Orthograde, Retrograde, and Turnaround Axonal Transport of Dopamine-β-Hydroxylase: Response to Axonal Injury

Reversal of the direction (turnaround) of orthograde axonal transport of dopamine-beta-hydroxylase (DBH) activity was studied at a ligature placed on rat sciatic nerve. DBH was allowed to accumulate at a ligature in vivo for selected intervals, at which time a second ligature was placed proximal to the first and turnaround transport measured just distal to the second tie after incubation in vivo or in vitro. Orthograde accumulation of DBH activity proximal to a ligature peaked at 2 days, and then rapidly decreased as a result of turnaround transport and injury-induced reduction of orthograde transport. Destruction of postganglionic sympathetic axon terminals in vivo with 6 hydroxydopamine resulted in a decrease in orthograde transport similar to that seen after axotomy and turnaround at or proximal to the site of chemical injury. Turnaround transport of DBH in vitro was blocked by incubation in the cold and in the presence of NaCN and vinblastine. Orthograde transport of DBH appeared to reverse direction within a few millimeters of a ligature.     

Death of an axon: studying axon loss in development and disease

The loss of axon branches is a common feature of both the developing and the diseased nervous system. Despite its fundamental importance, a clear mechanistic understanding is lacking on how axonal loss occurs. However, the first molecular inroads into post-traumatic (Wallerian) axon degeneration have recently been made. In parallel, imaging techniques that allow visualizing single axons in vivo are providing a first glimpse at the cellular mechanisms of active dismantling of superfluous or diseased axons. This gives hope that soon a clearer mechanistic understanding of axon loss will emerge: comparing different forms of axon loss will reveal the spectrum of axon loss mechanisms; studies aimed at integrating the known molecular and cellular players during axon loss will provide mechanistic insight into axon dismantling; finally—by understanding how axons are normally lost—we will hopefully find ways to protect them during neurological disease or after trauma. Note: Robert Feulgen Prize Lecture 2005 presented at the Joint meeting of The Histochemical Society and The Society for Histochemistry in Noordwijkerhout, The Netherlands. After completion of this review, a comprehensive and highly informative overview of axon loss events in development and disease was published (Luo and O’Leary 2005).     

Noradrenergic axon terminals in the substantia gelatinosa of the rat spinal cord

Summary The noradrenergic terminals in the substantia gelatinosa of the dorsal horn of the cervical spinal cord of the rat were investigated by means of the histofluorescence technique and electron-microscopic cytochemistry using the glyoxylic acid-KMnO₄ fixation technique. In accordance with the topographical distribution of fluorescent catecholaminergic fibers, noradrenergic terminals containing small granular vesicles were frequently observed electron microscopically in the outer layer of the substantia gelatinosa. These terminals were most frequently found to appose without showing typical synaptic features, small-caliber dendrites, spine apparatus, and rarely, large caliber dendrites. Only in a few cases, the noradrenergic terminals exhibited typical synaptic contacts with dendritic elements of small size. In addition, noradrenergic terminals apposed non-noradrenergic terminals containing small agranular vesicles. In rats bearing surgical lesions of the dorsal roots, no noradrenergic terminal were found in contact with the degenerated axon terminals in the substantia gelatinosa. These findings suggest that the noradrenergic afferents to the substantia gelatinosa may exert their influence on sensory transmission via dorsal horn cells.     

Inhibitory Smads differentially regulate cell fate specification and axon dynamics in the dorsal spinal cord

The roof plate resident BMPs have sequential functions in the developing spinal cord, establishing cell fate and orienting axonal trajectories. These activities are, however, restricted to the dI1–dI3 neurons in the most dorsal region of the spinal cord. What limits the extent of the action of the BMPs to these neurons? To address this question, we have examined both the distribution of the inhibitory Smads (I-Smads), Smad6 and Smad7 in the spinal cord and the consequence of ectopically expressing the I-Smads in chicken embryos. Our studies suggest that the I-Smads function in vivo to restrict the action of BMP signaling in the dorsal spinal cord. Moreover, the I-Smads have distinct roles in regulating the diverse activities of the BMPs. Thus, the ectopic expression of Smad7 suppresses the dI1 and dI3 neural fates and concomitantly increases the number of dI4–dI6 spinal neurons. In contrast, Smad6 most potently functions to block dI1 axon outgrowth. Taken together, these experiments suggest that the I-Smads have distinct roles in spatially limiting the response of cells to BMP signaling.     

