Immunohistochemical detection of calcium/calmodulin-dependent protein kinase II in the spinal cord of the rat and monkey with special reference to the corticospinal tract

Calcium/ calmodulin-dependent protein kinase II is a prominent enzyme in the mammalian brain that phosphorylates a variety of substrate proteins. In the present study, monoclonal antibodies that specifically recognize either the α or the β isoforms of this enzyme were used to determine the distribution of these isoforms within the rat and monkey spinal cord. In the rat, the corticospinal tract consists of two components: the dorsal corticospinal tract, which occupies the ventralmost aspect of the dorsal funiculus; and the ventral corticospinal tract, which occupies an area adjacent to the ventral median fissure. Both dorsal and ventral corticospinal tract fibers were strongly immunopositive for the α-antibody. Unilateral ablation of the sensorimotor cortex of the rat eliminated the α-immunoreactive staining in the contralateral dorsal corticospinal tract. The neuropil in the superficial laminae of the dorsal horn (Rexed's laminae I and II) was densely stained with the α-antibody, whereas the neuropil in laminae IV-X was immunonegative. Dense α-immunopositive neurons were also distributed in the head of the dorsal horn (laminae I-IV). In contrast to the strong α-immunoreactivity seen in the dorsal corticospinal tract fibers, only very weak β-immunoreactivity was observed in this tract. Moderate β-immunoreactive products were distributed homogenously throughout the neuropil of the gray matter, although the neuropil of the superficial laminae of the dorsal horn (laminae I and II) was stained more strongly than the other regions of the gray matter (laminae III-X). Neuronal components in all laminae were immunopositive for the β-antibody. Thus, motoneurons in the ventral horn, which were immunonegative for the α-antibody, were immunopositive for the β-antibody. This selective distribution pattern of immunoreactivity of α- and β-antibodies in the rat was also present in the monkey spinal cord, although the α-immunopositive corticospinal tract fibers in the monkey descended in the lateral funiculus as the lateral corticospinal tract instead of passing through the dorsal funiculus, as is the case in the rat. The differential distribution of immunoreactivity in the spinal cord suggests that these two isoforms of calcium/ calmodulin-dependent protein kinase II may have different functional roles in the spinal cord. © Wiley-Liss, Inc.     

Both dorsal and ventral spinal cord pathways contribute to overground locomotion in the adult rat

Identification of long tracts responsible for spontaneous locomotion is critical for spinal cord injury (SCI) repair strategies. We recently demonstrated that extensive demyelination of adult rat thoracic ventral columns, ventromedial, and ventrolateral white matter produces persistent, significant open-field hindlimb locomotor deficits. Locomotor movements resulting from stimulation of the pontomedullary locomotor region are inhibited by dorsolateral funiculus (DLF) lesions suggesting that important pathways for locomotion may also exist in the dorsal white matter. However, dorsal hemisections that interrupt dorsal columns/dorsal corticospinal tract (DC/CST) and DLF pathways do not produce persistent, severe locomotor deficits in the adult rat. We studied the contributions of myelinated tracts in the DLF and DC/CST to overground locomotion following complete conduction blockade of axons in the ventrolateral funiculus (VLF), a region important for locomotor movements and for transcranial magnetic motor-evoked potentials (tcMMEP). Animals received ethidium bromide plus photon irradiation to produce discrete demyelinating lesions sufficient to stop axonal conduction in the VLF, combined VLF + DLF, or combined VLF + DC/CST. Open-field BBB scores and tcMMEPs were studied at 1, 2, 3, and 4 weeks postlesion. VLF lesions resulted in mean BBB scores of 17 at 4 weeks. VLF + DC/CST and VLF + DLF lesions resulted in mean BBB scores of 15.9 and 11.1, respectively. TcMMEPs were absent in all lesion types confirming VLF conduction blockade throughout the study. Our data indicate that significant contributions to locomotion from myelinated pathways within the rat DLF can be revealed when combined with simultaneous compromise of the VLF.     

