Effects of experimental diabetes on axonal and Schwann cell changes in sciatic nerve isografts.

A reduced ability to regenerate peripheral axons may be partly responsible for diabetic neuropathy. The source of the impairment has not been narrowed down to axonal or Schwann cell failure. We used nerve grafts from control or diabetic donor rats transplanted into control or diabetic hosts to pursue this differential diagnosis. An isograft between the left sciatic nerves of inbred Lewis rats was performed 8 weeks after STZ treatment and on age-matched controls. The nerve exchanges were control-control, control-diabetic, diabetic-control and diabetic-diabetic. At postsurgical day 14, nerves were excised and analysed for levels of axonal markers, total and phosphorylated neurofilament, and Schwann cell receptors, ErbB2 and p75(NTR), using immunohistochemistry and Western blotting. The aim was to measure ingress of axonal markers into the graft and judge the appropriateness of Schwann cell phenotype changes. Transfer of nerve from diabetic to control rats resulted in a doubling in neurofilament, both phosphorylated and nonphosphorylated (both P<0.05). ErbB2 was decreased in grafts from diabetic rats (53% of control, P<0.05) and p75(NTR) levels were increased in both types of graft in diabetic rats (to 300-400% of controls, P<0.05). Schwann cells in diabetic nerve grafts showed receptor levels more similar to controls when placed into a normal environment and the converse also appeared to hold. TUNEL staining revealed increased apoptosis in diabetic nerve distal to the graft. The data show that alterations in Schwann cell phenotype in diabetes are reversed by transfer to control rats and develop in normal nerve after transfer to a diabetic host.

Pseudocapillarization and associated energy limitation in the aged rat liver.

Age-related impairment of drug metabolism by the liver is consistent with hepatocyte hypoxia, suggestive of the development of a diffusional barrier to oxygen supply. Because the effects of aging on the diffusional pathway (sinusoidal endothelium and space of Disse) have not been described, we performed comparative studies on the livers of Fischer F344 rats aged 4 to 7, 12 to 15, and 24 to 27 months. Light-microscopic examination revealed no evidence of fibrosis, cirrhosis, or other specific pathology. In contrast, scanning and transmission electron-microscopic examination revealed that aging is associated with pseudocapillarization of the sinusoidal endothelium, indicated by defenestration with reduced porosity, thickening of the endothelium, infrequent development of basal lamina, and only minor collagen deposits in the space of Disse. Furthermore, immunohistochemistry studies showed strong expression of collagen IV, moderate expression of factor VIII-related antigen, and weak expression of collagen I along the sinusoids of livers from old rats (P <.0001). In vitro (31)P magnetic resonance spectroscopy analysis showed that aging is associated with changes in high-energy phosphate and other metabolites, consistent with hepatocyte hypoxia. Aging in the liver is associated with changes in the sinusoidal endothelium and space of Disse that may restrict the availability of oxygen and other substrates.

The effect of acute oxidative stress on the ultrastructure of the perfused rat liver.

Ageing and liver disease are associated with ultrastructural changes in the hepatic sinusoid. Because of the possibility that reactive oxygen species could mediate these processes, we examined the effect of acute oxidative stress on the ultrastructure of the intact liver. Rat livers were perfused ex vivo, in situ with hydrogen peroxide via the portal vein. The livers were then fixed and the ultrastructure of the liver tissue examined with transmission and scanning electron microscopy. The effects of hydrogen peroxide were largely confined to the perisinusoidal areas. The sinusoidal endothelial cells became swollen and more porous, with large gaps replacing sieve plates. The space of Disse showed an increase in volume and the density of hepatocyte projections decreased. Kupffer cell activation was noted. Little or no ultrastructural change was observed within the hepatocytes. Oxidative stress delivered via the portal vein dramatically alters the ultrastructure of the perisinusoidal regions of the liver. This process may contribute to the pathogenesis of disease and age-related changes in the liver.