Effects of combining methylprednisolone with rolipram on functional recovery in adult rats following spinal cord injury

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.     

Developmental switch in axon guidance modes of hippocampal mossy fibers in vitro

Hippocampal mossy fibers (MFs), axons of dentate granule cells, run through a narrow strip, called the stratum lucidum, and make synaptic contacts with CA3 pyramidal cells. This stereotyped pathfinding is assumed to require a tightly controlled guidance system, but the responsible mechanisms have not been proven directly. To clarify the cellular basis for the MF pathfinding, microslices of the dentate gyrus (DG) and Ammon's horn (AH) were topographically arranged in an organotypic explant coculture system. When collagen gels were interposed between DG and AH slices prepared from postnatal day 6 (P6) rats, the MFs passed across this intervening gap and reached CA3 stratum lucidum. Even when the recipient AH was chemically pre-fixed with paraformaldehyde, the axons were still capable of accessing their normal target area only if the DG and AH slices were directly juxtaposed without a collagen bridge. The data imply that diffusible and contact cues are both involved in MF guidance. To determine how these different cues contribute to MF pathfinding during development, a P6 DG slice was apposed simultaneously to two AH slices prepared from P0 and P13 rats. MFs projected normally to both the host slices, whereas they rarely invaded P0 AH when the two hosts were fixed. Early in development, therefore, the MFs are guided mainly by a chemoattractant gradient, and thereafter, they can find their trajectories by a contact factor, probably via fasciculation with pre-established MFs. The present study proposes a dynamic paradigm in CNS axon pathfinding, that is, developmental changes in axon guidance cues.     

Complete sciatic nerve transection induces increase of neuropeptide Y-like immunoreactivity in primary sensory neurons and spinal cord of frogs

Neuropeptide Y (NPY) was immunohistochemically investigated in the frog spinal cord and dorsal root ganglia after axotomy. In normal ganglia, moderate NPY-like immunoreactivity (NPY-IR) prevailed in large and medium cells. In the spinal cord, the NPY-IR was densest in the dorsal part of the lateral funiculus. Other fibers and neurons NPY-IR were observed in the dorsal and ventral terminal fields and mediolateral band. NPY-IR fibers were also found in the ventral horn and in the ventral and lateral funiculi. The sciatic nerve transection increased the NPY-IR in large and medium neurons of the ipsilateral and contralateral dorsal root ganglia at 3 and 7 days, but no clear change was found at 15 days. In the spinal cord, there was a bilateral increase in the NPY-IR of the dorsal part of the lateral funiculus. In the ipsilateral side, the NPY-IR was increased at 3 and 7 days but was decreased at 15 days. In the contralateral side, a significant reduction at 15 days occurred. These findings seem to favor the role of NPY in the modulation of pain-related information in frogs, suggesting that this role of NPY may have appeared early in vertebrate evolution.     

Progesterone Effects on Neuronal Ultrastructure and Expression of Microtubule-associated Protein 2 (MAP2) in Rats with Acute Spinal Cord Injury

(1) Following acute spinal cord injury, progesterone modulates several molecules essential for motoneuron function, although the morphological substrates for these effects are unknown. (2) The present study analyzed morphological changes in motoneurons distal to the lesion site from rats with or without progesterone treatment. We employed electron microscopy to study changes in nucleus and cytoplasm and immunohistochemistry for the microtubule-associated protein 2 (MAP2) for changes in cytoskeleton. (3) After spinal cord injury, the nucleoplasm appeared more finely dispersed resulting in reduced electron opacity and the nucleus adopted an eccentric position. Changes of perikarya included dissolution of Nissl bodies and dissociation of polyribosomes (chromatolysis). After progesterone treatment for 3 days, the deafferented motoneurons now presented a clumped nucleoplasm, a better-preserved rough endoplasmic reticulum and absence of chromatolysis. Progesterone partially prevented development of nuclear eccentricity. Whereas 50% of injured motoneurons showed nuclear eccentricity, only 16% presented this phenotype after receiving progesterone. Additionally, injured rats showed reduced immunostaining for MAP2 in dendrites, pointing to cytoskeleton abnormalities, whereas progesterone treatment attenuated the injury-induced loss of MAP2. (4) Our data indicated that progesterone maintained in part neuronal ultrastructure, attenuated chromatolysis, and preclude the loss of MAP2, suggesting a protective effect during the early phases of spinal cord injury.     