Direct projection of the corticospinal tract to the superficial laminae of the spinal cord in the rat

The anterograde transport of both wheat germ agglutinin conjugated to horseradish peroxidase and the kidney bean lectin Phaseolus vulgaris leucoagglutinin was utilized to investigate the projection of primary sensorimotor corticospinal tract axons to the superficial laminae of the spinal dorsal horn in the rat. Both methods yielded qualitatively similar patterns of connectivity. Corticospinal tract axons were found to terminate within all laminae on the side contralateral to the injection site. Labeling was most dense within laminae III and IV and medial portions of laminae I, II, and V in the cervical and lumbar enlargements. Labeling in the ventral horn, though present, was relatively less dense. P. vulgaris leucoagglutinin-labeled axons within laminae I and II exhibited boutons en passant and terminaux; many of these axons also terminated or were collaterals of axons that terminated in deeper dorsal horn laminae. Results are discussed with reference to the somatotopic organization of the spinal cord and to a possible role for the cortex in the modulation of nociception within the spinal cord.     

Type II sodium channels in spinal cord astrocytes in situ: Immunocytochemical observations

The expression of sodium channel α-subunit isoforms in astrocytes in adult rat spinal cord and optic nerve was examined utilizing immunocytochemical methods with antibodies generated against conserved and subtype-specific sequences of the sodium channel. In adult rat spinal cord, astrocytes within the dorsal and ventral funiculi were immunolabelled with antibody SP20, which recognizes a conserved sequence within sodium channel types I, II, and III. In addition, astrocytes within these spinal cord white matter tracts were immunostained with antibody SP11-II, which recognizes sodium channel type II. Antibodies SP11-I and SP32-III, which are directed against subtype-specific sequences in sodium channel types I and III, respectively, did not label astrocytes in the dorsal and ventral funiculi of the spinal cord. In optic nerves, astrocytes were immunostained with antibody SP20. However, no detectable labelling of cells within the optic nerve was observed with antibodies SP11-I, SP11-II, and SP32-III. These observations demonstrate that sodium channel II is expressed by astrocytes in spinal cord white matter. Moreover, these data suggest that regional factors regulate the level of sodium channel isoform expression in astrocytes.     

Delayed glial cell death following wallerian degeneration in white matter tracts after spinal cord dorsal column cordotomy in adult rats

The devastating consequences of spinal cord injury (SCI) result primarily from damage to long tracts in the spinal white matter. To elucidate the secondary injury processes occurring after SCI, we investigated the relationship between apoptosis and Wallerian degeneration in spinal white matter tracts. In the rat spinal cord, the corticospinal tract (CST) and the dorsal ascending tract (DAT) are separated from each other in the dorsal column and relay information in opposite directions. A dorsal column cordotomy at the eighth thoracic (T8) level simultaneously induces Wallerian degeneration in the CST caudal to and in the DAT rostral to the injury. Using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, we demonstrate that apoptosis occurred in areas of Wallerian degeneration in both tracts throughout the length of the cord segments studied (from T3 to T12). This delayed cell death, more apparent in the DAT, began at 7 days after injury and peaked at 14 days for the DAT and 28 days for the CST. Although a few TUNEL+ cells, slightly above the noninjury control level, were found in intact areas of both tracts, statistically significant differences in the number of TUNEL+ cells were found between the intact and the lesioned tract segments (CST, F < 0.01; DAT, F < 0.001). Within a particular spinal segment, a mean number of 64 and 939 TUNEL+ cells in the degenerating CST and DAT, respectively, were estimated stereologically at 14 days postinjury. TUNEL+ cells in degenerating tracts outnumber their intact counterparts by 3.8:1 in the CST and 4.1:1 in the DAT, although a statistically significant difference between the two was only found in the DAT at this time point (P < 0.05). Finally, we demonstrated that oligodendrocytes, the myelin-forming cells in the central nervous system, constitute at least a portion of the cells undergoing apoptosis within areas of Wallerian degeneration.     

