Regeneration of perivascular adrenergic innervation in rat tibial nerve after nerve crush

Summary Adrenergic innervation of blood vessels in the rat tibial nerve during degeneration and regeneration was studied using the formaldehyde-induced fluorescence method. The left sciatic nerve was crushed with suture threads to produce a 4-mm length of crushed nerve. At 1, 3, 7, 14, 28, 56 and 84 days after nerve crush, degenerative and regenerative changes in the nerve were verified using light microscopy. At each time point, adrenergic innervation was examined in epi-perincurial whole mount and nerve cross-section preparations. One day after nerve crush, fluorescence of adrenergic nerve fibers in the endoneurium was absent. Fluorescent adrenergic nerve fibers reappeared in the endoneurium at day 56 and reached the control density by 84 days. In the epi-perineurium, adrenergic innervation of small and medium-size arterioles was absent at 3 days, in large arterioles at 7 days. At 56 days, all epi-perineurial arterioles were reinnervated by a faint, sparse adrenergic network, which reached the control density at 84 days. The results suggest that adrenergic innvervation in the rat peripheral nerve is lost during nerve degeneration, but recovers when the nerve has regenerated.     

Postnatal development of adrenergic innervation in the tibial and vagus nerves of Fischer-344 rats

Adrenergic innervation of rat tibial and vagus nerves was studied in male Fischer-344 rats between 1 and 84 days of age, using sucrose-phosphate-glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method. Adrenergic nerve fibers were found in epi-perineurial blood vessels of the vagus nerve at one day of age, whereas blood vessels in the tibial nerve received the first adrenergic nerve fibers at 3 days. A few adrenergic nerve fibers were seen in the endoneurium of both tibial and vagus nerves at 7 days. The densities of adrenergic innervation increased gradually during the first 4 postnatal weeks, and at 21 days the distributions of adrenergic innervation in both nerves resembled those in adult animals. The results suggest that development of adult adrenergic innervation in rat peripheral nerves occurs during the first postnatal month and that sympathetic innervation becomes available to regulate nerve blood flow within this period.     

Distribution of adrenergic innervation of blood vessels in peripheral nerve

The distribution of adrenergic innervation of microvessels in the extrafascicular and endoneurial compartments of rat tibial nerve was examined with glyoxylic acid-induced and formaldehyde-induced histofluorescence methods. Periarterial and arteriolar adrenergic nerves were present in the epineurium-perineurium suggesting that blood flow in the extrafascicular connective tissue is under neurogenic influence. In contrast, blood vessels in the nerve endoneurium were not associated with histofluorescent nerve fibers. The absence of perivascular adrenergic innervation in the endoneurium suggests that regulation of vascular function within the endoneurium is not under neurogenic control.     

Electromyographic evaluation of a novel surgical preparation to enhance nerve-muscle specificity that follows mammalian peripheral nerve trunk transection

Previous studies indicated that axons from proximal stumps of transected peripheral nerves "prefer" to grow through Silastic tubes attached to their native (originally associated) rather than foreign (not originally associated) distal stumps. We determined whether or not this specificity is expressed at the level of the neuromuscular junction. Proximal stumps of transected rat sciatic nerves (peroneal and tibial branches) were attached to single inlet ends of 6-mm-long, Y-shape Silastic implants. One outlet was attached to the distal peroneal and the other to the distal tibial stump. Ten weeks later, innervation of the anterior tibialis and interosseous muscles (normally innervated predominantly by peroneal and tibial nerve fibers, respectively) was assessed by measuring compound muscle action potential amplitudes and latencies that follow supramaximal peroneal and tibial nerve stimulation. Results showed higher amplitudes in anterior tibialis muscle, induced by "native" peroneal (vs. tibial) stimulation in four of five animals, and higher amplitudes in interosseous muscles after "native" tibial (vs. peroneal) stimulation in all cases examined. Preparations in which bridges between proximal and distal nerve stumps were bridged with unbranched tubes showed random patterns of muscle innervation. The results suggest that if allowed to express "specificity" at the level of nerve trunk transection, regenerating mammalian peripheral axons can grow into, and form functional connection with, native (vs. foreign) muscle groups. This finding has possible clinical significance.     

