Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration

Summary Adrenergic innervation of blood vessels in rat tibial nerve during Wallerian degeneration was examined, using the formaldehyde-induced histo-fluorescence method. The left sciatic nerve was transected at the level of the sciatic notch, whereas the right sciatic nerve was left intact and used as control. At 1, 3, 7, 14, 42, 56 or 84 days after transection, the tibial nerves of the transected and contralateral sides were exposed. Pieces of each nerve were used for light microscopy or for examination of adrenergic innervation with the fluorescence microscope. One day after transection, no adrenergic nerve fiber was observed in the endoneurium of the transected nerve. After 3 days, adrenergic innervation of small-and medium-sized arterioles in the epi-perineurium was absent, and after 7 days no fibers were visible around large arterioles. Fluorescent fibers were not detected even at 84 days post-surgery. It is concluded that adrenergic innervation of blood vessels in the rat tibial nerve is irreversibly lost after permanent axotomy, and that adrenergic regulation of nerve blood flow may also be lost.     

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.     

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.     

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.     

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.     

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     

Effect of rhTNF-alpha injection into rat sciatic nerve.

To assess whether TNF-alpha causes inflammatory demyelination or axonal degeneration, we injected into rat sciatic nerve saline, 100 U and 1000 U of rhTNF-alpha and studied the electrophysiological and pathological effects. At day 1 electrophysiology showed a slight reduction of proximal compound muscle action potential amplitude and pathology showed edema, inflammatory infiltration of vessel walls and endoneurium only in nerves injected with 1000 U of rhTNF-alpha. At day 5, there was no demyelination and a percentage of degenerated fibers similar in the three groups. To study the blood-nerve barrier, fluorescein isothiocyanate-labelled albumin was given intravenously after intraneural injection. The nerves injected with 1000 U rhTNF-alpha showed a leakage of the tracer in the endoneurium. TNF-alpha does not appear, at the doses used, to have myelinotoxic or axonopathic properties. The electrophysiological effect at day 1 may be due to mechanical compression of nerve fibers as a result of the blood-nerve barrier damage with consequent endoneurial edema.     

Laminin in rat sciatic nerve undergoing Wallerian degeneration. Immunofluorescence study with laminin and neurofilament antisera

Immunofluorescence with laminin antisera revealed a striking change in the localization of this basal membrane glycoprotein in rat sciatic nerve as a result of Wallerian degeneration. The staining was confined to the endoneurium in normal sciatic nerve and during the first days of degeneration. On day 11 endoneurial tubes were no longer identified in the distal stump of crushed nerves or of nerves that had been transected and tightly ligated to prevent regeneration. In both crushed and ligated nerves proliferating Schwann cells forming the cell-bands of Büngner were intensely laminin positive. With double-labeling experiments, laminin and neurofilament antisera revealed similar but not identical staining patterns in crushed nerves, which suggests a close relation between laminin and regenerating axons. Crushed nerves had recovered their normal appearance 18 days after operation while anti-laminin reactivity was decreased in parts of ligated nerves undergoing fibrosis. The localization of laminin in reactive Schwann cells was confirmed by electron microscopy using the indirect immunoperoxidase procedure. Axons did not contain reaction product.     

Thrombospondin expression in nerve regeneration I. comparison of sciatic nerve crush, transection, and long-term denervation

Patterns of expression of the extracellular matrix molecule thrombospondin (TSP) were examined during peripheral nerve regeneration following sciatic nerve crush or transection. In noninjured nerve, was present in the axoplasm, Schwann cells, endoneurium, and perineurium of the adult mouse sciatic nerve. Following nerve crush or nerve transection, levels of TSP rapidly increased distal to the trauma site. Elevated levels of TSP were present distal to regenerating axons, while expression gradually returned to normal proximal to the regenerating axons. When reinnervation was blocked, TSP levels remained high in the endoneurium in excess of 30 days, but TSP was absent by 60 days. Following reanastomosis of the proximal and distal segments after 60 days of denervation, TSP was re-expressed in the distal nerve stump. These results indicate that TSP, which is involved in neuronal migrations in the embryo and neurite outgrowth in vitro, appears to play a role in axonal regeneration in the adult peripheral nervous system.     

On the occurrence of the fenestrated vessels in Wallerian degeneration of the peripheral nerve

Summary Ultrastructural studies were made on the distal segments of the mouse phrenic nerve after crush injury. In the control, endoneurium contained only unfenestrated capillaries. In the experiment, from day 2 to day 6, endoneurial capillaries occasionally showed fenestrations with the attenuation of its cytoplasm. At this stage, axonal degeneration and myelin breakdown became evident showing early stage of Wallerian degeneration. In addition, detachment of the neighboring endothelial cells concomitant with the invasion of macrophage was also observed. These findings were previously unobserved changes of the endoneurial endothelium in Wallerian degeneration. The significance of the early occurrence of fenestrae was discussed briefly.     

