in high-K+ solution the denervated vessels showed an increased sensitivity to extracellular Ca2+. The results show that complete short-term denervation of the rat medial plantar artery in vivo causes a pronounced supersensitivity in the vascular smooth muscle. The supersensitivity appears not to be restricted to the sympathetic alpha-receptors but also associated with changes in the cellular excitation-contraction coupling. Such altered reactivity of the vascular smooth muscle may contribute to vascular disturbances observed in vivo after nerve damage or surgical denervation.

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

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