BDA皮质脊髓束神经顺行示踪在大鼠脊髓损伤模型中的应用

目的本研究采用生物素标记葡聚糖(Biotin Dextran Amine,BDA)顺行示踪技术来观察大鼠皮质脊髓束(CST)在中枢神经系统中的走行及脊髓损伤后的表现特征。方法20只雌性成年Sprague-Dawley大鼠,分为脊髓损伤组(n=10)和损伤对照组(n=10)。在相当于T7椎板水平用做好标记的显微剪刀剪断脊髓的后2/3。对照组动物术中仅咬除棘突、椎板,不切断脊髓。术后第15 d,所有动物通过立体定向开颅,将10％BDA溶液注入右侧的感觉运动区皮质内。BDA注射2周后,取出大脑和脊髓组织,采用自由漂浮法行BDA染色显影。实验动物于脊髓损伤术前、术后3d、1周、2周、4周采用Basso、Beatlie、Bresnahan(BBB)评分法测量运动功能,所得数据采用两组均数比较t检验进行统计学处理。结果1.脊髓损伤组动物双后肢瘫痪,BBB运动功能评分明显低于损伤对照组,统计学比较差异十分显著(P<0.01);2.BDA顺行示踪显示大脑皮层BDA注射区内见大脑皮层的锥体细胞及其发出的轴突呈阳性染色,BDA阳性染色的皮质脊髓束神经纤维在中脑、桥脑及延髓的腹侧面行走,但在锥体交叉后皮质脊髓束主要(约99％)在对侧脊髓白质的后索中行走。在致伤组动物中,位于脊髓白质后索中的皮质脊髓束纤维在脊髓损伤处终止;在对照组皮质脊髓束纤维染色可一直延伸至L1水平。结论BDA顺行神经     

Mice lacking L1 cell adhesion molecule have deficits in locomotion and exhibit enhanced corticospinal tract sprouting following mild contusion injury to the spinal cord

L1 is a member of the immunoglobulin superfamily of cell adhesion molecules that is associated with axonal growth, including formation of the corticospinal tract (CST). The present study describes the effects of L1 deletion on hindlimb function in locomotion, and examines the role of L1 in recovery and remodeling after contusive spinal cord injury (SCI) in mice. Uninjured adult L1 knockout (Y/-) mice had impaired performance on locomotor tests compared with their wild-type littermates (Y/+). Anterograde tracing demonstrated that CST axons project to thoracic, but not lumbar, levels of the spinal cord of Y/- mice, and revealed a diversion of these fibers from their position in the base of the dorsal columns. Retrograde tracing also revealed reduced numbers of descending projections from paraventricular hypothalamus and red nuclei to the lumbar spinal cord in Y/- mice. SCI at the mid-thoracic level produced a lesion encompassing the center of the spinal cord, including the site of the dorsal CST and surrounding gray matter (GM). The injury caused lasting deficits in fine aspects of locomotion. There was no effect of genotype on final lesion size or the growth of axons into the lesion area. However, injured Y/- mice demonstrated a robust expansion of CST projections throughout the GM of the cervical and thoracic spinal cord rostral to the lesion compared with Y/+ littermates. Thus, L1 is important for the development of multiple spinal projections and also contributes to the restriction of CST sprouting rostral to the site of a SCI in adults.     

The lateral corticospinal tract and spinal ventral horn in X-linked recessive spinal and bulbar muscular atrophy: a quantitative study

A quantitative study was performed on spinal cord lesions in seven patients with X-linked recessive spinal and bulbar muscular atrophy. The myelinated fiber density of the lateral corticospinal tracts at the T7 cord level was well preserved for both large and small myelinated fibers. On the other hand, neurons in the L4 ventral horn were markedly depleted; marked loss was noted of the large alpha and medium-sized gamma motor neurons located in the lateral and medial nuclei as well as the small neurons in the intermediate zones of the ventral horn. These results suggest that myelinated fiber density and fiber-size distribution in the corticospinal tract are well preserved and that neuronal loss in the ventral horns is not restricted to alpha and gamma motoneurons but also involves small interneurons. Received: 8 May 1996 / Accepted: 31 July 1996     

Early electrical field stimulation prevents the loss of spinal cord anterior horn motoneurons and muscle atrophy following spinal cord injury

