Distribution of some peptides (substance P, [Leu]enkephalin, [Met]enkephalin) in the brain stem and spinal cord of a lizard, Varanus exanthematicus

The distribution of substance P-like and [Leu]- and [Met]enkephalin-immunoreactive cell bodies, fibers and terminal structures in the brain stem and spinal cord of a lizard, Varanus exanthematicus, was studied with the indirect immunofluorescence technique, using antibodies to these peptides. Substance P-like immunoreactive cell bodies were found in the hypothalamus, in a periventricular cell group in the rostral mesencephalon, in the interpeduncular nucleus, in and ventral to the descending nucleus of the trigeminal nerve, in and directly ventral to the nucleus of the solitary tract, scattered in the brain stem reticular formation and in the trigeminal and spinal ganglia. A rather widespread distribution of substance P-like immunoreactivity was found in the brain stem and spinal cord, mainly concentrated in striatotegmental projections related to visceral and/or taste information (nucleus of the solitary tract, parabrachial region), in the descending nucleus of the trigeminal nerve and in the dorsal horn of the spinal cord (areas I and II). In the spinal cord also around the central canal (area X and adjacent parts of area V-VI) a distinct substance P innervation was found. The ventral horn receives only a very sparse substance P innervation. The distribution of [Leu]- and [Met]enkephalin in the brain stem and spinal cord of Varanus exanthematicus is less impressive than that of substance P. Enkephalinergic cell bodies were found particularly in the caudal hypothalamus. Small populations of enkephalinergic cell bodies were found in the vestibular nuclear complex, in the nucleus of the solitary tract, in and around the descending nucleus of the trigeminal nerve and throughout the rhombencephalic reticular formation. Enkephalins are likely to be present in efferent projections of the striatum, in projections related to taste and/or visceral information (nucleus of the solitary tract, parabrachial region) and in descending pathways to the spinal cord. Enkephalinergic fibers are present in the lateral funiculus and enkephalin-immunoreactive cell bodies are found in the reticular formation, particularly the inferior reticular nucleus which is known to project to the spinal cord. In the spinal cord enkephalinergic terminal structures were found especially in the superficial layer of the dorsal horn (areas I and II) and around the central canal. The ventral horn including the motoneuron area receives only a relatively sparse enkephalinergic innervation.     

Traumatic lower limb fractures following complete spinal cord injury

Patients with complete spinal cord injury experience a rapid bone mineral density loss that places them at an increased risk of lower limb fracture following trivial injury. We aimed to establish if spinal cord injured patients treated at our unit have an increased incidence of lower limb fracture, and if so to identify a cohort of patients who may benefit from prophylactic treatment to preserve bone mineral density. We reviewed 158 patients treated between January 1994 and July 2002. There was a significantly increased incidence of lower limb fracture in patients with complete spinal cord injury (5 fractures in 4 patients), when compared to the cohort of patients with spinal injury who subsequently attained mobility (no fractures). Prophylactic bisphosphonates reduce disuse osteopenia and warrant prospective assessment in patients with complete spinal cord injury in whom they may confer a protective effect against lower limb fracture.     

Localization of substance P and enkephalin by immunohistochemistry in the spinal cord of human fetus

The peroxidase-antiperoxidase method was used to study the distribution of substance P and enkephalin during development of the spinal cords of human fetuses. Thirty-seven cases were collected, ranging from 5- to 40-weeks-old (fetal ages). Both types of transmitters were present initially around the fifth week in the mantle layer of the base of the dorsal horn, around the tenth week at the anterior gray and the intermediate gray and around the sixth week at the marginal layer at the base of the ventrolateral funiculus. Substance P- and enkephalin-positive sites at the marginal layers at the base of the dorsolateral funiculus were evident in the same area at 5-6 weeks. The positive fibers in the dorsal horn were initially located in the superficial layers. By the eleventh week, the positive sites spread to other surface layers at the lateral sides of the dorsal horns bilaterally at all spinal levels above the sacral. In the sacral levels adjacent to the conus medullaris, the spreading to surface layers was not apparent bilaterally until the seventeenth week. By weeks 18-26 the positive sites penetrated deeper in the dorsal horn and by week 27 assumed the adult path. The enkephalin cell bodies were present in the Rexed layers I and II of the dorsal horn and the substance P-positive sites were apparent in the dorsal ganglia of the 28-40-week-old fetuses.     

