The sprouting of saphenous nerve terminals in the spinal cord following early postnatal sciatic nerve section in the rat

Transganglionic labelling of the saphenous nerve in rats with WGA-HRP revealed the central distribution of its terminals in the lumbar dorsal horn. The terminal field was clearly defined and consistent in rats aged between day 6 and day 90. If, however, the sciatic nerve was sectioned on day 1 of postnatal life, the saphenous terminal field expanded to occupy twice the normal area (measured between the L2 and L4 boundaries). The spread was caudal, medial, and lateral into areas normally occupied by sciatic nerve terminals. This sprouting was very weak if the sciatic nerve was sectioned later in postnatal life, on day 5, and nonexistent if sectioning took place on day 10. Crushing the sciatic nerve on day 1 also triggered the effect but the spread of the terminal field was less than that produced by section of the sciatic nerve. There was no evidence of sprouting from the contralateral intact sciatic nerve. The results demonstrate the necessity of intact afferent input during a critical period of neonatal life in order to maintain the precise somatotopic termination pattern of dorsal root afferents.     

Organization of hindlimb nerve projections to the rat spinal cord: A choleragenoid horseradish peroxidase study

The aim of the present study has been to investigate the projections of hindlimb muscle afferent fibers to the spinal cord with particular emphasis on the ventral horn and the column of Clarke. Following transections of the appropriate ventral roots, injections of the B-subunit of cholera toxin conjugated to horseradish peroxidase were made into the tibial, peroneal, hamstring, superior gluteal, femoral, and obturator nerves in one group of adult rats. In another group of rats, similar experiments were done with intact ventral roots in order to map the location in the ventral horn of the motoneuron cell columns supplying each investigated nerve. An extensive overlap was found for the different nerve projections to Rexed's laminae V-VII. A somatotopic organization of the nerve projections was seen in the lamina IX cell groups of the ventral horn as well as in the column of Clarke, even though an overlap existed. The densest primary afferent projection from each injected nerve was to its homonymous motoneurons. Only a small to moderate overlap between the projections of the tributary branches of the sciatic nerve was found in the ventral horn, whereas the obturator and femoral nerve projections showed more profound overlap. In the column of Clarke, hindlimb nerves innervating distal muscles projected medially, and nerves innervating proximal muscles projected laterally. © 1996 Wiley-Liss, Inc.     

Origin and distribution of phrenic primary afferent nerve fibers in the spinal cord of the adult rat

Previous studies from this laboratory have localized and morphologically characterized phrenic motor neurons in the rat spinal cord at light and electron microscopic levels. The present investigation used a modification of the TMB method for the retrograde transport of horseradish peroxidase (HRP) to describe at light microscope levels the origin and distribution of phrenic primary afferent axons in the adult rat spinal cord. Dry HRP crystals were applied to the central stump of the transected phrenic nerve in the neck to label spinal ganglion cell bodies and thus determine the levels of origin of afferent axons in the phrenic nerve. Camera lucida drawings were then made from serial sections through the appropriate spinal cord levels to determine the specific distribution of transganglionically labeled phrenic central axonal processes within the spinal cord. HRP-labeled phrenic primary neurons were observed in the C3 to C7 spinal ganglia. The camera lucida studies indicated that the transganglionically labeled central processes of phrenic primary afferent axons distributed into the dorsal horn at the C4 and C5 levels of the spinal cord. Furthermore, central processes distributing to C5 were more numerous than those that distributed to C4. Afferent axons were never seen in the dorsal horn at C3, C6, or C7. As spinal ganglion cells were labeled at C3 above and C6 and C7 below, it follows that central processes of phrenic afferent fibers descend and ascend in the dorsal columns of the spinal cord before distributing into the dorsal horn. Specifically, the labeled primary afferent axons and their collateral branches were found in the fasciculus cuneatus, and in laminae I, II, III, and IV of the dorsomedial aspect of the dorsal horn. The function of these central axonal processes is unknown, but based on a comparison of our morphologic data with previous physiological and anatomical studies, we suggest that phrenic afferent fibers may arise from proprioceptors (muscle spindles and Golgi tendon organs), nociceptors, or rapidly adapting mechanoreceptors (Pacinian corpuscles) within the diaphragm.     