Our previous study revealed that early application of electrical field stimulation(EFS) with the anode at the lesion and the cathode distal to the lesion reduced injury potential, inhibited secondary injury and was neuroprotective in the dorsal corticospinal tract after spinal cord injury(SCI). The objective of this study was to further evaluate the effect of EFS on protection of anterior horn motoneurons and their target musculature after SCI and its mechanism. Rats were randomized into three equal groups. The EFS group received EFS for 30 minutes immediately after injury at T_(10). SCI group rats were only subjected to SCI and sham group rats were only subjected to laminectomy. Luxol fast blue staining demonstrated that spinal cord tissue in the injury center was better protected; cross-sectional area and perimeter of injured tissue were significantly smaller in the EFS group than in the SCI group. Immunofluorescence and transmission electron microscopy showed that the number of spinal cord anterior horn motoneurons was greater and the number of abnormal neurons reduced in the EFS group compared with the SCI group. Wet weight and cross-sectional area of vastus lateralis muscles were smaller in the SCI group to in the sham group. However, EFS improved muscle atrophy and behavioral examination showed that EFS significantly increased the angle in the inclined plane test and Tarlov's motor grading score. The above results confirm that early EFS can effectively impede spinal cord anterior horn motoneuron loss, promote motor function recovery and reduce muscle atrophy in rats after SCI.     

Bladder and urethral sphincter responses evoked by microstimulation of S2 sacral spinal cord in spinal cord intact and chronic spinal cord injured cats

Urinary bladder and urethral sphincter responses evoked by bladder distention, ventral root stimulation, or microstimulation of S2 segment of the sacral spinal cord were investigated under alpha-chloralose anesthesia in cats with an intact spinal cord and in chronic spinal cord injured (SCI) cats 6-8 weeks after spinal cord transection at T9-T10 spinal segment. Both SCI and normal cats exhibited large amplitude reflex bladder contractions when the bladder was fully distended. SCI cats also exhibited hyperreflexic bladder contractions during filling and detrusor-sphincter dyssynergia during voiding, neither was observed in normal cats. Electrical stimulation of the ventral roots revealed that the S2 sacral spinal cord was the most effective segment for evoking large amplitude bladder contractions or voiding in both types of cats. Microstimulation with a stimulus intensity of 100 microA and duration of 30-60 s via a single microelectrode in the S2 lateral ventral horn or ventral funiculus evoked large amplitude bladder contractions with small urethral contractions in both normal and SCI cats. However, this stimulation evoked incomplete voiding due to either co-activation of the urethral sphincter or detrusor-sphincter dyssynergia. Stimulation in the S2 dorsal horn evoked large amplitude sphincter responses. The effectiveness of spinal cord microstimulation with a single electrode to induce prominent bladder and urethral sphincter responses in SCI animals demonstrates the potential for using microstimulation techniques to modulate lower urinary tract function in patients with neurogenic voiding dysfunctions.     

Reorganization of descending motor tracts in the rat spinal cord

Following lesion of the central nervous system (CNS), reinnervation of denervated areas may occur via two distinct processes: regeneration of the lesioned fibres or/and sprouting from adjacent intact fibres into the deafferented zone. Both regeneration and axonal sprouting are very limited in the fully mature CNS of higher vertebrates, but can be enhanced by neutralizing the neurite outgrowth inhibitory protein Nogo-A. This study takes advantage of the distinct spinal projection pattern of two descending tracts, the corticospinal tract (CST) and the rubrospinal tract (RST), to investigate if re-innervation of denervated targets can occur by sprouting of anatomically separate, undamaged tracts in the adult rat spinal cord. The CST was transected bilaterally at its entry into the pyramidal decussation. Anatomical studies of the RST in IN-1 antibody-treated rats showed a reorganization of the RST projection pattern after neutralization of the myelin associated neurite growth inhibitor Nogo-A. The terminal arborizations of the rubrospinal fibres, which are normally restricted to the intermediate layers of the spinal cord, invaded the ventral horn but not the dorsal horn of the cervical spinal cord. Moreover, new close appositions were observed, in the ventral horn, onto motoneurons normally receiving CST projections. Red nucleus microstimulation experiments confirmed the reorganization of the RST system. These observations indicate that mature descending motor tracts are capable of significant intraspinal reorganization following lesion and suggests the expression of cues guiding and/or stabilizing newly formed sprouts in the adult, denervated spinal cord.     

