Conduction studies in peripheral cat nerve using implanted electrodes: II. The effects of prolonged constriction on regeneration of crushed nerve fibers

Arrays of chronically implanted electrodes were used to examine the time course of elongation and maturation of peripheral nerve fibers in the cat after crush of the tibial nerve in the proximal calf. Regeneration after crush alone was compared with crush 5 mm proximal to a tight constriction of the nerve. Regeneration was monitored by the progression of excitability along the electrode arrays on the tibial and plantar nerves. The sensitivity was sufficient to record the averaged activity in single nerve fibers allowing detection of the earliest regeneration. The diameters of the fastest regenerating fibers were estimated from the conduction velocity proximal to the site of crush. Both after crush alone, and after crush constriction, small myelinated fibers regenerated in front of large fibers. The rate of elongation after crush alone was 3.2 mm/day, whereas it was slower (P less than 0.02) distal to crush + constriction (2.2 mm/day). In both lesions, the extrapolated delay to onset of regeneration was 8 days. In observations up to 300 days after crush, maturation was delayed or impaired by the constriction, and the compound nerve action potential had a smaller amplitude and a dispersed shape. Transverse sections of nerves after crush + constriction showed a diminished number of large and an increased number of small fibers compared with crush alone, possibly due to persistent branching of regenerated fibers. After both crush alone and crush + constriction, regenerated fibers had similar g ratios, suggesting that myelination developed fully in fibers of diminished diameters.     

Conduction studies in peripheral cat nerve using implanted electrodes: I. Methods and findings in controls

Silicone rubber cuff and patch electrodes with multiple contacts were implanted along the sciatic-tibial-plantar nerves in cat for repeated studies of conduction properties of normal peripheral nerve over periods of time. The contacts were used in various combinations for precise localization of changes in conduction velocities and excitability along the extent of normal nerves. In this paper the particular characteristics and limitations associated with cuff-electrode recordings of neural activity are discussed. The nerve action potential was recorded using a tripolar configuration with a central lead flanked by two shunted leads at symmetrical distances. This configuration records the spatial derivative of the action potential and rejects potentials from sources outside the cuff. The voltage changes are restricted by the silicone cuff, and the dynamic range is therefore very high, allowing detection from single myelinated fibers to whole nerve responses. The electrodes are well suited for following the development of regeneration and degeneration following experimental lesions.     

Neurobehavioral studies of forced swimming: The role of learning and memory in the forced swim test

West A. Preston: Neurobehavioral studies of forced swimming: The role of learning and memory in the forced swim test. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1990, 863–877.

1. 1. Immobility in the forced swim test (“behavioral despair test”) has often been regarded as an animal model of despair or depression.

2. 2. Behavioral studies of forced swimming (“behavioral despair”) are reviewed and compared with certain behavioral effects of exposure to inescapable shock (i.e., “learned helplessness”).

3. 3. Exposure to inescapable shock clearly impairs subsequent coping responses. However, detailed behavioral studies of forced swimming indicate that immobility during forced swimming is not a failure of coping but instead reflects a relatively successful coping strategy that employs energy conserving behaviors.

4. 4. Certain neurobiological studies of forced swimming are reinterpreted in light of the behavioral evidence that immobility during forced swimming reflects effects of learning and memory rather than effects of despair or depression.

5. 5. Some implications for future neurobehavioral studies of forced swimming and uncontrollable shock are discussed.

Neurofibromatosis 2 with peripheral neuropathies: Electrophysiological,pathological and genetic studies of a Taiwanese family