Motor and sensory neurons of the rat sural nerve: A horseradish peroxidase study

Neurons of origin of the rat sural nerve were labelled with horseradish peroxidase. Dorsal root ganglionic cells were located in the L4 and L5 ganglia, and occasionally at the L6 level. Most of these sensory neurons measured under 35 μm in diameter. In keeping with previous electrophysiological studies suggesting the presence of motor fibers to plantar muscles in the rat sural nerve, motoneurons were identified at the caudal end of the L5 spinal segment, intermingled in the posterior aspect of the ventral horn with posterior tibial motor cells supplying the foot muscles. A quantitative analysis of HRP-labelled motoneurons revealed no difference between normal (average 67) and deafferented animals (average 70), the values being only marginally lower than counts of motor axons in deafferented sural nerves (average 80).     

Spread of saphenous somatotopic projection map in spinal cord and hypersensitivity of the foot after chronic sciatic denervation in adult rat

The left sciatic nerve was cut and ligated in adult rats (chronic denervation). Twenty-one days later the right sciatic nerve was cut and ligated (acute denervation). The somatotopic maps of the surviving intact saphenous nerves (left and right) were compared on day 21 by recording from single interneurons in the dorsal horn of the spinal cord. On the acute side saphenous mediated natural responses were observed only as far caudally as L3, while no natural responses were found in L4 and 5 (this silent zone in L4 and 5 had previously been sciatic territory). In contrast, after chronic sciatic denervation, L4 and L5 were not silent to natural stimulation as the saphenous natural responses had spread into the sciatic territory. Saphenous inputs always won the sciatic territory in L4 and L5 over competing thigh afferents after chronic sciatic denervation. Electrical stimulation of the saphenous nerve on the acute side produced unit responses all the way down to S1 (with no silent areas in L4 and 5). These electrically evoked unit responses in L4 to S1 of the acute side were called ‘long range’ pathways. There were no differences in ‘long range’ electrical responses in the acutely and chronically denervated cord. The caudal boundary for electrically evoked saphenous responses was S1 on both sides of the cord, and post stimulus histograms of unit responses were not statistically different on the two sides. Thus after chronic sciatic denervation natural responses mediated by saphenous spread caudally into sciatic territory, but electrically evoked responses did not change.Behaviorally, there was the expected spread of saphenous mediated responses from the medial toe and foot to more lateral regions after chronic sciatic denervation. Unexpectedly, there was an increase in sensitivity (hyperalgesia) of the medial toe and foot. We postulate that this increased sensitivity might be mediated by the spread of saphenous projections into L4 and L5 of the cord after chronic sciatic denervation. Perhaps post traumatic neuralgia in humans could be due to increased number of spinal cord cells responding to stimulation of the receptive field of the surviving nerve.     

Corneal sensory pathway in the rat: a horseradish peroxidase tracing study

The methods of transganglionic transport of horseradish peroxidase (HRP) and horseradish peroxidase--wheat germ agglutinin (HRP-WGA) were used to determine the location within the trigeminal ganglion of the primary afferent neurons that innervate the rat central cornea, and the brainstem and spinal cord termination sites of these cells. In each of 18 animals, solutions of HRP or HRP-WGA were applied to the scarified corneal surface and allowed to infiltrate into the corneal epithelium and stroma for 15 minutes. Postmortem examination of the corneal whole mounts from the experimental animals, and of corneas and neural tissues from several control animals, showed that the HRP/HRP-WGA remained confined to the central cornea with no spread into adjacent intra- or extraorbital tissues. HRP-labeled corneal afferent somata were located in the dorsal part of the ophthalmic region of the ipsilateral trigeminal ganglion. The central fibers of the corneal afferent neurons projected very heavily to interstitial nuclei of Cajal in the spinal tract of V at the level of caudal pars interpolaris and rostral pars caudalis, lightly to the pars caudalis/C1 transition zone, and sparsely to the dorsal horn of spinal cord segments C1-C3. The trigeminal main sensory nucleus, pars oralis, the rostral three-fourths of pars interpolaris, and an extensive midregion of pars caudalis were totally devoid of reaction product. Terminal fields in caudal pars caudalis and in the spinal cord dorsal horn were concentrated largely in the outer half of lamina II, with lesser accumulations in lamina I, the deeper half of lamina II, and in lamina III. The present study demonstrates for the first time by means of an anatomical tracing procedure the brainstem termination sites of corneal afferent neurons in the rat. The patchy, discontinuous nature of the corneal afferent projection to the caudal trigeminal brainstem nuclear complex (TBNC), and the total lack of corneal projections to rostral subdivisions of the TBNC, provide an exception to the general rule of trigeminal organization in which most areas of the head and face are represented as continuous columns throughout the rostrocaudal extent of the ipsilateral TBNC.     