Bilateral cervical contusion spinal cord injury in rats

There is increasing motivation to develop clinically relevant experimental models for cervical SCI in rodents and techniques to assess deficits in forelimb function. Here we describe a bilateral cervical contusion model in rats. Female Sprague–Dawley rats received mild or moderate cervical contusion injuries (using the Infinite Horizons device) at C5, C6, or C7/8. Forelimb motor function was assessed using a grip strength meter (GSM); sensory function was assessed by the von Frey hair test; the integrity of the corticospinal tract (CST) was assessed by biotinylated dextran amine (BDA) tract tracing. Mild contusions caused primarily dorsal column (DC) and gray matter (GM) damage while moderate contusions produced additional damage to lateral and ventral tissue. Forelimb and hindlimb function was severely impaired immediately post-injury, but all rats regained the ability to use their hindlimbs for locomotion. Gripping ability was abolished immediately after injury but recovered partially, depending upon the spinal level and severity of the injury. Rats exhibited a loss of sensation in both fore- and hindlimbs that partially recovered, and did not exhibit allodynia. Tract tracing revealed that the main contingent of CST axons in the DC was completely interrupted in all but one animal whereas the dorsolateral CST (dlCST) was partially spared, and dlCST axons gave rise to axons that arborized in the GM caudal to the injury. Our data demonstrate that rats can survive significant bilateral cervical contusion injuries at or below C5 and that forepaw gripping function recovers after mild injuries even when the main component of CST axons in the dorsal column is completely interrupted.     

Some observations on early myelination in the human spinal cord. Light and electron microscope study.

Segments of cervical spinal cord from a 23-week-old human foetus have been examined by light and electron microscopy. Myelinated fibres were found in the dorsal, ventral and peripheral lateral tracts, while the lateral corticospinal tract was completely unmyelinated. Myelin sheaths appeared to be formed by spiral wrapping of elongated mesaxons which originated from the apposition of the plasma membranes of oligodendrocytes. Preparations stained with Sudan red and Sudan black revealed the occurrence of lipid inclusions in the interfascicular glia. The topographical relation and the ultrastructural features of these inclusions are described. The possible significance of the inclusion bodies is discussed.     

Distribution of substance P and vasoactive intestinal polypeptide neurons in the chicken spinal cord, with notes on their postnatal development

The distribution of substance P (SP) and vasoactive intestinal polypeptide (VIP) was investigated by immunohistochemistry in the adult chicken spinal cord. By using colchicine treatment, populations of neurons containing either SP or VIP was observed in several regions of the spinal cord. SP neurons were found dorsal to the central canal (CC) and in lamina IV throughout the cord. However, at the thoracic level, numerous relatively larger SP perikarya were located ventral to the CC and aligned on either side of the midline. The distribution of SP fibers is very similar to that reported previously in mammals: they were mostly observed in laminae I and II, in Lissauer's tract, in the dorsolateral funiculus, and dorsal to the CC. In addition, two dense plexuses of SP fibers were noticed in lamina IV. VIP neurons were located mainly in lamina I, in the nucleus of the dorsolateral funiculus, and in the lateral portion of the neck of the dorsal horn throughout the spinal cord. At the thoracic level, many also were located lateral to the CC. Occasionally, single VIP neurons also were encountered dorsal to the CC, in laminae II-IV, and in the intermediate zone. VIP fibers were observed in similar numbers at all spinal levels, occurring mainly in laminae II (probably I) and III, dorsal to the CC, and in the intermediate zone. In addition, examination of the developing chick spinal cords showed similar results as in adult chickens.     

A reassessment of whether cortical motor neurons die following spinal cord injury

Over the past century, the question of whether the cells of origin of the corticospinal tract (CST) die following spinal cord injury (SCI) has been debated. A recent study reported an approximately 20% loss of retrogradely labeled cortical motoneurons following damage to their axons resulting from SCI at T9 (Hains et al. [2003] J. Comp. Neurol. 462:328-341). In follow-up studies, however, we failed to find any evidence of loss of CST axons in the medullary pyramid, which must occur if CST neurons die. Here, we seek to resolve the discrepancy by re-evaluating possible loss of CST neurons using the same techniques as Hains et al. (quantitative analysis of retrograde labeling and staining for cell death markers including TUNEL and Hoechst labeling of the nuclei). Following either dorsal funiculus lesions at thoracic level 9 (T9) or lateral hemisection at cervical level 5 (C5), our results reveal no evidence for a loss of retrogradely labeled neurons and no evidence for TUNEL staining of axotomized cortical motoneurons. These results indicate that CST cell bodies do not undergo retrograde cell death following SCI, and therefore targeting such cell death is not a valid therapeutic target.     