Functional and structural abnormalities in the nerves of type I diabetic baboons: aminoguanidine treatment does not improve nerve function.

AIMS/HYPOTHESIS: To improve understanding of the pathophysiology of diabetic neuropathy and to establish a primate model for experimental studies, we examined nerve changes in baboons with Type I (insulin-dependent) diabetes mellitus. We also examined the effect of aminoguanidine (an inhibitor of the formation of advanced glycation end products) on nerve function. METHODS: Male baboons (Papio hamadryas) were assigned to four groups; control, diabetic, control and diabetic treated with aminoguanidine. Diabetes was induced with streptozotocin (60 mg/kg, intravenous). Insulin and aminoguanidine (10 mg/kg) were injected subcutaneously daily. Motor and sensory nerve conduction velocity was measured using standard techniques. Autonomic function was examined by measuring heart rate response to positional change. Sural nerve morphometry was analysed in the diabetic group (mean duration 5.5 years) along with their age-matched controls. RESULTS: The diabetic groups were smaller in size with a mean HbA1c of 8.9 +/- 1.2%. The nerve conduction velocity and heart rate response was reduced in the diabetic groups. Morphometric analysis of the diabetic sural nerve showed smaller axon diameter (2.99 +/- 0.06 microns vs 3.29 +/- 0.06 microns; p < 0.01) accompanied by thinner myelin (1.02 +/- 0.02 microns vs 1.15 +/- 0.02 microns, p < 0.01) with no change in the axon density. Treatment with aminoguanidine for 3 years had no effect on glycaemic control and did not restore conduction velocity or autonomic dysfunction in the diabetic animals, contrary to the studies in rats. CONCLUSIONS/INTERPRETATION: These results show that the primate is a good model to study diabetic neuropathy and suggest that the accumulation of advanced glycation end products are not an early mechanism of nerve damage in this disorder.

Glutaminergic and adrenergic receptors expressed on adult guinea pig Schwann cells in vitro.

We have investigated the responsiveness of adult guinea pig Schwann cells to a range of neuroligands, using ratiometric calcium imaging. The majority of cells responded to ATP (90 +/- 4%), adrenaline (57 +/- 5%), and noradrenaline (61 +/- 5%), as well as glutamate (60 +/- 5%). The number of cells responding to glutamate increased significantly (90 +/- 4%; p < 0.01) when the cells were grown in excitatory amino acid (EAA) free medium, indicating EAA-induced downregulation. Only a small number of cells (9 +/- 2%) responded to acetylcholine. Agonist and antagonist experiments show that these adult Schwann cells predominantly express ionotropic glutaminergic receptors (N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isooxazolepropionic acid (AMPA), and kainate) as well as alpha1-, alpha2-, and beta-adrenoreceptors. We conclude that Schwann cells derived from adult guinea pigs express a variety of neuroligand receptors when established in culture and are particularly rich in glutamate receptors. This probably reflects a de-differentiated state important to development and regeneration.

Peripheral nerve regeneration through nerve guides seeded with adult Schwann cells.

This study tested the usefulness of Schwann cells in the repair of a severed nerve with a biosynthetic bridge or guide. Reinforced collagen nerve guides were used to bridge an 18 mm gap in the sciatic nerve of 21 young adult rats. The animals were divided into three groups and the guides were filled with: (i) more than 0.5 x 10(6) cultured syngeneic adult Schwann cells (group L, n = 12); (ii) less than 0.5 x 10(6) Schwann cells (Group S, n = 6); and (iii) phosphate buffered saline (control, n = 3). Schwann cells were pre-labelled with Hoechst dye. Regeneration was assessed functionally and histologically at 1, 2, 3 and 6 + months after surgery. Group L animals showed numerous regenerated axons surrounded by implanted Schwann cells within the first month. The total number of myelinated fibers (12.5 x 10(3)) remained above normal unoperated values (7 x 10(3)) in long-term animals. Regenerated axons were found in Group S in the third month, but no Hoechst labelled cells were found. The number of myelinated fibers (3.9 x 10(3)) remained below normal values in long-term animals. Control guides failed to support axonal regeneration. Functional recovery was evident at week 20 (Group L) and week 30 (Group S) after surgery, with no difference in function between the two groups by the end of the study. Supplementing guides with Schwann cells enhances regeneration of peripheral axons over a distance normally prohibitive. This effect is greatest in the early stages of regeneration (1-3 months) and is dependent on the number of cells implanted.