The objective of this study was to assess peripheral nerve involvement and DNA mutation of the neurofibromatosis type 2 (NF2) gene (NF2) in a Taiwanese family with classic NF2. Eleven members (six symptomatic and five asymptomatic) of a family carrying NF2 underwent clinical examination, neuroimaging, and electrophysiological analysis. Mutation and linkage analyses were conducted on DNA samples prepared from peripheral blood (all individuals), a sural nerve biopsy specimen (one symptomatic member), and a tumor specimen (another symptomatic member). Six of the 11 members were diagnosed with classic NF2. DNA sequencing of the tumor specimen demonstrated a frameshift mutation with 756delC on exon 8 of NF2. Three affected subjects showed clinical variability of the neuropathic disorders. Electrophysiological studies demonstrated variation in the disease pattern and severity of peripheral nerve involvement in five affected subjects. The morphometric assessment of the sural nerve biopsy specimen showed a marked reduction in both large myelinated and unmyelinated fibre density and increased density of non‐myelinating Schwann cell nuclei. Apart from numerous pathological nuclei of isolated Schwann cells, multiple profiles of non‐myelinating Schwann cell subunits were apparent in the endoneurium. Schwann cell proliferation in association with first‐hit mutation of the merlin gene might be responsible for the NF2‐associated neuropathy. Sural nerve biopsy showed a progressive neuropathy in the disease. Further, we suggest nonmyelinating Schwann cells are involved in NF2 neuropathy.     

A magnetic evaluation of peripheral nerve regeneration: II. The signal amplitude in the distal segment in relation to functional recovery

Motor and sensory function in a healthy nerve is strongly related to the number of neuronal units connecting to the distal target organs. In the regenerating nerve the amplitudes of magnetically recorded nerve compound action currents (NCACs) seem to relate to the number of functional neuronal units with larger diameters regenerating across the lesion. The goal of this experiment was to compare the signal amplitudes recorded from the distal segment of a reconstructed nerve to functional recovery. To this end, the peroneal nerves of 30 rabbits were unilaterally transected and reconstructed. After 6, 8, 12, 20, and 36 weeks of regeneration time the functional recovery was studied based on the toe-spread test, and the nerve regeneration based on the magnetically recorded NCACs. The results demonstrate that the signal amplitudes recorded magnetically from the reconstructed nerves increase in the first 12 weeks from 0% to 21% of the amplitudes recorded from the control nerves and from 21% to 25% in the following 23 weeks. The functional recovery increases from absent to good between the 8th and the 20th week after the reconstruction. A statistically significant relation was demonstrated between the signal amplitude and the functional recovery (P < 0.001). It is concluded that the magnetic recording technique can be used to evaluate the quality of a peripheral nerve reconstruction and seems to be able to predict, shortly after the reconstruction, the eventual functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:750–755, 1998.     

Long-term observation on the changes of somatotopy in the facial nucleus after nerve suture in the cat: Morphological studies using retrograde labeling

To examine the time course of plasticity of the cranial nucleus during axonal regeneration, we followed the topographical reorganization of the cat facial nucleus (FN) up to 24 months after facio-facial nerve suture using retrograde labeling methods. The trunk of the temporal-zygomatico-orbital and both superior and inferior buccolabial branches (defined as main branch) of the facial nerve was cut and sutured again under ketamine hydrochloride anesthesia. At 11-722 days after nerve suture, Fast Blue (FB) and 1,1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (Dil) or horseradish peroxidase (HRP) were injected into the distal part of the sutured main branch and the unoperated posterior auricular branch, respectively. Until about 3 months after suture, the topographical pattern in FN was similar to that observed in normal cats. At about 4 months after suture, FB-labeled motoneurons were distributed not only in the lateral part (including intermediate, dorsal and ventrolateral divisions) but also in the medial subdivision of FN. After a survival period of 18-24 months, FB-labeled neurons were found all over the FN, and their number increased significantly. Interestingly, in the longer survival cases, we noticed that the Dil- or HRP-labeled posterior auricular branch motoneurons also showed a tendency to distribute outside the medial region. The present study showed that somatotopic disorganization starts at around 4 months after suture, which seems to be somewhat slower than that in rats, and continues until a much later postoperative period. Furthermore, we suggested a possibility that the regeneration of one branch may affect the somatotopy of the unoperated nerve branch. These phenomena may contribute to aberrant facial nerve functions such as abnormal associated movement and facial spasm observed after nerve injury.     

