Miquel Butí

Influence of Collagen and Laminin Gels Concentration on Nerve Regeneration after Resection and Tube Repair

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

Comparison of Regenerative and Reinnervating Capabilities of Different Functional Types of Nerve Fibers

Functional reinnervation of sweat glands (SGs), skin, and muscle in the mouse paw after sciatic nerve lesions was evaluated to allow comparisons of the regeneration efficiency of different functional types of nerve fibers. In four groups of mice the sciatic nerve was crushed, sectioned, and left unrepaired or repaired by suture or tubulization. Reappearance of SG secretion and pinprick responses occurred slightly earlier than recordings of compound muscle and nerve action potentials in all groups. The degree of reinnervation, with respect to preoperative control values, of SGs and skin nociceptors was higher than the amplitude of the action potentials, mainly when the nerve injury was severe. The chances for recovery progressively decreased with the severity of the lesion, affecting the larger nerve fibers most. These results indicate that, after injuries of peripheral nerves, all types of nerve fibers are able to regenerate in the mouse, although small size fibers (sudomotor and nociceptive) allow for a higher degree of functional recovery than large myelinated fibers (skeletomotor and sensory).

Influence of physical parameters of nerve chambers on peripheral nerve regeneration and reinnervation.

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

Histologic assessment of sciatic nerve regeneration following resection and graft or tube repair in the mouse.

The present study determines the number and morphology of myelinated fibers that regenerate after resection of the mouse sciatic nerve. In different groups of mice, a resection of 4 or 6 mm of the sciatic nerve was left unrepaired, repaired with silicone or collagen guides or by an autologous nerve graft of the same or smaller calibre. Regeneration was examined, under light microscopy, 3 months after operation and quantified by morphometric analysis of light micrographs of cross-sectional nerve fibers. The results show that, without repair, few nerve fibers reach the distal nerve stump, while tubulization or autografts allowed better regeneration. Tube repair allowed a comparable degree of regeneration to that of an autograft with 4 mm gaps, but lower with 6 mm gaps. Regeneration was limited with a gap of 6 mm in silicone tubes, but was successful in half the mice with collagen tubes. The size and myelination of regenerated fibers were below normal values in all experimental groups, although they were closer to normal with sciatic autografts than after smaller grafts and tubulization. There were no signs of secondary degeneration in the nerve regenerates within silicone and collagen tubes.

The effect of aging on efferent nerve fibers regeneration in mice

This study evaluates the influence of aging on nerve regeneration and reinnervation of target organs in mice aged 2, 6, 9, 12, 18 and 24 months. In animals of each age group the sciatic nerve was subjected to crush, section or section and suture. Reinnervation of plantar muscles and sweat glands (SG) was evaluated over three months after operation by functional methods. Reappearance of SG secretion and motor responses occurred slightly earlier in young than older mice. The degree of motor and sudomotor reinnervation, with respect to preoperative control values, was also significantly higher in young than old animals. The differences were more pronounced after 12 months of age. The degree of recovery progressively decreased with the severity of the lesion, differences being more marked in older mice. Neurorraphy improved recovery, comparatively more in older than in young mice. These results indicate that, after injuries of peripheral nerves, axonal regeneration and reinnervation are maintained throughout life, but tend to be more delayed and slightly less effective with aging.

Autotomy prevention by amitriptyline after peripheral nerve section in different strains of mice

This study evaluates the degree of autotomy induced by anesthesia dolorosa after transection of the sciatic and saphenous nerves in four different strains of mice, and the effectiveness of amitriptyline administration in two of them. The self-mutilating lesions were assessed by means of an autotomy score for one month after denervation. The onset of lesions generally occurred during the first week, starting in the nails and progressing proximally. Autotomy behavior developed differently in the mouse strains studied, involving 88% of the paw areas in OF1 mice, 61% in Balb-C, 35% in NMRI, and 15% in B6CBAF1. Two selected strains, OF1 and NMRI, were treated with amitriptyline (8 mg/kg/day, p.o.) from different intervals pre-operation. Administration starting 14 days before nerve lesion was the most effective treatment schedule for reducing autotomy in both strains.