Ingrowth of optic nerve fibers and onset of myelin ensheathment in the optic tectum of the trout (Salmo gairdneri)

Summary The early differentiation of the optic pathway of the trout was studied by means of autoradiography, silver impregnation and electron microscopy. Ingrowth of optic nerve fibers into the optic tectum was consistently shown by tracer application and Golgi studies to occur at stage 28, about one week before hatching. Fibers being arranged in discrete bundles were rapidly growing through the longitudinal axis of tectum and at stage 33 reached its posterior end. Cross sections of these fiber bundles at different positions revealed myelin ensheatment to be initiated at the end of stage 34 at the anterior pole of the tectum. Since in the optic nerve of the trout the onset of myelination occurred even earlier (stage 33), it is assumed that this differentiation process follows a rostro-caudal gradient during development of the optic pathway.     

Origins of nerve fibers containing nitric oxide synthase in the rat celiac-superior mesenteric ganglion

The origin of nitric oxide synthase-containing nerve fibers in rat celiac-superior mesenteric ganglion was examined using retrograde tracing techniques combined with the immunofluorescence method. Fluoro-Gold was injected into the celiac-superior mesenteric ganglion. Neuronal cell bodies retrogradely labeled with Fluoro-Gold in the thoracic spinal cord, the dorsal root ganglia at the thoracic level, the nodose ganglion, and the intestine from the duodenum to the proximal colon were examined for nitric oxide synthase immunoreactivity. About 60% of sympathetic preganglionic neurons in the intermediolateral nucleus projecting to the celiac-superior mesenteric ganglion were immunoreactive for nitric oxide synthase, as were approximately 27% of nodose ganglion neurons and about 65% of dorsal root ganglion neurons projecting to the cceliac-superior mesenteric ganglion. Neurons projecting to the celiac-superior mesenteric ganglion were found in the myenteric plexus of the small and large intestine. In the proximal colon, about 23% of such neurons were immunoreactive for nitric oxide synthase. However, in the small intestine, no immunoreactivity was found in these neurons.     

Immunohistochemical study on the distribution of serotonin fibers in the spinal cord of the dog

Summary Distribution of serotonin fibers in the spinal cord of the dog was investigated by means of a modified PAP method; a rabbit anti-serotonin serum prepared in the laboratory of the authors was used in this study. Serotonin fibers were revealed as PAP-positive dark-brown elements displaying dot-like varicosities (0.5–2.0 m in diameter). In the spinal cord of the dog, the distribution of serotonin fibers is extensive. These fibers occur more densely in more caudal segments and are most prominent at the sacrococcygeal level. From the level of the cervical spinal cord to the upper lumbar region, the descending serotonin fibers are located immediately under the pia mater in the ventrolateral portion of the lateral funiculus. In more caudal segments, serotonin fibers are dispersed throughout the ventral and lateral funiculi. These longitudinal en passage-fibers send numerous transverse collaterals to the gray matter. Serotonin fibers are distributed abundantly in the laminae I and III of the posterior column, while only a few fibers are found in the lamina II (substantia gelatinosa). In the intermediate zone, two descending serotonin pathways, i.e., lateral and medial longitudinal bundles, are observed to coincide topographically with the nucleus intermediolateralis at C8(T1)-L3(L4) and the nucleus intermediomedialis at C1-Co respectively. The former is particularly prominent and communicates with the contralateral bundle via commissural bundles at intervals of 300–500 m. The large motoneurons in the anterior column, especially those in the nucleus myorabdoticus lateralis within the cervical and lumbar enlargements, are closely surrounded by fine networks of serotonin fibers and terminals.Supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Preparation of Neurofilament Protein from Guinea Pig Peripheral Nerve and Spinal Cord