The effects of nerve growth factor and its antibodies on axonal numbers in the medial gastrocnemius nerve of the rat

A double fluorescence labeling technique was developed to study the specificity of dye-coupling among frog spinal neurons. A pool of motoneurons known to be electrically coupled was prelabeled with a large molecule (rhodamine conjugated to horseradish peroxidase) that was not expected to pass through gap junctions. Then a single sensory or motor neuron within or outside this pool was injected with lucifer yellow to see if the dye spread specifically among neurons that are electrically coupled. We observed almost no examples of specific dye-coupling.     

The rat's postero-orbital sinus hair: II. Normal morphology and the increase in peripheral innervation with adjacent nerve section.

The morphology and innervation of the postero-orbital (PO) sinus hair has been studied in normal rats and in adult animals in which an adjacent nerve, the infraorbital nerve, was sectioned on postnatal day 0 or day 7. The normal morphology of the follicle was similar to that of mystacial sinus hairs. However, the normal innervation differed from mystacial follicles in three respects: (1) instead of a separate innervation, the deep vibrissal nerve (DVN) and dermal plexus were supplied by a common follicle and skin nerve, named here the postero-orbital cutaneous nerve, a branch of the zygomaticofacial nerve; (2) the entry of the DVN through the capsule was highly variable; in some cases fascicles entered in close proximity, but in others they were widely distributed around the capsule; and (3) two or three small nerves, called here anastomosing nerves, were found to leave the PO follicle. These arose from the DVN after it had passed through the capsule to the cavernous sinus. The anastomosing nerves passed back through the capsule and ascended on the outer surface of the follicle to join the dermal plexus. Each nerve contained 1-4 myelinated fibres and 11-35 unmyelinated fibres. Infraorbital (IO) nerve section on day 0 caused a 19% (P less than 0.001, n = 8) increase in numbers of fibers to the DVN on the lesioned side. Most of the increase was due to unmyelinated fibres with no significant change in myelinated axons. No change in axon numbers in the DVN occurred after day 7 lesions. Labelling of the mystacial pad and the PO follicle did not result in any double labelling of cells in the trigeminal ganglion, in either normal or lesioned animals, making it improbable that the increased numbers of unmyelinated axons arose from rerouting of infraorbital fibres. It is suggested that the increased innervation of the PO follicle may arise by the rescue of ganglion cells from developmentally programmed cell death.     

Differential effects of lentiviral vector-mediated overexpression of nerve growth factor and glial cell line-derived neurotrophic factor on regenerating sensory and motor axons in the transected peripheral nerve

Even after reconstructive surgery, major functional impairments remain in the majority of patients with peripheral nerve injuries. The application of novel emerging therapeutic strategies, such as lentiviral (LV) vectors, may help to stimulate peripheral nerve regeneration at a molecular level. In the experiments described here, we examined the effect of LV vector-mediated overexpression of nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) on regeneration of the rat peripheral nerve in a transection/repair model in vivo. We showed that LV vectors can be used to locally elevate levels of NGF and GDNF in the injured rat peripheral nerve and this has profound and differential effects on regenerating sensory and motor neurons. For sensory neurons, increased levels of NGF and GDNF do not affect the number of regenerated neurons 1 cm distal to a lesion at 4 weeks post-lesion but do cause changes in the expression of markers for different populations of nociceptive neurons. These changes are accompanied by significant alterations in the recovery of nociceptive function. For motoneurons, overexpression of GDNF causes trapping of regenerating axons, impairing both long-distance axonal outgrowth and reinnervation of target muscles, whereas NGF has no effect on these parameters. These observations show the feasibility of combining surgical repair of the transected nerve with the application of viral vectors. Furthermore, they show a difference between the regenerative responses of motor and sensory neurons to locally increased levels of NGF and GDNF.     