Extracellular ATP increases intracellular calcium in cultured adult Schwann cells.

We have previously reported that extracellular ATP causes a transient rise in intracellular calcium concentration ([Ca2+]i) in cultured Schwann cells derived from adult animals [Ansselin A. D. et al. (1994) Int. J. Neurosci. 74, 148]. In this study, the receptor mediating this response has been characterized. Established adult rat and rabbit Schwann cell cultures were loaded with fura-2 (acetoxymethyl ester, 10 micromol/l, 40 min, 37 degrees C). which indicated, by fluorescence imaging, a resting [Ca2+]i of 34.7 +/- 1.4 nmol/l (mean S.E., n=591). The cells were exposed to 100 micromol/l ATP, ADP, AMP, UTP and adenosine in defined medium for 1-2 min, and the change in [Ca2+]i was observed as a change in the Fura-2 ratio. Seventy-seven percent of adult rat Schwann cells (n=235) and 88% adult rabbit Schwann cells (n=356) responded to the presence of extracellular ATP (100 mmol/l) with a transient increase in [Ca2+]i (41 and 90 nmol/l from resting value, respectively), independent of the presence of [Ca2+]o. Calcium waves were observed in one experiment. The following order of agonist potency was observed: UTP= ATP>>ADP>AMP=adenosine. The agonists alpha,beta-methylene-ATP and 2-methylthio-ATP had a small effect on the cells, similar to AMP, and were mutually desensitizing. The ATP antagonist suramin blocked the response. We conclude that adult Schwann cells express a purinergic ATP receptor belonging to the G-protein-coupled P2u alpha subtype [O'Connor S. et al. (1991) Trends pharmac. Sci. 12, 137-141].

The regeneration of axons through normal and reversed peripheral nerve grafts.

The effect of proximo-distal orientation of peripheral nerve grafts upon axonal regeneration has been investigated using the sciatic nerve of the rat as a model. To test the hypothesis that the presence of nerve branches within a graft will cause misdirection of axons in normally oriented grafts but not in reversed grafts, all grafts studied contained branches. Qualitative electron microscopic examination of graft ultrastructure revealed no differences in nerve structure related to graft orientation. In most normally oriented grafts, branches persisted up to 12 months after surgery. These branches contained axons which terminated at the end of the branch. In all reverse oriented grafts, and in a small number of normally oriented ones, the branches could not be seen after two or more months of regeneration. Axons sprouting outside of the epineurium of the graft caused the branch to be incorporated into the nerve structure. Axon counts in the distal stump of grafted nerves after twelve months recovery revealed that normally oriented grafts with persistent branches led to poorer peripheral regeneration, especially of unmyelinated fibers. The results indicate that regeneration of axons to their peripheral targets may be facilitated by reversing the graft orientation.

Immunosuppression in nerve allografting: is it desirable?