Human spinal neurons: innervation by both substance P and enkephalin

The distribution of the peptides substance P and methionine-enkephalin in relation to neurons in the human thoracic and lumbar spinal cord has been studied with the unlabeled peroxidase anti-peroxidase method employing antibodies directed against substance P and methionine-enkephalin. The peptides have been localized in varicosities and terminal-like processes in close apposition to the soma and proximal dendrites of neurons in the marginal zone, the intermediolateral nucleus, intermediomedial nucleus, the reticular nucleus of lamina V and the ventral horn; some cells in all these regions are innervated by both peptides.The findings provide morphological evidence of these peptides in the human spinal cord. The demonstration of innervation by both peptides of single neurons in laminae I and V provides some morphological basis to models that postulate the interaction of substance P and enkephalin in pain modulation. The innervation of other neuron types indicates that these neuropeptides may be involved in other spinal functions as well.     

Morphine and substance P release in the spinal cord

Summary In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive substance P (irSP) in the lumbar dorsal horn during noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. The major region of irSP release detected was centred on the substantia gelatinosa, with lesser release at the dorsal cord surface. Release at these sites was unchanged by systemic administration of morphine, or of morphine followed by naloxone. During superfusion of the dorsal cord surface with high concentrations of morphine, irSP release in the substantia gelatinosa region was slightly reduced and surface release was not observed, effects not reversed by systemic naloxone administration. The results suggest that the analgesic action of morphine does not involve reduced release of SP in the spinal cord.     

Edinger-Westphal neurons that project to spinal cord contain substance P

Combined retrograde transport and immunocytochemical methods were used to determine whether Edinger-Westphal neurons projecting to spinal cord also demonstrate substance P-like immunoreactivity (SPLI). Large injections of horseradish peroxidase (HRP) into cervical and lumbar enlargements retrogradely labeled cells throughout the length of the Edinger-Westphal complex (EW). Nearly all HRP-labeled EW neurons also stained for SPLI, evidence that EW is the origin of a direct substance P pathway linking rostral mesencephalon with spinal cord.     

Vasopressin and oxytocin in the rat spinal cord: distribution and origins in comparison to [Met]enkephalin, dynorphin and related opioids and their irresponsiveness to stimuli modulating neurohypophyseal secretion

Immunoreactive-vasopressin, -oxytocin, -dynorphin, -dynorphin-(1-8), -alpha-neo-endorphin and -[Met]enkephalin were, in each case, present in greater concentrations in dorsal as compared to ventral, and lumbo-sacral as compared to cervico-thoracic, spinal cord. These differences were significantly more pronounced for vasopressin and oxytocin than for the other peptides. Lesions of the hypothalamic paraventricular nucleus depleted levels of immunoreactive-vasopressin and -oxytocin throughout the cord whereas levels of the opioid peptides therein were unaffected. In contrast, destruction of either the supraoptic or suprachiasmatic nucleus failed to change the content of immunoreactive-vasopressin, -oxytocin or any of the opioid peptides in the cord. Dehydration for 3 days depressed levels of immunoreactive-vasopressin, -oxytocin and -dynorphin in the neurointermediate lobe of the pituitary. In distinction, the levels of these were not modified in the spinal cord. Further, treatment with the synthetic corticosteroid, dexamethasone, elevated levels of immunoreactive-vasopressin, -oxytocin and -dynorphin in the neurointermediate pituitary whereas these were unaffected in the spinal cord. It is concluded that vasopressin and oxytocin in the spinal cord are predominantly derived from the paraventricular nucleus, localized in dorsal lumbo-sacral regions of the cord and insensitive to endocrinological manipulations. These pools may, thus, be modulated differently from their counterparts in the neurohypophysis and have a differing role, possibly in the control of the primary processing, autonomic or motor junctions. Further, there is no evidence from these or our prior studies for a close interrelationship of spinal cord vasopressin with dynorphin-related peptides (or oxytocin with [Met]enkephalin), likewise in contrast to the neurohypophysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury

Upon noxious stimulation, substance P (SP) is released from primary afferent fibers into the spinal cord where it interacts with the SP receptor (SPR). The SPR is located throughout the dorsal horn and undergoes endocytosis after agonist binding, which provides a spatial image of SPR-containing neurons that undergo agonist interaction. Under normal conditions, SPR internalization occurs only in SPR+ cell bodies and dendrites in the superficial dorsal horn after noxious stimulation. After nerve transection and inflammation, SPR immunoreactivity increases, and both noxious as well as nonnoxious stimulation produces SPR internalization in the superficial and deep dorsal horn. We investigated the primary afferent fibers that contribute to enhanced SPR internalization in the spinal cord after nerve transection and inflammation. Internalization evoked by electrical stimulation of the sciatic nerve was examined in untreated animals, at 14 days after sciatic nerve transection or sham surgery and at 3 days after hindpaw inflammation. Electrical stimulation was delivered at intensities to excite Abeta fibers only, Abeta and Adelta fibers or A and C fibers as determined by the compound action potential recorded from the tibial nerve. Electrical stimuli were delivered at a constant rate of 10 Hz for a duration of 5 min. Transection of the sciatic nerve and inflammation produced a 33.7 and 32.5% increase in SPR and immunoreactivity in lamina I, respectively. Under normal conditions, stimulation of Adelta or C fibers evoked internalization that was confined to the superficial dorsal horn. After transection or inflammation, there was a 20-24% increase in the proportion of SPR+ lamina I neurons that exhibited internalization evoked by stimulation of Adelta fibers. The proportion of lamina I SPR+ neurons that exhibited internalization after stimulation of C-fibers was not altered by transection or inflammation because this was nearly maximal under normal conditions. Moreover, electrical stimulation sufficient to excite C fibers evoked SPR internalization in 22% of SPR+ lamina III neurons after nerve transection and in 32-36% of SPR+ neurons in lamina III and IV after inflammation. Stimulation of Abeta fibers alone never evoked internalization in the superficial or deep dorsal horn. These results indicate that activation of small-caliber afferent fibers contributes to the enhanced SPR internalization in the spinal cord after nerve transection and inflammation and suggest that recruitment of neurons that possess the SPR contributes to hyperalgesia.     

Temporal changes in spinal cord expression of mRNA for substance P,dynorphin and enkephalin in a model of chronic pain

We have used a partial sciatic nerve ligation model to examine the time course for changes in the expression of mRNA for three peptides related to pain transmission at spinal sites (dynorphin, enkephalin and substance P), during the development of allodynia. Enhanced expression of mRNA for dynorphin and substance P was observed in the dorsal horn on the same side as the partial nerve ligation. Increased expression of dynorphin mRNA was biphasic. The initial increases in expression of dynorphin mRNA occurred at 3 h, and a secondary peak was observed 1–3 days after surgery. The secondary increases coincided roughly with increased substance P mRNA expression. However, both dynorphin and substance P mRNA returned to control values after 1 week despite continuing allodynia. No significant changes in expression of mRNA for enkephalin were observed. The elevation of substance P mRNA in intrinsic spinal cord neurons may be secondary to changes in immediate early genes c-fos and jun-B, whereas the expression of dynorphin and enkephalin mRNA is differently regulated. The results also suggest that changes in the expression of the three neuropeptides are not critically involved in the development and maintenance of chronic pain or allodynia.     

Light inhibits the release of both [Met5]enkephalin and [Met5]enkephalin-containing peptides in chicken retina, but not their syntheses

The levels of native and cryptic [Met5]enkephalin in the chicken retina were found to vary during a 12:12 h light-dark cycle, both rising in the light and falling during the dark. Such variations could conceivably arise from (a) changes in the rate of release and subsequent degradation of native and/or cryptic [Met5]enkephalin, (b) changes in the rate of proenkephalin A synthesis, or (c) changes in the rate of proenkephalin A processing. Measurement of the rate of release of native and cryptic [Met5]enkephalin in vitro indicated that the increased rate of release of both of these forms of [Met5]enkephalin during the dark quantitatively accounted for the fall in their retinal levels during the dark. This indicated that the biosynthesis of proenkephalin A was not activated during the light-dark cycle. Molecular weight fractionation of retinal extracts also supported this idea, since the pool of high molecular weight precursors did not vary in size, suggesting that processing was not modulated during the light-dark cycle. Instead, the fall in both cryptic and native [Met5]enkephalin during the dark was due to their increased rate of release together with a rate-limiting conversion of high molecular weight [Met5]enkephalin-containing peptides to low molecular weight [Met5]enkephalin-containing peptides. The enkephalinergic cells of the retina seem to cope with physiological variations in demand by accumulating a large pool of peptide during periods of low stimulation (light), so that when stimulation and release is high (dark), the decrease in pool levels does not compromise the function of the cells and their postsynaptic targets.     

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.     

Transendothelial vesicular transport of protein following compression injury to the spinal cord.