Ascending myelinated axons of primary sensory neurons of the sciatic nerve in the posterior column of the rat spinal cord

The present study was undertaken to obtain morphologic data about the posterior column of the spinal cord to characterize ascending myelinated axons of primary sensory neurons of the sciatic nerve. By applying doxorubicin to the right sciatic nerve in eight male Wistar rats, selective degeneration of centrally directed axons of these neurons in the posterior column was produced. Epon-embedded transverse sections of the posterior column at spinal cord segments C1, C3, C8, T6, L3 and L5 showed a circumscribed area (R) that contained a cluster of degenerated myelinated fibers. To characterize area R, its size and distances between various defined points on transverse sections of the posterior column were measured and compared at several spinal segments. The location of area R was illustrated in representative rats. The posterior intermediate septum corresponded to the lateral border of area R at C8 and T6. To characterize the putatively degenerating and degenerated myelinated fibers, area L in the left posterior column, corresponding to area R, was defined, and subsequently the number and size distribution of normal-appearing myelinated fibers in areas R and L were evaluated at C3, T6 and L3 in four rats. After comparative evaluation of these data, it was concluded that large myelinated fibers degenerated preferentially in area R. The number of putatively degenerating and degenerated myelinated fibers in area R at segments C3 and T6 was estimated to be 38.6% and 50.1%, respectively, of that at segment L3. Received: 25 August 1995 / Revised, accepted: 25 January 1996     

A comparative study of the effects of chronic axotomy, crush lesion and re-anastomosis of the rat sural nerve on horseradish peroxidase labelling of primary sensory neurons

Chronic axotomy is detrimental to the incorporation of horseradish peroxidase (HRP) by neurons of the central and peripheral nervous system. Using the rat sural nerve as a model, this study aimed to determine the effects of other types of nerve injury on the peroxidase labelling of dorsal root ganglion (DRG) cells. Compared to the decreased labelling occurring shortly after permanent transection of the sural axons at the ankle, crush injury of the nerve had no effect on the number and size distribution of peroxidase-stained cells. Re-anastomosing the sural nerve to its own distal segment or to the tibial nerve delayed the changes in HRP neuronal labelling, which subsequently were less severe in neurons allowed to reinnervate their own nerve. It also sustained the incorporation of HRP by many large DRG neurons, a function which is lost shortly after these cells are chronically axotomized. Nerve re-anastomosis also prevented the retrograde atrophy of myelinated and unmyelinated nerve fibers which is triggered by permanent transection. Based on the preservation of fiber counts in the sural nerves proximal to the site of surgery, with no evidence of degeneration, our observations possibly reflect alterations in the peroxidase metabolism of DRG neurons depending on the type of axonal injury they sustained and the possibility they had upon regeneration to contact endoneurial tubes and ultimately their original end-organs.     