Immunologically incompatible sciatic nerve grafts were inserted into the severed sciatic nerves of Wistar rats. In an attempt to induce graft tolerance, low-dose cyclosporin A (CsA) was administered to some animals for 20 weeks, then gradually withdrawn. Behavioural, electrophysiological and histological studies indicated that some degree of regeneration took place in all animals regardless of treatment. Neither a daily dose of 5 mg/kg nor 10 mg/kg was sufficient to prevent the rejection and subsequent disruption of allograft structure, and as a consequence reinnervation of the distal stump was limited. This was manifest both in the poor functional recovery of the denervated foot, and in the large number of regenerated axons found outside of the perineurial membranes of the transplanted fascicles. Therefore, tolerance was not induced at these doses. Furthermore, the significant decrease in the amplitude of electromyographs recorded from experimental and unoperated (control) animals suggests CsA may have a deleterious effect on unlesioned nerve even at these low doses. It would be prudent, therefore, to exercise caution in the combined use of nerve allografts and CsA immunosuppression, until the neurotoxicity of CsA has been investigated further. This is particularly important since CsA is sometimes used in the treatment of certain neuropathic autoimmune diseases.

Labelling of restricted numbers of axons by solid rhodamine implantation into nerve trunks.

Solid rhodamine B isothiocyanate microsurgically implanted into mammalian peripheral nerve labels a small number of axons within that nerve. These axons can be observed in whole mounts or slices following fixation of the tissue approximately 16 h after the rhodamine is applied. The labelling appears to involve axoplasmic transport in both directions, for axons can be followed over long distances in the whole mounts. Since only some axons are labelled, their structure can be observed within large populations of cells in a whole nerve. This is valuable in interpreting axon counts in developmental and regenerative studies, and in observing the pattern of sprouting at regenerating axon terminals.

Low dose, short term cyclosporin A does not protect the Schwann cells of allogeneic nerve grafts.

The question of whether Cyclosporin A, at the low doses reported to allow survival of nerve allografts, allows the allogeneic Schwann cells to survive has been investigated. Nerve allografts were inserted by microsurgical techniques into trembler mice treated with 10 mg/kg or 15 mg/kg Cyclosporin A, daily for 12 weeks post-operatively. Electrophysiological recordings were made one week prior to surgery, and at the end of the immunosuppression period, after which the grafts were processed for electron microscopy. All grafts showed signs of rejection and donor Schwann cells had been replaced by host cells. The results suggest that Cyclosporin A in these doses is insufficient to overcome rejection problems or to induce tolerance to donor Schwann cells.

Patterns of functional innervation in the auditory nuclei of the chick brainstem following early unilateral removal of the otocyst.

Neurons in the third-order auditory nuclei in the brainstem of chicks (nuclei laminares, NL) receive functional innervation from the ipsilateral and contralateral second-order nuclei (nuclei magnocellulares, NM) which is restricted to the dorsal and ventral dendrites respectively. This pattern of innervation in NL is established by embryonic stage 40 (day 15 of incubation). We have examined the distribution of this innervation in both NL at this age or older in embryos from which one otocyst had been removed or damaged on day 3 of incubation. The distribution of functional synapses was determined by analysis of the changes in polarity of field potentials evoked by electrical stimulation of either the ipsilateral or contralateral NM. The distribution of field potential polarity in NL of 40% of recordings in operated embryos and in all sham-operated embryos was the same as that observed in unoperated embryos. However, in the remaining operated embryos, the time course of the field potentials and the changes in the polarity of the responses as the recording electrode penetrated NL were abnormal. The abnormal complexity of responses and the abnormal distribution of field potential amplitude in NL in the operated embryos suggests that loss or damage to the first-order auditory innervation can result in (a) the formation of novel, functional synapses between second-order auditory neurons, and/or (b) disruption of processes that produce segregated innervation of the dendrites of the third-order auditory neurons.

Immunopathological factors in peripheral nerve allograft rejection: quantification of lymphocyte invasion and major histocompatibility complex expression.