Patterns of adrenergic and peptidergic innervation in human olfactory mucosa: Age-related trends

The distribution and targets of nerves containing the adrenergic markers tyrosine hydroxylase, dopamine β-hydroxylase, and neuropeptide Y in the human olfactory mucosa were investigated by immunohistochemistry. Tissue was obtained at autopsy from the nasal cleft of 16 adults ranging in age from 24 to 90 years, and from one spontaneously aborted 16-week-old fetus. The presence of olfactory receptor neurons in nasal mucosa was confirmed by staining with the antibody to olfactory marker protein. Targets of adrenergic innervation were blood vessels, including the vasa nervorum within the sheaths of olfactory nerve bundles, and Bowman's glands in the lamina propria. Adrenergic fibers penetrated the adventitia of blood vessels and terminated near the media, and were in close proximity to Bowman's glands but did not enter the acini. In the fetal tissue, the vasa nervorum were the major targets of adrenergic fibers. Age-related differences in the pattern and statistically significant differences in the density of innervation of blood vessels were noted between adults under and over 60 years of age. In the younger group, plexuses of nerve fibers containing colocalized dopamine β-hydroxylase and neuropeptide Y occurred adjacent to arterioles and large bundles of fibers adjacent to venules; in older individuals, few fiber plexuses occurred adjacent to arterioles and thin bundles of fibers adjacent to venules. The distribution of adrenergic innervation suggests that vasomotor tone and secretion are regulated by adrenergic nerves. The decrease in adrenergic innervation in older individuals, with resultant effects on perireceptor processes, may be associated with age-related declines in olfactory function. © 1993 Wiley-Liss, Inc.     

Adrenergic innervation in the tibial and vagus nerves of rats with streptozotocin-induced diabetes

Adrenergic innervation of tibial and vagus nerves was studied after 1-16 weeks duration of streptozotocin (STZ)-induced diabetes in rats. Sucrose-phosphate glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method were used to demonstrate adrenergic nerve fibers in the epi-perineurial and endoneurial compartments. Densities of innervation were quantitated with fluorescence microscopy. The density of periarteriolar adrenergic innervation in the epi-perineurium of the tibial and vagus nerves was increased 5 and 12 weeks after STZ injections as compared with control. At 16 weeks, mean densities of periarteriolar innervation in epi-perineurium had returned to or below control levels in both nerve types. In the endoneurium, however, the mean density of adrenergic nerve fibers decreased gradually at 5 weeks after induction of diabetes in both nerves, and was totally absent at 12 weeks. At 16 weeks no sign of recovering innervation in the endoneurium was seen. In conclusion, adrenergic innervation goes through similar pathological alterations both in tibial and vagus nerves shortly after the induction of streptozotocin diabetes. These changes may contribute to diabetic peripheral neuropathy by impairing the regulation of nerve blood flow.     

Directional specificity of regenerating primary sensory neurons after peripheral nerve crush or transection and epineurial suture A sequential double-labeling study in the rat

Clinical and experimental observations have demonstrated that peripheral nerve transection generally results in lasting disturbed sensory discrimination whereas nerve crush is followed by more or less complete functional restoration. This has been explained by an increased misdirection of regenerating fibers after transection as compared to crush injury. In the present study, sequential double-labeling was used to investigate the relative proportions of peripherally misdirected sensory fibers in the sural and tibial nerve branches after crush or transection of the parent sciatic nerve in the rat. Control experiments showed that 0.21% ± 0.12 (mean ± S.D.) of all labeled tibial and sural neurons normally send axons to both nerves. After sciatic nerve crush or transection, 1.31% ± 0.78 and 3.79% ± 3.01, respectively, of all labeled tibial and sural axons were double-labeled indicating previously sural axons now having an axon in the tibial. Statistically significant differences in the percentages of bidirectional sciatic sensory neurons were found between the normal controls and after crush injury (P < 0.01) or transection injury (P < 0.001), respectively, but not between transection and crush (P > 0.05). The results indicate that the number of sensory neurons having an axon in two peripheral nerves is normally very small, that a substantial number of sensory axons become misdirected after both crush and transection with resuture, and that the number of misdirected fibers in the major sciatic branches after these types of injury is similar.     