Binding of the neurotrophic peptide Org 2766 to rat spinal cord sections is affected by a sciatic nerve crush

The binding of the neurotrophic peptide, [3H]Org 2766 (55 nM), to rat spinal cord sections was studied, employing quantitative autoradiography. The binding was unevenly distributed over spinal cord structures and was displaceable by non-labelled Org 2766 to a limited extent (35%). Binding could not be displaced by the opiate antagonist, naloxone, indicating that [3H]Org 2766 binding sites are distinct from opiate receptors. However, the exact nature of the binding sites remains to be elucidated. A marked left-right difference in [3H]Org 2766 binding in the dorsal horns of the spinal cord at level L2 was observed, 6 days after unilateral crush lesioning of the sciatic nerve. No such effect was found at level T10. After 28 days, when sensorimotor functioning had completely recovered, the [3H]Org 2766 binding pattern was comparable to that in sham-operated rats again. It is suggested that Org 2766 binds to axonal sprouts or glia in the dorsal horn of the spinal cord.     

Morphology of sympathetic preganglionic neurons in the neonatal rat spinal cord: an intracellular horseradish peroxidase study

Understanding the central neural control of autonomic functions requires a knowledge of the morphology of the preganglionic neurons, for the location of the dendritic arborizations of these neurons will indicate which central pathways may have access to them. In the present study, individual sympathetic preganglionic neurons in the neonatal rat spinal cord have been examined by the intracellular injection of horseradish peroxidase (HRP) in an in vitro preparation. Seventeen HRP-labeled preganglionic neurons in thoracic segments T1-T3 were examined in detail; of these, 12 somata were located in the intermediolateral cell column (IML), one in the lateral funiculus (LF), two in the intercalated nucleus (IC), and two at the border between IML and IC. All of the neurons had extensive dendritic arborizations arising from an average of six primary dendrites; the average total dendritic length for these cells was 2,343 microns. The morphology of preganglionic neurons differed depending on the location of their cell bodies. Preganglionic neurons located in the IML were essentially two-dimensional: the cells had some dendrites that coursed rostrocaudally for 300-500 microns within the IML and others that coursed mediolaterally, extending to the lateral surface of the cord and close to the central canal. Axons of these cells coursed ventrally from the cell body and exited from the spinal cord at the first ventral root caudal to the cell body. No intraspinal axon branches were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Labeling of cutaneous sensory nerve endings with axonally transported horseradish peroxidase and wheat germ agglutinin-horseradish peroxidase conjugate: a methodological study in the rat

Practical aspects on the use of horseradish peroxidase (HRP) and wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) to trace peripheral cutaneous nerve endings have been studied. The parameters studied included application site of the tracer, post-application survival time, tracer concentration and tracer volume. These parameters were examined in the glabrous skin of the rat hindpaw. The best results were obtained with injections of 1 microliter WGA-HRP (20 micrograms/microliter) in dorsal root ganglia innervating the examined cutaneous region and a postinjection survival time of 18-36 h. With this approach extensive and heavy labeling was achieved of epidermal nerve endings, nerve endings in Merkel cell-neurite complexes and Meissner corpuscles. Useful, but less extensive labeling of these types of peripheral nerve endings, was obtained with injections of HRP in the lumbar spinal cord dorsal horn.     

Recurrent potentials in human peripheral sensory nerve: possible evidence of primary afferent depolarization of the spinal cord.

To study slowly conducted components of the orthodromic compound sensory action potential (CSAP), the response evoked at the lateral malleolus in the sural nerve was recorded through near-nerve needles at two to four sites along the nerve at midcalf. When 500 to 2000 responses were averaged at high gain, components with latencies of 30 to 80 ms were often recorded. In contrast to the main component and late components with latencies of less than 15 to 20 ms, the latencies of these extremely late components diminished the closer to the spinal cord that they were recorded. This suggested that the components were conducted antidromically from proximal to distal. This assumption was supported by abolishing the components by local anesthesia of the nerve proximal to the recording electrodes. These antidromic potentials therefore appear to be due to recurrent discharges in the sural nerve. Recurrent discharges were recorded from 65% of 60 subjects (18 normal subjects and 42 patients with peripheral or central nervous system disorders). The latencies of the recurrent discharges allowed conduction to and back from the spinal cord. Although the origin of these potentials remains unknown, we suggest that they are due to dorsal root reflexes within the spinal cord. In this case, the responses may be a direct expression of primary afferent depolarization (PAD) seen in presynaptic inhibition, and may be of value in further studies on the physiology and pathophysiology of presynaptic inhibition of cutaneous fibers in man.     