Abstract: A simple and rapid method for preparation of enriched neurofilament protein from mammalian peripheral nerve or spinal cord is described. Tissue extracts from guinea pig nerve or spinal cord are fractionated by ammonium sulfate fractionation, chromatography on Sepharose 4B, and precipitation with ethanol. Molecular exclusion chromatography on Sepharose 4B, in which the neurofilament protein elutes quantitatively in the exclusion volume of the column, with little contamination by other proteins, is found to be a highly effective purification step. The protein is found to precipitate in ammonium sulfate fractions over a wide range of salt concentration, from 20 to 80% saturation. It is found to be quantitatively precipitated in 40% v/v ethanol-water. The preparative method described yields 0.25 mg of neurofilament protein per gram of nerve or spinal cord, with a purity of approximately 50%. The three principal neurofilament polypeptides, which have molecular weights by SDS-polyacrylamide gel electrophoresis of 200K, 145K, and 68K, are found to be present in the preparation in a molar ratio of 1:2:6. A variant form of neurofilament protein occurring in approximately 20% of Hartley strain guinea pigs is described, which has the polypeptide composition: 200K, 192K, 145K, 68K.     

Androgen regulation of axon growth and neurite extension in motoneurons

Androgens act on the CNS to affect motor function through interaction with a widespread distribution of intracellular androgen receptors (AR). This review highlights our work on androgens and process outgrowth in motoneurons, both in vitro and in vivo. The actions of androgens on motoneurons involve the generation of novel neuronal interactions that are mediated by the induction of androgen-dependent neurite or axonal outgrowth. Here, we summarize the experimental evidence for the androgenic regulation of the extension and regeneration of motoneuron neurites in vitro using cultured immortalized motoneurons, and axons in vivo using the hamster facial nerve crush paradigm. We place particular emphasis on the relevance of these effects to SBMA and peripheral nerve injuries.     

Preemptive effects of intrathecal cyclooxygenase inhibitor or nitric oxide synthase inhibitor on thermal hypersensitivity following peripheral nerve injury

The present study provides an important implication for the management of chronic neuropathic pain, focusing on prostaglandin (PG) and nitric oxide (NO) in the spinal cord. To determine if spinally administered cyclooxygenase (COX) inhibitor or nitric oxide synthase (NOS) inhibitor had preemptive analgesia on thermal hypersensitivity induced by chronic constrictive nerve injury, Sprague-Dawley rats were chronically implanted with an intrathecal (i.t.) catheter. The left sciatic nerve was loosely ligated with 2-mm polyethylene tubing to produce painful mononeuropathy. Animals received tenoxicam (7.5, 15 or 30 micromol/10 microl, i.t.), NS-398 (15 or 30 micromol), or L-NAME (30, 150 or 300 micromol) immediately before the nerve injury, followed by daily injection extending into the four postoperative days. The hindpaw was immersed into a hot (42 degrees C, 44 degrees C and 46 degrees C) or cold (10 degrees C) water bath. The paw immersion test revealed significant thermal hyperalgesia and allodynia 5 day after nerve injury in vehicle control animals. Tenoxicam (7.5, 15 or 30 micromol) or L-NAME (30, 150 or 300 micromol) dose-dependently attenuated hyperalgesia and allodynia. Equimolar dose of NS-398 (15 or 30 micromol) also diminished these nociceptive behaviors. Higher dose of either drug primarily produced longer duration of inhibition. The inhibitory effect of tenoxicam (30 micromol) on hyperalgesia was more effective than that of an equimolar dose of NS-398 or L-NAME. These results demonstrated that intrathecally administered COX inhibitor or NOS inhibitor provides preemptive analgesia on thermal hypersensitivity following chronic constrictive nerve injury in rats.     

ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve

Adrenocorticotropin (ACTH)-treated adrenalectomized rats subjected to crush denervation recover sensation and functional movement sooner than saline-treated rats. Axonal regeneration is accelerated, the number of large endplates and the frequency of preterminal branching are increased. ACTH has no effect on either intact or denervated muscles. The ameliorative action of ACTH during regeneration is apparently neurogenic and independent of corticoids.     

Localization of substance P-like immunoreactive fibers in the thoracic spinal cord of guinea pig