A ventral uncrossed corticospinal tract in the rat

By studying cross-section autoradiograms of the spinal cord with dark field microscopy we demonstrated a ventral uncrossed corticospinal tract in the rat. Corticospinal fibers were labeled by the slow axoplasmic flow of a minute volume of high specific activity tritiated proline injected directly into the motor sensory cortex. The uncrossed ventral corticospinal tract was small but easily identifiable in the cervical region. More caudally the tract became less distinct and could not be traced below midthoracic levels. Only two corticospinal tracts were identified in this study: the well-known crossed dorsal corticospinal tract and the ventral uncrossed corticospinal tract described in this study.     

Intraoperative neurophysiological monitoring of the spinal cord during spinal cord and spine surgery: a review focus on the corticospinal tracts.

Recent advances in technology and the refinement of neurophysiological methodologies are significantly changing intraoperative neurophysiological monitoring (IOM) of the spinal cord. This review will summarize the latest achievements in the monitoring of the spinal cord during spine and spinal cord surgeries. This overview is based on an extensive review of the literature and the authors' personal experience. Landmark articles and neurophysiological techniques have been briefly reported to contextualize the development of new techniques. This background is extended to describe the methodological approach to intraoperatively elicit and record spinal D wave and muscle motor evoked potentials (muscle MEPs). The clinical application of spinal D wave and muscle MEP recordings is critically reviewed (especially in the field of Neurosurgery) and new developments such as mapping of the dorsal columns and the corticospinal tracts are presented. In the past decade, motor evoked potential recording following transcranial electrical stimulation has emerged as a reliable technique to intraoperatively assess the functional integrity of the motor pathways. Criteria based on the absence/presence of potentials, their morphology and threshold-related parameters have been proposed for muscle MEPs. While the debate remains open, it appears that different criteria may be applied for different procedures according to the expected surgery-related morbidity and the ultimate goal of the surgeon (e.g. total tumor removal versus complete absence of transitory or permanent neurological deficits). On the other hand, D wave changes--when recordable--have proven to be the strongest predictors of maintained corticospinal tract integrity (and therefore, of motor function/recovery). Combining the use of muscle MEPs with D wave recordings provides the most comprehensive approach for assessing the functional integrity of the spinal cord motor tracts during surgery for intramedullary spinal cord tumors. However, muscle MEPs may suffice to assess motor pathways during other spinal procedures and in cases where the pathophysiology of spinal cord injury is purely ischemic. Finally, while MEPs are now considered the gold standard for monitoring the motor pathways, SEPs continue to retain value as they provide specificity for assessing the integrity of the dorsal column. However, we believe SEPs should not be used exclusively--or as an alternative to motor evoked potentials--during spine surgery, but rather as a complementary method in combination with MEPs. For intramedullary spinal tumor resection, SEPs should not be used exclusively without MEPs.     

Neurogenic period of ascending tract neurons in the upper lumbar spinal cord of the rat