Routes of vascular leakage resulting in trauma-induced edema have not been clarified. To explore the problem we followed the fate of intravascular horseradish peroxidase (HRP) after compression injury to the thoracic cord (cats). At 90 seconds and 15 minutes, HRP was confined to the gray matter, occupying perivascular spaces, unexpanded extracellular channels, and cytoplasmic compartments of injured cells. Adjoining segments contained similar but lesser deposits. At 4 hours, tracer occupied the expanded extracellular spaces of the lesion's white matter; gray matter deposits were present up to 4 cm. distal. Vessels revealed no evidence of rupture. Open interendothelial junctions were not found. Counts of HRP-labeled vesicles in the endothelium of gray matter capillaries revealed a significant intensification of vesicular activity in the lesion and in adjacent areas up to 9 cm. caudal. Morphologically, labeled vesicles exhibited a wide diversity in shape and size. Typical pinocytotic (700A) and tubular forms measured 400 to 700 A in width; vacuolar forms measuring up to 0.7 mum. across were frequently observed. Continuity between the three types was often evident. Where basement membrane and perivascular clefts were not yet inundated with HRP, sites of vesicular emptying of HRP at the tissue front were identified. Serial sections revealed that vesicles may be contiguous from luminal to abluminal surfaces, thus providing facilitated transport pathways. The data suggest that vesicular transport plays a role in the genesis of trauma-induced edema.     

Increased calpain I-mediated proteolysis, and preferential loss of dephosphorylated NF200, following traumatic spinal cord injury.

We investigated the hypothesis that the Ca2+-activated protease calpain is involved in the pathophysiology of spinal cord injury, and is linked to the proteolytic degradation of cytoskeletal proteins. We report here that levels of calpain I (mu-calpain)-mediated spectrin breakdown products are increased by 15 min post-injury, with peak levels reached by 2 h post-injury. The dephosphorylated form of the neurofilament protein NF200 is substantially lost over the same time-period. A 35-g compressive injury was applied to the midthoracic rat spinal cord for 1 min, and animals were killed at 15 min, 1, 2, 4, 8, 16, and 24 h post-injury. Calpain I-mediated spectrin breakdown products accumulated post-injury, with peak levels reached at 2 h. Secondly, we have demonstrated a progressive loss of the 200,000 mol. wt neurofilament protein NF200, a cytoskeletal calpain substrate, which began within 1-2 h post-injury. Densitometric analyses confirmed that loss of NF200 is a substrate-specific phenomenon, since (i) dephosphorylated NF200 was preferentially lost while phosphorylated NF200 was relatively spared, and (ii) actin, which is not a substrate for calpain, was relatively spared following spinal cord injury. Finally, we demonstrated calpain I-mediated spectrin breakdown within NF200-positive neuronal processes post-injury. We conclude that the accumulation of spectrin breakdown products is temporally and spatially correlated with loss of dephosphorylated NF200 after spinal cord injury.     

A re-examination of the localization of immunoreactive dynorphin(1–8), [Leu]enkephalin and [Met]enkephalin in the rat neurohypophysis

We addressed in this study, with immunocytochemical methods, the following questions:

(1) are immunoreactive enkephalins in the rat neurohypophysis stored in nerves distinct from neurosecretory nerves;

(2) where is [Met]enkephalin immunoreaction localized;

(3) does immunoreactive [Leu]enkephalin coexist with pro-enkephalin or with pro-dynorphin fragments; and

(4) are the interpretations of localization studies influenced by the choice of pre-embedding or post-embedding immunocytochemical techniques? We compared immunoreactions due to antibodies which had been used by others in previous studies, examined both lyophilized and conventionally fixed specimens, and applied pre- and post-embedding protocols. Both pre- and post-embedding stainings confirmed co-storage of immunoreactive dynorphin(1–8)-like materials with vasopressin. Immunoreactive [Met]enkephalin-like material always coexisted with oxytocin. Most of the immunoreactive [Leu]enkephalin-like material appeared to occur in oxytocin nerves; only in larger vasopressin varicosites was there some dot-like [Leu]enkephalin immunoreaction. This indicates that neural lobe [Leu]enkephalin predominantly is cleaved from a precursor which also contains [Met]enkephalin.

When pre-embedding methods were modified in order to block diffusion and to enhance penetration of antibodies, enkephalin immunoreactivity was always found in typical neurosecretory varicosities with large granules. Structures previously interpreted as enkephalinergic nerve terminals contacting pituicytes most likely are neurosecretory varicosities.     