Degeneration and regeneration of peripheral nerve in the rat trigeminal system: III. Abnormal sensory reinnervation of rat guard hairs following nerve transection and crush

The present study was undertaken in an attempt to better understand the abnormalities of cutaneous sensibility that are present in patients following nerve injury with concomitant cutaneous denervation and subsequent reinnervation. Reinnervated intervibrissal pelage of the rat mystacial pad was studied in silver-impregnated sections 3 and 5 months after transecting and 2 and 5 months after crushing the infraorbital nerve. The sensory terminals on guard and vellus hairs were analyzed in serial paraffin sections and in thick frozen sections. In normal rat mystacial skin, approximately nine/ten of innervated guard hairs have a typical piloneural complex consisting of a palisade of highly regular lanceolate terminals surrounded by circularly arranged Ruffini terminals and free nerve endings (FNEs). The remaining one of ten innervated guard hairs has only circularly arranged presumptive FNEs and Ruffini terminals. Vellus hairs, either singly or in clusters, typically have only circularly arranged terminals that in many cases are simple FNEs. We first recognized abnormalities in innervation of hairs following nerve transection and fully expected nerve terminals to be completely normal following nerve crush. Almost all reinnervated sensory nerve terminals associated with guard hairs were markedly abnormal following nerve transection and quantitatively abnormal following nerve crush. Following nerve transection, lanceolate terminals were almost completely absent, and they were remarkably reduced in number following nerve crush. Vellus hairs when reinnervated typically lacked the complex circular presumptive Ruffini terminals. These findings may be in part the basis for the abnormal cutaneous sensory perceptions (dysasthesias and paresthesias) noted in human subjects following damage to nerves with subsequent sensory reinnervation of the skin.(ABSTRACT TRUNCATED AT 250 WORDS)     

A magnetic evaluation of peripheral nerve regeneration: I. The discrepancy between magnetic and histologic data from the proximal segment

Histologic techniques can quantify the number of axons in a nerve, but give no information about electrical conductibility. The number of functional myelinated neuronal units in a nerve can be quantified based on a magnetic recording technique. When studying reconstructed peripheral nerves a significant difference between the results found with these two techniques can be observed. A comparison was made between the long-term changes in the number of histologically and magnetoneurophysiologically measured neuronal units proximal to a nerve reconstruction. This study was performed on 6 New Zealand White rabbits, 20 weeks after the peroneal nerve had been reconstructed. The contralateral nerves were used as a control. Histologic examination demonstrates a statistically significant decrease of approximately 5% in the number of myelinated fibers. The magnetoneurophysiological results demonstrate a decrease which is estimated to be caused by the loss of approximately 50% of the functional myelinated neuronal units in the nerve. Therefore we conclude that of the initially available myelinated neuronal units, 5% degenerate completely, 45% are vital but lose their signal conducting capability, and the remaining 50% are vital and continue to conduct signals. Apparently, only this latter group of 50% of the initially available functional neuronal units appears to remain available for functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:739–749, 1998.     

Primary neurons maintain their central axonal arbors in the spinal dorsal horn following peripheral nerve injury: an anatomical analysis using transganglionic transport of horseradish peroxidase

This study in adult cats demonstrates that primary neurons of all sizes survive following the transection and capping with a polyethylene tube of their peripheral processes in the superficial radial nerve. The central axonal arbors of these injured primary neurons remain intact and maintain their normal topographic position across laminae I–VI of the cervical (C6–C8) dorsal horn. In addition, they maintain their synaptic vesicles, some of their synaptic connections and their ability to transport horseradish peroxidase transganglionically.     

Transforming growth factor-beta and forskolin attenuate the adverse effects of long-term Schwann cell denervation on peripheral nerve regeneration in vivo