Retrograde axonal transport in rat sciatic nerve after nerve crush injury

We investigated the quantitative alterations in retrograde transport of proteins following a nerve crush injury using the 3H N-succinimidyl propionate (3H NSP) method in rat sciatic nerve. After subepineurial injection of 3H NSP into the nerve the amount of radioactively labeled proteins accumulating in the cell bodies of the motor and sensory neurons was determined 1 day or 7 days later in nerves which had been crushed distal to the injection site 1, 3, 5, 7, or 33 days prior to 3H NSP labeling. One day accumulation in the DRG and spinal cord was not altered by nerve crush. Seven day accumulation in the DRG was initially slightly increased, then fell to 73% of control by 7 days, remaining reduced 33 days after crush. Seven day accumulation in the spinal cord was reduced to 25% of control 1 day after crush and remained at that low level except for 5 days post-crush when a normal amount of labeled protein was transported to the spinal cord. The time course of these changes suggests that quantitative alterations in retrograde transport may be involved in the long-term trophic interactions between the cell body and periphery, but are too slow to account for the earliest perikaryal responses to injury. In addition, the difference between the alterations of retrograde transport in motor and sensory neurons may reflect fundamental differences in the composition of retrograde transport in those different systems.     

Adrenergic innervation of the vasa nervorum in the cranial nerves and spinal roots in the subarachnoid space

Fluorescence microscopy revealed that the innervation of the vasa nervorum of both cranial and spinal nerve roots in the subarachnoid space in the monkey is adrenergic in nature. The endoneurium of the cranial nerve roots contained free adrenergic nerve fibers which were not related to the vessel wall.     

Alpha-melanocyte-stimulating hormone stimulates the outgrowth of myelinated nerve fibers after peripheral nerve crush

The effect of alpha-melanocyte-stimulating hormone on peripheral nerve regeneration was studied by monitoring functional recovery and quantifying histologic changes that follow crush lesion of the rat sciatic nerve. The results showed that such treatment of rats with a crushed sciatic nerve resulted in a reduction of the recovery period and in an increase in the number of regenerating nerve fibers.     

Toxin-induced inhibition of nerve terminal growth

An in vivo model to permit the quantitative study of the effects of neurotoxins upon nerve terminal growth has been developed and used in practice to test the ability of methyl mercury chloride, leptophos, ethanol, morphine and acrylamide to inhibit nerve terminal growth. The model utilized the sympathetic innervation of rat submaxillary salivary glands. Nerve terminal growth was induced following crush of the sympathetic axons which innervated the submaxillary gland and quantified by measuring the return towards normal values of the density of sympathetic innervation in denervated glands. The nerve crush reduced the density of sympathetic innervation to a very low value but nerve growth began within 9 days and was sufficient to restore the density of innervation to approximately 70% of normal values in three weeks. Restoration of the original density of innervation took between 40 and 60 days. The major advantage of the model is its ability to detect and quantify 30% - 100% inhibition of nerve terminal growth. The major disadvantage is the time required to measure the density of sympathetic innervation though this can be minimized in a number of ways which are discussed. Acrylamide inhibited sympathetic nerve terminal growth without causing degeneration of sympathetic neurones. This demonstrates that toxins can selectively inhibit nerve terminal growth which will result in altered neuronal circuitry and abnormal function. Methyl mercury chloride, leptophos, ethanol and morphine did not inhibit sympathetic nerve terminal growth.     

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.     

The effect of aging on the morphological nerve changes during muscle reinnervation after nerve crush

The extent of the muscle endplate reinnervation that followed crush injury of the sciatic nerve was compared between young adult (4 and 5 months old) and aged (24 months old) animals. The time course of regeneration in the muscular nerve bundle, its ramification, and the nerve terminal was immunohistochemically estimated using an antibody against the neuron specific enolase (NSE), a neuronal marker. During early phases of regeneration (7, 21 and 28 days post-crush) in the young adult animal, there were tortuosity, vacuolation and/or unfasciculation in the nerve bundle and its ramification, along with immature nerve terminals and multiple innervation. Following a subsequent advancement in reinnervation to the denervated motor endplates, the adult type of single motor innervation was common on the day 56. The old muscles basically followed the course of reversible axotomy alike the young adult ones. The age difference accounted for as follows: a reduced rate of reinnervation as indicated by a greater frequency of abnormal nerve bundles and immature nerve terminals at 28 days and 56 days post-crush, as well as unusual pathways or striking tortuosity represented by the NSE-labeled processes between day 7 and 56; late in the reinnervation period, abnormal regeneration characterized by damage of the nerve bundle, and poorly developed terminal architectures. These results suggest that despite the capability of the nerve from the old animals to extend its process, re-establishment of normal single motor innervation is reduced due to some age-related deficits, which may be related to the impaired Schwann cell-axon interactions.     