Immunohistochemical localization of substance P in anencephalic human fetus spinal cord

The distribution of substance P (SP) in human anencephalic fetus spinal cord has been studied with the indirect immunofluorescence technique. The SP-like immunoreactivity was detected within plexuses of fibres localized in the superficial layers of the dorsal grey including the marginal zone and substantia gelatinosa, and also the dorsal funicular grey. The other spinal cord areas were devoid of SP-like immunoreactivity. Comparison with normal fetus spinal cord reveals that in both normal and anencephalic fetuses the dorsal SP-patterns are comparable. This study indicates that in human fetus spinal cord most of the SP-fibres dorsally localized, occur even if the brain itself does not develop.     

Plasticity in sublesionally located neurons following spinal cord injury

Neuronal plasticity has been traditionally associated with learning and memory processes in the hippocampal regions of the brain. It is now generally accepted that plasticity phenomena are also associated with other kinds of cellular changes and modifications occurring in all areas of the CNS after injury or intense neuronal activity. For instance, spinal cord injuries have been associated with a series of cellular modifications and adaptations taking place distally in sublesional areas. Some of these modifications include changes in the expression of immediate early genes (e.g., c-fos and nor-1), TNF-alpha, preprodynorphin, neurotrophic factors (e.g., BDNF and NT-3), and several subtypes of transmembranal receptors (e.g., 5-HT(1A) and 5-HT(2A)). This review constitutes an update of the current knowledge regarding this broadly defined plasticity phenomenon that occurs spontaneously or can be modulated by training in sublesional segments of the spinal cord. Spinal cord plasticity is an increasingly popular field of research, believed by many as being a complex phenomenon that may contribute to the development of innovative therapeutics and rehabilitative approaches for spinal cord injured patients.     

小鼠脊髓损伤模型的行为学评估方法

目前已存在多种针对小鼠脊髓损伤(spinal cord injury,SCI)模型的行为学评分方法,常用的包括Basso Mouse Scale(BMS)法、单帧运动分析、爬梯实验和斜面实验等。其中,BMS和单帧运动分析属运动学分析,前者的评分与损伤程度有较高一致性,但是较为复杂,后者观察指标简单,但是只能做粗略的评估;爬梯实验和斜面实验是观察小鼠完成某一任务的能力而评分,指标客观,但前者应用前提是小鼠已恢复到可负重行走,并需要事先对小鼠进行训练,后者的敏感性可能有品系差异。     

Increased oxidative-related mechanisms in the spinal cord injury in old rats

In the present study, we evaluated the effect of age, in a model of spinal cord injury that was induced by the application of vascular clips to the dura via a four-level T5-T8 laminectomy. Spinal cord injury in old rats resulted in severe trauma characterized by edema and neutrophil infiltration. Immunohistochemical examination demonstrated an increase in immunoreactivity for nitrotyrosine. In contrast, the degree of: (a) spinal cord inflammation and tissue injury (histological score), (b) nitrotyrosine, (c) PARS, and (d) neutrophils infiltration was markedly reduced in spinal cord tissue obtained from young rats. We have also demonstrated that ageing significantly worsened the recovery of limb function and caused an increase in mortality rate when compared with young rats.     

Interlimb reflexes following cervical spinal cord injury in man

Summary The reflex interconnection of lower and upper extremity muscles was investigated in subjects with chronic (> 1 year post-injury) lesions to the cervical spinal cord. Lower extremity mixed nerves were stimulated with single shocks or with brief trains of high-frequency stimuli of varying intensities. EMG from a number of lower and upper extremity muscles was recorded on magnetic tape for later analysis. In one population of spinal cord injury (SCI) subjects, single stimuli to lower extremity nerves resulted in muscle responses in both ipsi- and contralateral upper extremity muscles. The minimal response latency to a single shock was typically much less in muscles on the ipsilateral side than for contralateral upper extremity muscles. Application of brief trains of stimuli (for example, 2 stimulus pulses at 500 Hz) typically resulted in a large reduction in latency to the contralateral motor response, such that it was now approximately equal to the ipsilateral motor response latency. This decline in response latency was not gradual with increasing afferent input. Instead, the response occurred either early or late, but not at intermediate latencies. Stimuli which were subthreshold for evoking M-waves or H-reflexes were sometimes still adequate to evoke upper extremity motor responses. Once the threshold had been exceeded, the magnitude of the evoked response appeared to be independent of the stimulus magnitude. These reflex interconnections of lower and upper extremities were obtained only from subjects with chronic and motor-complete cervical spinal cord injury. No such interlimb responses were seen in control subjects, or in subjects who had recovered some motor function below the level of their injury, and were now considered to be motor-incomplete quadriplegics.