Chronic constriction model of rat sciatic nerve: nerve blood flow,morphologic and biochemical alterations

We evaluated the nerve blood flow (NBF), light and electron microscopy, and adrenergic innervation of rat sciatic nerve at 2–45 days after the application of four loose ligatures. Ischemia developed at the lesion edge, creating an endoneurial dam. Calcitonin gene-related peptide, norepinephrine and NBF were increased within the lesion. Morphologic alterations consisted of early endoneurial edema, followed by myelinated fiber degeneration, with relative sparing of small myelinated and unmyelinated nerve fibers, and leukocyte adhesion to microvessels. Axonal degeneration predominated over demyelination. At 45 days, profuse regeneration of small myelinated fibers was seen. The mechanism of lesional sensitization is discussed. Received: 30 May 1996 / Revised, accepted: 13 August 1996     

Neuronal growth factors produced by adult peripheral nerve after injury

Dorsal root ganglion neurons from embryonic rats, co-cultured with endoneurial explants from transected, adult rat sciatic nerve, extended neurites in the absence of exogenous nerve growth factor (NGF). The effect was seen with endoneurial explants from normal adult sciatic nerves or from nerves which had been permanently transected up to 51 days prior to explantation. The rate of outgrowth decreased at 5 and 7 days and reached a minimum at 14 days after transection. A second phase of increased neurite-promoting activity appeared in 28-, 35-, 41- and 51-day posttransection tissue. The early phase, but not the late phase, was partially inhibited by antisera to NGF.     

The longitudinal epineural incision and complete nerve transection method for modeling sciatic nerve injury

Injury severity, operative technique and nerve regeneration are important factors to consider when constructing a model of peripheral nerve injury. Here, we present a novel peripheral nerve injury model and compare it with the complete sciatic nerve transection method. In the experimental group, under a microscope, a 3-mm longitudinal incision was made in the epineurium of the sciatic nerve to reveal the nerve fibers, which were then transected. The small, longitudinal incision in the epineurium was then sutured closed, requiring no stump anastomosis. In the control group, the sciatic nerve was completely transected, and the epineurium was repaired by anastomosis. At 2 and 4 weeks after surgery, Wallerian degeneration was observed in both groups. In the experimental group, at 8 and 12 weeks after surgery, distinct medullary nerve fibers and axons were observed in the injured sciatic nerve. Regular, dense myelin sheaths were visible, as well as some scarring. By 12 weeks, the myelin sheaths were normal and intact, and a tight lamellar structure was observed. Functionally, limb movement and nerve conduction recovered in the injured region between 4 and 12 weeks. The present results demonstrate that longitudinal epineural incision with nerve transection can stably replicate a model of Sunderland grade IV peripheral nerve injury. Compared with the complete sciatic nerve transection model, our method reduced the difficulties of micromanipulation and surgery time, and resulted in good stump restoration, nerve regeneration, and functional recovery.     

Endoneurial microvasculature of chronically transected sciatic nerves in diabetic rats

To characterize the morphology of the endoneurial microvasculature of degenerating nerves under hyperglycemia, the morphology of endoneurial microvessels in transected sciatic nerves was examined in normal and streptozotocin-induced diabetic rats. Three months after transection, the fascicular area and median vascular luminal area at the proximal level of the distal stump were significantly larger in diabetic than in control animals, whereas the number of vessels per fascicle was the same in the two groups. Arterioles in various stages of development were found in the centrifascicular region in some transected nerves. Serial sections revealed that these vessels originated from transperineural arterioles. The frequency and magnitude of vascular wall thickening were both greater in diabetic rats. These results suggest that the endoneurial microvasculature responds abnormally to nerve injury under hyperglycemia.     

Contralateral non-operated nerve to transected rat sciatic nerve shows increased expression of IL-1beta,TGF-beta1, TNF-alpha,and IL-10

Recent reports indicate that after a peripheral nerve injury, the uninjured contralateral nerve is also affected. Because cytokines play an important role in the peripheral nerve injury, we studied the expression of five different mRNAs (interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), transforming growth factor-beta1 (TGF-beta1) and interleukin-4 (IL-4)) in the contralateral, non-operated, left sciatic nerve when the right rat sciatic nerve was transected. This study extended up to 42 days after the transection. No IL-4 expression was noted. During the first 3 days, high expression of the other studied cytokines was noted in the endoneurium. At day 7, the expression diminished to the control levels. After this, a cyclic expression pattern appeared, which was most pronounced in the endoneurium at 35 days. We also show that the expression pattern in the endoneurium is different from that in the surrounding epi- and perineurium. Also, our present study shows clearly that contralateral nerves are poor controls after injury.     