Compensatory projection of primary nociceptors and c-fos induction in the spinal dorsal horn following neonatal sciatic nerve lesion

The sciatic nerve was cut in newborn rats, and prevented from regenerating for 8 weeks. The number of dorsal root ganglion (DRG) neurons in L4 and L5, the distribution of central axon terminals of primary nociceptors, and the activity of secondary nociceptors were examined in the lumbar dorsal horn. The neonatal sciatic lesion caused about 60% reduction of DRG neurons. The central terminal field of the sciatic primary nociceptors negatively labeled by in situ binding of Bandeiraea simplicifolia isolectin B4 (BsIB4) markedly shriveled. Instead, the central representation of the saphenous nerve and the posterior cutaneous nerve of the thigh (PC) expanded. The laminae I/II neuropil in the medialmost (1/4) of the L3 dorsal horn and in the second lateral (1/4) around the L4/5 junction was occupied by the BsIB4 binding sites derived from the saphenous and the PC primary neurons, respectively. Noxious stimuli applied to the receptive fields of the saphenous and the PC nerves induced c-Fos-like immunoreactivity in many neurons in the expanded central terminal fields of the nerves. The collateral sprouts of uninjured primary nociceptors did not only invade the deafferented area of the dorsal horn but also established functional synaptic connections.     

Effect of 17 beta-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-alpha and estrogen receptor-beta localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-alpha and estrogen receptor-beta mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-alpha and -beta mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i). axotomized motor neurons increase expression of estrogen receptors-alpha and -beta mRNA, (ii). estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii). estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.     

Morphine acts via mu-opioid receptors to enhance spinal regeneration and synaptic reconstruction of primary afferent fibers injured by sciatic nerve crush

The present study investigated whether morphine can promote regeneration and synaptic reconstruction of the terminals of injured primary afferent fibers in lamina II of the spinal cord in rats following sciatic nerve injury. Fluoride-resistant acid phosphatase (FRAP)-positive terminals in lamina II of the L4 spinal segment after sciatic nerve injury were assessed after treatment with vehicle, morphine, and naloxone plus morphine. Under the electron microscope, types I and II complex terminals of unmyelinated afferent fibers from the dorsal root, simple terminals of interneuronal axons, and terminals of descending axons at lamina II of the L4 spinal segment were documented in the different groups after injury. FRAP-positive terminals in lamina II were depleted after sciatic nerve injury in the vehicle group. Treatment with morphine increased the numbers of FRAP-positive terminals, and this was prevented by naloxone. The present study demonstrates that morphine may promote the regeneration and synaptic reconstruction of the terminals of injured primary unmyelinated afferent fibers in lamina II of spinal cord, by a process mediated by mu-opioid receptors.     

Differences in horseradish peroxidase labeling of sensory, motor and sympathetic neurons following chronic axotomy of the rat sural nerve

In an attempt to clarify the ultimate fate of permanently axotomized adult primary neurons, horseradish peroxidase (HRP) was used as a cell marker to label the motor, sensory and postganglionic sympathetic neurons of rat sural nerves which had been sectioned at the ankle and prevented from regenerating for periods of up to 80 weeks. Axotomy did not affect sympathetic neurons, but resulted 4 weeks later in a sudden reduction in the number of labeled sensory and motor cells which persisted to the end of the study. The missing neuronal population amounted to 44.4% and 45.9% respectively of the normal sensory and motor contingent and included most of the large afferent and efferent neurons. However, examination of sural nerves at the thigh, 30 mm proximal to the neuroma, revealed marked axonal atrophy but no change in the number of myelinated and unmyelinated fibers up to 52 weeks after axotomy. Such prolonged survival of the peripheral processes is indirect evidence that axotomized neurons can endure long-term detachment from their end organs and suggests that the lack of HRP labeling in certain sensory and motor neurons does not imply their degeneration, but expresses one of many retrograde dysfunctions triggered by axotomy.