Functional changes of the peripheral nervous system with aging in the mouse

The influence of aging on peripheral nerve and target organ function was investigated in six groups of mice aged 2, 6, 9, 12, 18, and 24 months. Sudomotor, motor, and sensory functions mediated by the sciatic nerve were evaluated by silicone imprints, electrophysiological recordings and pinprick test from the distal hindpaw. Nerve conduction was also studied in the caudal nerves. The results showed that the number of sweat glands reactive to pilocarpine does not change significantly with aging, but the size of the sweat droplets is smaller in aged mice than in young mice. The amplitude of muscle and nerve action potentials evoked by stimulation of sciatic and caudal nerves decreased progressively from 2 to 24 months, while the latencies decreased from 2 to 6 months, remained unchanged until 12 months and increased thereafter. All the animals of the six groups studied showed positive, indistinguishable responses to pinprick. These results indicate that neurophysiological responses mediated by large diameter nerve fibers deteriorated with age, while those dependent of small fibers were preserved.

Peripheral nerve repair : role of agarose matrix density on functional recovery

This study presents quantitative data on the functional reinnervation of sweat glands, muscle and skin in the mouse paw after resection of the sciatic nerve and repair by entubulation with silicone tubes prefilled with agarose gels of different concentrations. The rate and the degree of functional recovery of animals with chambers filled with 0.5% agarose gels were slightly higher than with 1% agarose, and significantly higher than with 2% agarose. All these three groups recovered significantly better than a fourth group with an unrepaired resection. We conclude that peripheral nerves can regenerate through agarose gels in vvio and reinnervate distal target organs, but the concentration and the density of the exogenous intratubular matrix are important factors in determining the success of nerve regeneration and reinnervation.

Peripheral nerve regeneration through microelectrode arrays based on silicon technology.

This paper describes some developments, made to obtain a chronic neural interface to record signals from regenerated peripheral nerves. Microperforated silicon dices, fabricated by techniques compatible with CMOS processes, were coupled in silicone nerve chambers and implanted between the severed ends of peripheral nerves in rats. Three configurations of perforated dices with 25 via-holes of 100 μm diameter, 121 via-holes of 40 μm and 400 via-holes of 10 μm were assessed. The feasibility of axonal regeneration through the dices via-holes was proved by histological and physiological methods over 3 months post-implantation. The regenerated nerves were organized in fascicles corresponding to the grid pattern of the via-holes. However, nerve regeneration was difficult and distal re-innervation delayed with respect to simple tubulization repair. The size of the via-holes and the total open area are determinants of the degree and quality of regeneration. Further improvements are needed in both the microelectrode dice design and in neurobiological stimulation of regeneration.

Abnormalities of sympathetic sudomotor function in experimental acrylamide neuropathy

The possible involvement of sympathetic sudomotor function by acrylamide intoxication was investigated in the mouse, and compared with nerve conduction studies and global motor tests. Acrylamide (40 mg/kg, 3 days per week, 8 weeks) was given per os to a group of mice (A1). Their motor ability to stand on the rotarod was impaired from day 11, reaching a minimum between 46 and 60 days. The number of pilocarpine reactive sweat glands (SG), evaluated by the silicone mold technique, was similar to controls at 40 days and slightly decreased at 54 days. Another group of mice (A2), given acrylamide at a higher dose (50 mg/kg, 5 days per week, 5 weeks), showed abnormalities on the rotarod by 11 days, a progressive decrease of muscle action potential (CMAP) amplitude, and significantly decreased number of reactive SG from 15 days, with respect to controls. Comparatively, sudomotor dysfunction was milder and appeared later in time than alphamotor involvement, being noticeable only after severe poisoning. The decrease in SG response is attributable to damage by acrylamide intoxication of postganglionic sudomotor nerve fibers, which are unmyelinated sympathetic efferents.