Increased density of perivascular adrenergic innervation in tibial and vagus nerves of spontaneously hypertensive rats

Adrenergic innervation of epiperineurial arterioles and of the endoneurium of the tibial and vagus nerves of spontaneously hypertensive rats (SHR) and normotensive controls (WKY) was studied by glyoxylic acid-induced fluorescence and paraformaldehyde-induced fluorescence methods. Adrenergic perivascular innervation of epiperineurial arterioles in both nerves was denser in SHR than in controls. Mean density in the endoneurium also was higher in SHR in both nerve types. These results suggest that adrenergic perivascular innervation, which may influence nerve blood flow, becomes greater in density in peripheral nerves during chronic hypertension.     

Regeneration after nerve transection: effect of inhibition of collagen synthesis

Dense collagen scar forms at the cut ends of transected nerves, and impedes nerve regeneration. To minimize scarring, we treated rats with the proline analog, cis-hydroxyproline, from day 4 to 21 after sciatic nerve transection and reanastomosis. Twenty-eight days after surgery, there was 47% less collagen in the distal nerve stump of treated rats than of controls. Seventy days after surgery, posterior tibial nerve content of the myelin lipids, sulfatide, and unesterified cholesterol, was 40% higher in treated rats than in controls.     

The role of a barrier between two nerve fascicles in adjacency after transection and repair of a peripheral nerve trunk

Aberrant reinnervation of target organs caused by misdirected axonal growth at the repair site is a major reason for the poor functional outcome usually seen after peripheral nerve transection and repair. The following two studies investigate whether criss-crossing of regenerating rat sciatic nerve axons between tibial and peroneal nerve fascicles can be reduced by using a barrier at the coaption site. The left sciatic nerve was transected and repaired at mid-thigh as follows: epineural sutures (group A, A-II), fascicular repair of tibial and peroneal nerve fascicles (group B, B-II), fascicular repair of tibial and peroneal nerve fascicles separating the two fascicles with a pedicled fat flap (group C), Integra (group D) or non-vascularized autologous fascia (group C-II). In the control groups E and D-II, only the left tibial fascicle was transected and repaired. Four and 5 months postoperatively, the outcome of regeneration was evaluated by histology, by retrograde tracing, and by assessment of the muscle force of the gastrocnemius and tibial anterior muscles. The tracing experiments showed that specificity of muscle reinnervation significantly improved when a barrier was employed, which significantly or clearly improved muscle twitch tension in groups C and D. However, muscle contraction force was not better when fascia was used as barrier. The histological picture indicated that this inferior result in group C-II was due to nerve compression caused by fibrotic scar tissue at the site of the fascia graft. Results of this study show that a pedicle fat flap and Integra used as barrier significantly prevent aberrant reinnervation between two sutured nerve fascicles in adjacency resulting in improved motor recovery in rats. Non-vascularized autologous fascia however, reduces also criss-crossing of regenerating axons between the fascicles, but causes significant nerve compression.     

Effects of delayed nerve repair on regeneration of rat sciatic nerve

The optimal time for nerve suture in transected rat sciatic nerve was investigated. The sciatic nerve on both side nerves were transected and repaired by an epineurial suture technique 0-28 days after nerve transection. The regeneration distance was measured by the sensory pinch reflex test 2-6 days after nerve repair. The initial delay period and the regeneration rate were calculated by using a new mathematical model. One day delayed repair was sufficient to reduce the initial delay period and the largest reduction on the initial delay period was observed in the three day delayed repair group. The initial delay period was reduced to 0.87 days as compared to 2.31 days for nerves repaired immediately (controls). The seven day and ten day delayed repair had similar effects but they also showed a decrease of the regeneration rate. The regeneration rate was not affected except for these two time points. The results suggest that the repair delay period can influence the initial delay as well as the regeneration rate. In our model an optimal effect was achieved on both these measures with a one or three day delay between transection and repair.     