Summary A dorsal-horn fiber system is revealed in the thoracic spinal cord of guinea pig by means of substance P immunocytochemistry. This system has repeated craniocaudal and/or caudo-cranial extensions and possesses five main components: (1) a superficial network, situated beneath the dorsolateral surface of the spinal cord. This network is connected with the dorsal root fibers and the accumulations of substance P-like immunoreactive (SP-LI) fibers in the Lissauer's tract; (2) an accumulation of SP-LI fibers in the Lissauer's tract at the border of the dorsal horn; (3) two collateral SP-LI fascicles (one lateral and one medial) emerging from the SP-LI fiber accumulation in the Lissauer's tract; (4) a transversal fascicle running through laminae III–V, and (5) an SP-LI network in the region of the lateral spinal cord nucleus. These components of the dorsal-horn fiber system show widespread connections with ipsi-and contralateral spinal cord areas, connecting them in cranio-caudal and/or caudo-cranial directions. The SP-LI dorsal-horn system has close relationship with groups of preganglionic sympathetic cells in the intermediate zone of the spinal cord, respective with the vegetative network of this zone. It is suggested that some fibers of the dorsal-horn system that originate from dorsal-root ganglia may represent primary sensory or visceral afferents. It is likely that the dorsal-horn fiber system and the vegetative network of the thoracic spinal cord may represent the morphological basis for the integration of (1) the central and peripheral vegetative nervous systems, and (2) the somatic and vegetative nervous system.     

Synaptic loss following removal of serotoninergic fibers in newly hatched and adult chickens

Neurotransmitters such as serotonin (5HT) may have nontransmitter, trophic-like functions in the developing and adult nervous system. In order to examine this possibility in the avian spinal cord, we have quantified synapse numbers on spinal neurons following treatment with drugs that result in the destruction of 5HT positive axons. Either p-chlorophenylalanine or reserpine was injected into newly hatched or adult chickens. Following treatment for 7 days the density of nonserotoninergic synapses was considerably decreased in the targets of 5HT fibers. By contrast, neither change was observed in the dendritic structures of spinal motoneurons or in the distribution of substance P and enkephalin positive fibers. These data suggest that 5HT may play an important role in the normal increase and maintenance of synapses in developing and adult animals. A lesion of 5HT neurons may not only alter neurochemistry but also alter the general synaptic structures of the brain. While 5HT containing fibers were depleted in a dose-dependent fashion we cannot rule out the possibility that other neurotransmitter systems were depleted at higher dose of PCPA and reserpine. © 1993 John Wiley & Sons, Inc.     

缺血预处理对兔脊髓缺血再灌注损伤水通道蛋白-4表达的影响

目的探讨缺血预处理（IPC）对兔脊髓缺血再灌注损伤后水通道蛋白-4（AQP-4）表达的影响。方法日本大耳白兔72只,随机分为3组：假手术组（S组）、脊髓缺血再灌注损伤组（I／R组）和缺血预处理组（IPC组）。I／R组和IPC组阻断腹主动脉30min造成脊髓缺血再灌注损伤,IPC组在损伤前短暂阻断腹主动脉5min二次实施预处理,S组暴露肾动脉下腹主动脉但不阻断。分别于再灌注损伤后4h和24h进行神经功能评分,并取L4—6脊髓缺血节段,计算脊髓组织含水量,免疫组化法测定脊髓组织中AQP-4表达水平。结果与S组比较,I／R组神经运动功能评分降低,脊髓组织含水量增加,AQP-4表达增加（P〈0．05）。与I／R组比较,IPC组神经运动功能评分增高,脊髓组织含水量降低,AQP-4表达减少（P〈0．05）。结论IPC可抑制脊髓损伤后AQP-4的表达,进而减轻脊髓水肿,保护缺血再灌注损伤的脊髓。     

Transganglionic degenerative atrophy in the substantia gelatinosa of the spinal cord after peripheral nerve transection in rhesus monkeys

Summary The effect of sciatic nerve transection on its centrally located terminals in the spinal cord was analyzed by electron microscopy in adult rhesus monkeys one and three months following lesion. Although the peripheral and intermediate portions of the dorsal roots, where the axons are enveloped by Schwann cells were normal, their central portion and their terminals in the substantia gelatinosa were remarkably altered. Transganglionic degenerative atrophy (TDA) is characterized by three distinct types of electronmicroscopic alterations. The first type exhibits a conspicuous electron density of the terminal and pre-terminal axoplasm. Importantly, shrinkage replaces fragmentation and glial engulfement of the terminal seen in the course of Wallerian degeneration. The second type is characterized by the disappearance of synaptic vesicles from the terminals. The third type of TDA consists of intricate labyrinthine structures, composed of flattened profiles of axonal, dendritic and glial elements. The complex and diverse cellular changes that occur in the upper dorsal horn following peripheral nerve injury may provide the structural basis of plasticity of the primary nociceptive system.