Transforming growth factor-beta (TGF-beta) plays a central role in the regulation of Schwann cell (SC) proliferation and differentiation and is essential for the neurotrophic effects of several neurotrophic factors (reviewed by Unsicker and Krieglstein, 2000; Unsicker and Strelau, 2000). However, its role in peripheral nerve regeneration in vivo is not yet understood. Our studies were carried out to characterize (1) the effects of duration of regeneration, and chronic SC denervation on the number of tibial (TIB) motor neurons that regenerated axons over a fixed distance (25 mm into distal common peroneal [CP] nerve stumps), and (2) the effect of in vitro incubation of 6-month chronically denervated sciatic nerve explants with TGF-beta and forskolin on their capacity to support axonal regeneration in vivo. TIB--CP cross-suture in Silastic tubing was used, and regeneration into 0-24-week chronically denervated CP stumps was allowed for either 1.5 or 3 months. Chronically denervated rat sciatic nerve explants (3 x 3 mm(2)) were incubated in vitro with either DMEM and 15% fetal calf serum (D-15) plus TGF-beta/forskolin or D-15 alone for 48 h and placed into a 10-mm Silastic tube that bridged the proximal and distal nerve stumps of a freshly cut TIB nerve. The number of tibial motor neurons that regenerated axons through the explants and 25 mm into the distal nerve stump after 6 months, and TIB regeneration into the CP nerve stumps, were assessed using retrograde tracers, fluorogold, or fluororuby. We found that all tibial motor neurons regenerate their axons 25 mm into 0-4-week denervated CP nerve stumps after a regeneration period of 3 months. Reducing regeneration time to 1.5 months and chronic denervation, reduced the number of motor neurons that regenerated axons over 25 mm. Exposure of 6-month denervated nerve explants to TGF-beta/forskolin increased the number of motor neurons that regenerated through them from 258 +/-13; mean +/- SE to 442 +/- 22. Hence, acute treatment of atrophic SC with TGF-beta can reactivate the growth-permissive SC phenotype to support axonal regeneration.     

Effects of electromagnetic field (PEMF) exposure at different frequency and duration on the peripheral nerve regeneration: in vitro and in vivo study

Purpose: The purpose was to clarify the influence of frequency and exposure time of pulsed electromagnetic fields (PEMF) on the peripheral nerve regeneration. Materials and methods: Immortalized rat Schwann cells (iSCs) (1 × 10²/well) were exposed at four different conditions in 1 mT (50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12h/d). Cell proliferation, mRNA expression of S100 and brain-derived neurotrophic factor (BDNF) were analyzed. Sprague-Dawley rats (200–250 g) were divided into six groups (n = 10 each): control, sham, 50 Hz 1 h/d, 50 Hz 12 h/d, 150 Hz 1 h/d and 150 Hz 12 Hr/d. Mental nerve was crush-injured and exposed at four different conditions in 1 mT (50 Hz 1 Hr/d, 50 Hz 12 Hr/d, 150 Hz 1 h/d and 150 Hz 12 h/d). Nerve regeneration was evaluated with functional test, histomorphometry and retrograde labeling of trigeminal ganglion. Results: iSCs proliferation with 50 Hz, 1 h/d was increased from fourth to seventh day; mRNA expression of S100 and BDNF was significantly increased at the same condition from first week to third week (p < .05 vs. control); difference score was increased at the second and third week, and gap score was increased at the third under 50 Hz 1 h PEMF compared with control while other conditions showed no statistical meaning. Axon counts and retrograde labeled neurons were significantly increased under PEMF of four different conditions compared with control. Although there was no statistical difference, 50 Hz, 1 h PEMF showed highest regeneration ability than other conditions. Conclusion: PEMF enhanced peripheral nerve regeneration, and that it may be due to cell proliferation and increase in BDNF and S100 gene expression.     

Axonal elongation through long acellular nerve segments depends on recruitment of phagocytic cells from the near-nerve environment. Electrophysiological and morphological studies in the cat

The distal nerve stump plays a central role in the regeneration of peripheral nerve but the relative importance of cellular and humoral factors is not clear. We have studied this question by freezing the tibial nerve distal to a crush lesion in cat. The importance of constituents from the near-nerve environment was assessed by modification of the contact between the tibial nerve and the environment. Silicone cuffs, containing electrodes for electrophysiological assessment of nerve regeneration, were placed around the tibial nerve distal to the crush site. The interaction between long acellular frozen nerve segments (ANS) and the near-nerve environment was ascertained by breaching the silicone cuff to allow access of cellular or humoral components. Tibial nerves were crushed and frozen for 40 mm and enclosed in nerve cuffs with 0.45-microm holes or 2.0-mm holes to allow access of humoral factors or tissue ingrowth, respectively. In a second set of experiments, tibial nerves were crushed and either frozen for 20+20 mm, leaving a 10 mm segment with viable cells in the center (stepping-stone segment) or frozen for 50 mm. These nerves were enclosed in cuffs with 2.0 mm holes corresponding to the viable nerve segment. The regeneration was monitored electrophysiologically by implanted electrodes and after 2 months the nerves were investigated by light and electron microscopy. The results indicate that soluble substances in the near-nerve environment, such as nutrients, oxygen or tropic substances did not exert any independent beneficial effect on the outgrowing axons. However, phagocytic cells entering the acellular segment from the near-nerve environment were crucial for axonal outgrowth in long ANS.     