Local administration of vasoactive intestinal peptide after nerve transection accelerates early myelination and growth of regenerating axons

Our goal was to determine whether local injections of vasoactive intestinal peptide (VIP) promote early stages of regeneration after nerve transection. Sciatic nerves were transected bilaterally in 2 groups of 10 adult mice. In the first group, 15 microg (20 microL) of VIP were injected twice daily into the gap between transected ends of the right sciatic nerve for 7 days (4 mice) or 14 days (6 mice). The same number of mice in the second group received placebo injections (20 microL of 0.9% sterile saline) in the same site, twice daily, for the same periods. After 7 days, axon sizes, relationships with Schwann cells and degree of myelination were compared in electron micrographs of transversely sectioned distal ends of proximal stumps. Fourteen days after transection, light and electron microscopy were used to compare and measure axons and myelin sheaths in the transection gap, 2-mm distal to the ends of proximal stumps. Distal ends of VIP-treated proximal stumps contained larger axons 7 days after transection. More axons were in 1:1 relationships with Schwann cells and some of them were surrounded by thin myelin sheaths. In placebo-treated proximal stumps, axons were smaller, few were in 1:1 relationships with Schwann cells and no myelin sheaths were observed. In VIP-treated transection gaps, measurements 14 days after transection showed that larger axons were more numerous and their myelin sheaths were thicker. Our results suggest that in this nerve transection model, local administration of VIP promotes and accelerates early myelination and growth of regenerating axons.     

Relationship between functional deficiencies and the contribution of myelin nerve fibers derived from L-4, L-5, and L-6 spinolumbar branches in adult rat sciatic nerve

The distribution and relative intrafascicular contribution of myelin fibers derived from spinal segments L-4 to L-6 were analyzed in adult rat sciatic nerve and its main branches, using 200-kDa neurofilament subunit immunodetection in previously injured nerve sections in the L-4 or L-5 spinal branch or both. These branches' functional contribution was evaluated 16 days after the injury, using the method of J. Bain, S. Mackinnon, and D. Hunter (1988, Plast. Reconstr. Surg. 83: 129-136). A common topographic intrafascicular distribution was found in 69% of cases, with notable segregation of L-4 and L-5 fibers and a random distribution for L-6 fibers. At sciatic nerve main branch level, L-4 contributes almost entirely to the peroneal nerve, L-5 to the tibial nerve, and L-6 and other branches to the sural nerve. After injury to L-4, a significant reduction in peroneal nerve functional index (PFI) was observed, as was a reduction in print length (PL). Injury to L-5 caused a significant reduction in the sciatic (SFI) and tibial (TFI) functional nerve indices, an increase in PL, and a reduction in the spread between opposite toes (TS). Finally, transection of both L-4 and L-5 was followed by a significant reduction in all functional indices measured, an increase in PL, and a reduction in intermediate toe (ITS) and opposite toe spread (TS). The results indicate a direct relationship between the distribution and contribution of the spinal nerve fibers forming the sciatic nerve and the alteration in functional indices for sciatic, tibial, and peroneal nerves.     

The role of a barrier between two nerve fascicles in adjacency after transection and repair of a peripheral nerve trunk

Abstract

Aberrant reinnervation of target organs caused by misdirected axonal growth at the repair site is a major reason for the poor functional outcome usually seen after peripheral nerve transection and repair. The following two studies investigate whether criss-crossing of regenerating rat sciatic nerve axons between tibial and peroneal nerve fascicles can be reduced by using a barrier at the coaption site. The left sciatic nerve was transected and repaired at mid-thigh as follows: epineural sutures (group A, A-II), fascicular repair of tibial and peroneal nerve fascicles (group B, B-II), fascicular repair of tibial and peroneal nerve fascicles separating the two fascicles with a pedicled fat flap (group C), Integra^® (group D) or non-vascularized autologous fascia (group C-II). In the control groups E and D-II, only the left tibial fascicle was transected and repaired. Four and 5 months postoperatively, the outcome of regeneration was evaluated by histology, by retrograde tracing, and by assessment of the muscle force of the gastrocnemius and tibial anterior muscles. The tracing experiments showed that specificity of muscle reinnervation significantly improved when a barrier was employed, which significantly or clearly improved muscle twitch tension in groups C and D. However, muscle contraction force was not better when fascia was used as barrier. The histological picture indicated that this inferior result in group C-II was due to nerve compression caused by fibrotic scar tissue at the site of the fascia graft. Results of this study show that a pedicle fat flap and Integra^® used as barrier significantly prevent aberrant reinnervation between two sutured nerve fascicles in adjacency resulting in improved motor recovery in rats. Non-vascularized autologous fascia however, reduces also criss-crossing of regenerating axons between the fascicles, but causes significant nerve compression.     

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.