Although the neurogenic period for neurons in the lumbar spinal cord has been clearly established (Days 12 through 16 of gestation), it is not known when the neurogenesis of ascending tract neurons is completed within this period. The purpose of the present study was to determine the duration of the neurogenic period for projection neurons of the ascending tracts. To label neurons undergoing mitosis during this period, tritiated thymidine was administered to fetal rats on Embryonic (E) Days E13 through E16 of gestation. Ascending tract neurons of the lumbar cord were later (Postnatal Days 40-50) labeled in each animal with a retrograde tracer, Fluoro-Gold, applied at the site of a hemisection at spinal cord segment C3. Ascending tract neurons which were undergoing mitosis in the upper lumbar cord were double labeled, i.e., labeled with both tritiated thymidine and Fluoro-Gold. On Day E13, 89-92% of the ascending tract neurons were double labeled; on Day E14, 35-37%; and on Day E15, 1-4%. Results showed, then, that some ascending tract neurons were double labeled through Day E15 and were, therefore, proliferating in the final one-third of the neurogenic period. Ascending tract neurons proliferating on Day E15 were confined to laminae III, IV, V, and X and the nucleus dorsalis. Long tract neurons in the superficial dorsal horn (laminae I and II), on the other hand, were found to have completed neurogenesis on Day E14 of gestation. Results of the present study show that spinal neurogenesis of ascending projection neurons continues throughout most of the neurogenic period and does not completely follow the well-established ventral to dorsal gradient.     

Calcium-binding proteins, parvalbumin- and calbindin-D 28k-immunoreactive neurons in the rat spinal cord and dorsal root ganglia: a light and electron microscopic study

The distribution of two calcium-binding proteins, parvalbumin (PV) and calbindin-D 28K (CaBP), was studied by the peroxidase-anti-peroxidase immunohistochemical method at the light and electron microscopic level in the rat spinal cord and dorsal root ganglia. The possible coexistence of these two proteins was also investigated. PV-positive neurons were revealed in all layers of the spinal cord, except lamina I, which was devoid of labelling. Most of the PV-positive cells were found in the inner layer of lamina II, lamina III, internal basilar nucleus, central gray region, and at the dorsomedial and ventromedial aspects of the lateral motor column in the ventral horn. Neuronal processes intensely stained for PV sharply delineated inner lamina II. With the electron microscope most of them appeared to be dendrites, but vesicle containing profiles were also found in a smaller number. CaBP-positive neurons appeared to be dispersed all over the spinal gray matter. The great majority of them were found in laminae I, II, IV; the central gray region; the intermediolateral nucleus; and in the ventral horn just medial to the lateral motor column. Laminae I and II were densely packed with CaBP-positive punctate profiles that proved to be dendrites and axons in the electron microscope. A portion of labelled neurons in lamina IV and on the ventromedial aspect of the lateral motor column in the ventral horn disclosed both PV- and CaBP-immunoreactivity. All of the funiculi of the spinal white matter contained a large number of fibres immunopositive for both PV and CaBP. The highest density of CaBP-positive fibres was found in the dorsolateral funiculus, which was also densely packed with PV-positive fibres. PV-positive fibres were even more numerous in the dorsal part of the dorsal funiculus. The territory of the gracile funiculus in the brachial cord and that of the pyramidal tract in its whole extent were devoid of labelled fibres. In the thoracic cord, the dorsal nucleus of Clarke received a large number of PV-positive fibres. Dorsal root ganglia displayed both PV- and CaBP-immunopositivity. The cell diameter distribution histogram of PV-positive neurons disclosed two peaks--one at 35 microns and the other at 50 microns. CaBP-positive cells in the dorsal root ganglia corresponded to subgroups of small and large neurons with mean diameters of 25 microns and 45 microns, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

C-fos expression in the spinal cord of rats exhibiting allodynia following contusive spinal cord injury

Contusive spinal cord injury (SCI) may result in central neuropathic pain marked by allodynia-like features in the dermatomes close to the level of injury. The aim of this study was to compare the laminar distribution of activated neurons (as determined by c-fos immediate early gene expression) in the spinal cord immediately above the level of a SCI in rats with or without allodynia-like features. Non-noxious mechanical stimulation was applied to half the animals in the dermatomes corresponding to the level of injury prior to perfusion. Stimulation resulted in a significant increase in c-fos labelling in all laminae of the spinal dorsal horn in the segment immediately above the level of injury only in allodynia animals. Animals that had allodynia also demonstrated a significant increase in the level of c-fos labelling in lamina III, IV and V of the dorsal horn without stimulation. Thus, allodynia following SCI is associated with significant increases in basal and evoked c-fos expression ("neuronal activity") in response to non-noxious mechanical stimulation. The data also suggest that allodynia-like behaviour following SCI cannot be accounted for solely by changes occurring at a spinal level.