The numbers of helper T and cytotoxic T lymphocytes and macrophages were quantified, and the expression of major histocompatibility complex (MHC) class I and class II molecules was examined in rat peripheral nerve allografts from 1 to 14 days after implantation, using the indirect immunoperoxidase method for light and electron microscopy. Two centimetre segments of peripheral nerve freshly obtained from inbred Dark Agouti strain rats were inserted in a gap created in n. fibularis or n. tibialis of young adult inbred Wistar strain rats, using fascicular nerve repair techniques under general anaesthesia. There was a gradual increase in the number of helper T and cytotoxic/suppressor T cells from day 2 with peak numbers of both types of T cells observed around day 7. The results suggest that the critical time for T cell proliferation is between day 6 and day 7 post-operatively. The number of macrophages increased over 10 days, with peak numbers being observed at day 10 post-operatively. This is in accord with the pattern of rejection observed in allografts of other tissue. Schwann cells were found to express MHC class I and class II molecules by day 2 post-operatively, which is well before there is any substantial T cell and macrophage infiltration. It may be that the donor Schwann cells act as antigen presenting cells, triggering the immune response and finally becoming a target of the rejection process.

Development of functional innervation in the second and third order auditory nuclei of the chick.

The neurones that constitute the auditory nuclei of the brainstem in the chick (nuclei magnocellularis, NM, and laminaris, NL) are generated between days 2 and 4 of incubation. These neurones migrate towards the dorsal surface of the brainstem over the next few days and reach their final destination at about day 9 of incubation. We have examined the development of functional connections between the auditory nerve and neurones in NM and between neurones in NM and NL in embryos from stage 34 (day 8 of incubation) using electrophysiological techniques and electron and light microscopy. The earliest extracellular recordings of electrically evoked field and spike potentials were made in NM with stimulation of the ipsilateral auditory nerve and in NL with stimulation of the ipsilateral NM at stage 35 (day 9). No activity could be recorded in NL with stimulation of the auditory nerve at this stage. By stage 37 (day 11), direct stimulation of the contralateral NM evoked responses in NL and by stage 38 (day 12) stimulation of the auditory nerves evoked stable field potentials in NL. These potentials changed polarity as the electrode penetrated NL in a direction that was perpendicular to the laminar arrangement of neurone somas and parallel to the dendritic axes of these neurones. In 18 of 26 analyses of current-source density in NL of 12 preparations between stages 38 and 40 there was a sink of current associated with synaptic activity at levels both above and below the source of current (cell somas) following stimulation of the ipsilateral auditory nerve. In the remaining analyses, and in all 15 analyses from preparations older than stage 40, stimulation of the ipsilateral input evoked only a single sink of current above the level of the cell somas. In all preparations from embryos at stage 38 and older, stimulation of the contralateral auditory nerve was associated with a single sink of current below the level of the cell somas. The axon projections to the ipsilateral NL from neurones in NM were examined using HRP labelling between stages 38 and 40. The presence of terminal fields of single axons in both the dorsal and ventral dendritic regions of the ipsilateral NL at these ages was confirmed. Furthermore, dense vesicles within synaptic terminals in both the dorsal and ventral dendritic fields could be identified in preparations at stage 36 following injection of HRP into NL and stimulation of the ipsilateral NM.(ABSTRACT TRUNCATED AT 400 WORDS)

Axonal regeneration through peripheral nerve grafts: the effect of proximo-distal orientation.

Peripheral nerve transplants are used for the surgical repair of nerve loss. If normally oriented grafts contain branches, regenerating axons may be lost into them, limiting re-innervation and restoration of function in the denervated area. Since axons entering a reversed graft may not be lost in this way, we have investigated whether reverse implantation could enhance the number of axons that reach the distal stump. In young adult rats, a section of sciatic nerve, including one major branch, was removed and reinserted with either the normal or reversed proximo-distal orientation, using microsurgical techniques. After signs of recovery of function, the operated and contralateral unoperated nerves of each animal were fixed and processed for electron microscopy. Regenerated axons were seen to enter branches in normally orientated grafts, and there was substantial loss in the cross-sectional area of the graft distal to the branch termination. Reverse grafts are as well reinnervated by regenerating axons as normally oriented grafts and show a smaller loss in the cross-sectional area, even though the branches of these implants seemed to disappear.