The influence of protein-calorie malnutrition on the development of internodal segments. A study on peripheral nerve and spinal roots in rats

Summary The current investigation comprised normal young rats as well as rats submitted to a 50% food reduction or severe protein restriction. Isolated nerve fibres from lumbar spinal roots and sciatic nerves were investigated with reference to the relation between length and diameter of internodes as well as variation of internodal length along single nerve fibres. The present results do not support the view that protein-calorie malnutrition should cause neuropathy. Internodal segments were, on an average, shorter in relation to their thickness in young rats submitted to severe protein restriction or a 50% food reduction. The deviation was most marked in low-protein animals and particularly among coarser internodal segments. An inhibition of longitudinal growth was considered to be the main factor behind the difference between malnourished and normal rats.     

The effects of peripheral nerve injury of the masseteric nerve on the levels of calcium binding proteins and neuropeptide Y, and their correlation in the mesencephalic trigeminal nucleus of the rat

Combined retrograde neuronal tracing with FluoroGold (FG) and a double immunofluorescence method was performed to examine the effects of peripheral nerve injury of the masseteric nerve (MassN) on the levels of two calcium binding proteins (CaBPs), parvalbumin (PV) and calbindin D28k (CB), and neuropeptide Y (NPY) in the mesencephalic trigeminal nucleus (MesV) in the rat. In the normal MesV, many medium- to large-sized unipolar PV-like immunoreactive (-IR) cells were detected through the entire rostrocaudal extent, but CB-IR cells were rarely observed. No NPY-IR cells were observed in the normal MesV. The distributions of these three neurochemical markers in the MesV contralateral to the transection of Mass were almost identical to those observed in the normal MesV. Four days following transection and application of FG to the MassN, approximately 52% (572/1104) and 38% (414/1104) of FG-labeled cells (FG cells) in the MesV displayed PV-like immunoreactivity (-LI) and NPY-LI, respectively; Approximately 24% (265/1104) of FG cells showed both PV-LI and NPY-LI. Approximately 47% (265/572) of FG cells with PV-LI showed NPY-LI or 64% (265/414) of FG cells with NPY-LI displayed PV-LI. Fourteen days following transection and application of FG, the percentage of FG cells with PV-LI significantly decreased to 36% (365/1024) compared to that observed 4 days post-injury; approximately 44% (448/1024) of FG cells displayed NPY-LI; approximately 38% (141/365) of FG cells with PV-LI showed NPY-LI and approximately 31% (141/448) of FG cells with NPY-LI displayed PV-LI. In contrast, FG cells showing CB-LI were very rare on 4 days (1%; or 14 days (1%; 16/1085) following MassN transection. The present results indicate that the levels of PV in the MesV decreased 14 days following the MassN injury compared to those observed 4 days post-injury and rapid induction of NPY in the injured MesV neurons, and that the correlation between CaBP and NPY in the MesV following the MassN transection is different from that observed in the trigeminal ganglion, which is equivalent to the MesV, following peripheral nerve injury of the inferior alveolar nerve.     

The origins of the sciatic nerve and changes in neuropeptides after axotomy: a double labelling study using retrograde transport of Tru blue and vasoactive intestinal polypeptide immunohistochemistry

One month after sciatic nerve section, only dorsal root ganglion cells which take up True blue applied to the cut end of the nerve show vasoactive intestinal polypeptide immunoreactivity. This indicates that VIP expression is only in cells with damaged axons. Motor axons with True blue-positive perikarya in the ventral horn originate from the fourth to sixth lumbar segments whereas unmyelinated and small myelinated sensory nerves terminate in the third to fifth lumbar segments of the spinal cord.