Effect of pulsed magnetic field on regenerating rat sciatic nerve: An in-vitro electrophysiologic study

Some experimental studies report that low-frequency pulsed electromagnetic field (PEMF) stimulation may accelerate regeneration in peripheral nerves. In the present study, effects of PEMF on the regeneration of the crushed rat sciatic nerves were investigated with histological and in-vitro electrophysiological methods (sucrose-gap). After crush injury of the sciatic nerves, rats were divided into 5, 15, 25, 38 day-groups and exposed to PEMF (1.5 h/day, intensity; 1.5 mT, consecutive frequency; 10-10-100 Hz). In the 15th day post crush, compound action potential (CAP) amplitude was measured as 5.5 ± 1 mV (crush group) and 5.4 ± 1.2 mV (crush + PEMF group). In addition, half width of CAP extended ～ 3 fold in both groups and frequency-dependent amplitude inhibition (FDI) decreased -20% at 100 Hz. In the 38th day, amplitude of CAP, half width of CAP and FDI were measured nearly intact nerve values in both groups. In histological examinations, Wallerian degeneration was observed similar progress between both groups. The results were compared between crush and crush + PEMF groups, it was found that the effect of PEMF was not significant. The authors conclude that PEMF were ineffective on rat sciatic nerve regeneration.     

Effect of pulsed magnetic field on regenerating rat sciatic nerve: an in-vitro electrophysiologic study

Some experimental studies report that low-frequency pulsed electromagnetic field (PEMF) stimulation may accelerate regeneration in peripheral nerves. In the present study, effects of PEMF on the regeneration of the crushed rat sciatic nerves were investigated with histological and in-vitro electrophysiological methods (sucrose-gap). After crush injury of the sciatic nerves, rats were divided into 5, 15, 25, 38 day-groups and exposed to PEMF (1.5 h/day, intensity; 1.5 mT, consecutive frequency; 10-40-100 Hz). In the 15th day post crush, compound action potential (CAP) amplitude was measured as 5.5+/-1 mV (crush group) and 5.4+/-1.2 mV (crush+PEMF group). In addition, half width of CAP extended ~3 fold in both groups and frequency-dependent amplitude inhibition (FDI) decreased approximately 20% at 100 Hz. In the 38th day, amplitude of CAP, half width of CAP and FDI were measured nearly intact nerve values in both groups. In histological examinations, Wallerian degeneration was observed similar progress between both groups. The results were compared between crush and crush + PEMF groups, it was found that the effect of PEMF was not significant. The authors conclude that PEMF were ineffective on rat sciatic nerve regeneration.     

The effect of chronic nicotine and withdrawal on intra-axonal transport of acetylcholine and related enzymes in sciatic nerve of the rat

Summary The content of acetylcholine (ACh) and activities of the cholinergic enzymes choline acetyltransferase (CAT) and ACh-esterase (AChE) were studied in intact and crushed rat sciatic nerve afterchronic nicotine administration andwithdrawal 2 days before the final experiment. Nicotine was given in the drinking water during 8–10 weeks and the final dose reached was about 8 mg/kg/day, i.e. equivalent to that of the heavy cigarette smoker.In thechronic nicotine group, ACh levels and AChE activity of uncrushed nerve were significantly decreased as compared to the controls. The accumulation of ACh and AChE proximal to a single crush was also somewhat decreased, but significant only for AChE at 18 hours postoperatively. Afterwithdrawal of nicotine for 2 days the ACh content of both uncrushed and 12 hours crushed nerves were further decreased, while AChE was instead increased to control (uncrushed) or even supranormal (18-hour crush) levels.     

Trophic influence of the distal nerve segment on GABAA receptor expression in axotomized adult sensory neurons

The depolarizing action of gamma-aminobutyric acid (GABA), or the GABAA receptor agonist muscimol, on rat dorsal root (L4 and L5) fibers is attenuated following transection, but not crush, of the sciatic nerve. Following discrete nerve crush, axons actively regenerate and contact both the distal nerve segment and the peripheral target tissues. The aim of the present study was to distinguish between these two regions as possible sources of trophic support for retrograde maintenance of dorsal root GABA receptor sensitivity. A surgical procedure was employed to permit a delimited segment of axonal regeneration while prohibiting reestablishment of end organ innervation; the sciatic nerve was crushed and a ligature was placed 3 cm distal to the crush site. Under these conditions, the injury-induced decrement in the dorsal root GABA response, observed between 12 and 21 postoperative days, was significantly attenuated relative to that of ligated nerves, in which regeneration into the distal stump does not occur. The data suggest that nerve transection by ligation restricts trophic support for maintenance of GABA receptor expression in dorsal root ganglion (DRG) neurons. Furthermore, during regeneration the denervated distal nerve segment assumes a neurotrophic role in the maintenance of dorsal root GABA sensitivity, consistent with the hypothesis that growth factors derived from reactive Schwann cells may positively regulate the expression of receptors on axotomized sensory neurons.