T13 Level Research Articles

Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats

In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCKB receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCKB receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20mg/kg). Intrathecal CI-988 (100, 200 and 500μg) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCKB receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain.

Contribution of Commissural Projections to Bulbospinal Activation of Locomotion in the In Vitro Neonatal Rat Spinal Cord

Commissural projections are required for left-right coordination during locomotion. However, their role, if any, in rhythm production is unknown. This study uses the neonatal rat in vitro brain stem-spinal cord model to examine the rostrocaudal distribution of locomotor-related commissural projections and study whether commissural connections are needed for the generation of hindlimb rhythmic activity in response to electrical stimulation of the brain stem. Midsagittal lesions were made at a wide range of rostrocaudal levels. Locomotor-like activity persisted in some preparations despite midsagittal lesions extending from C(1) to the mid-L(1) level or from the conus medullaris to the T(12/13) junction. In some preparations, midsagittal lesions throughout the entire spinal cord had no effect on locomotor-like activity if two or three contiguous segments remained intact. Those bridging segments had to include the T(13) and/or L(1) levels. These observations suggested that commissural projections in the thoracolumbar junction region were critical. However, locomotor-like activity was also elicited in preparations with limited midsagittal lesions focused on the thoracolumbar junction (T(12) through L(1) or L(2) inclusive). In other experiments, locomotor-like activity was evoked by bath-applied 5-hydroxytryptamine (5-HT) and N-methyl-d-aspartate (NMDA). Appropriate side-to-side coordination was observed, even when only one segment remained bilaterally intact. Commissural projections traversing the thoracolumbar junction region were most effective. In combination, these results suggest that locomotor-related commissural projections are redundantly distributed along a bi-directional gradient that centers on the thoracolumbar junction. This commissural system not only provides a robust left-right coordinating mechanism but also supports locomotor rhythm generation in response to brain stem stimulation.

Neuropathic pain and temporal expression of preprodynorphin, protein kinase C and N-methyl-d-aspartate receptor subunits after spinal cord injury

Central neuropathic pain is refractory to conventional treatment and thus remains a therapeutic challenge. In this work, we used a well-recognized model of central neuropathic pain to evaluate time-dependent expression of preprodynorphin (ppD), protein kinase C gamma (PKCγ) and NMDA receptor (NMDAR) subunits NR1, NR2A and NR2B, all critical players in nociceptive processing at the spinal level. Male Sprague-Dawley rats were subjected to spinal hemisection at T13 level and sham-operated rats were included as control animals. The development of hindpaw mechanical allodynia was assessed using the von Frey filaments test. Real time RT-PCR was employed to determine the relative mRNA levels of NMDAR subunits, ppD and PKCγ in the dorsal spinal cord 1, 14 and 28 days after injury. Our results show that, coincident with the allodynic phase after injury, there was a strong up-regulation of the mRNAs coding for ppD, PKCγ and NMDAR subunits in the dorsal spinal cord caudal to the injury site. The present study provides further evidence that these molecules are involved in the development/maintenance of central neuropathic pain and thus could be the target of therapeutic approaches.

Simple measurement of spinal cord evoked potential: A valuable data source in the rat spinal cord injury model

Measurement of spinal cord evoked potentials (SCEPs) is proposed as a means of predicting locomotion outcome in the rat spinal cord injury (SCI) model. Using 55 rats, three reproducible peak waves (waves I, II and III) were observed during stimulation at the C7 level with recording at the L1 epidural space. Hemisection at the T13 level showed three wave loss patterns: wave III loss only, loss of both wave II and III, and loss of all three waves. Defining an ideal SCI model as establishment of stable monoparesis or paraparesis, all animals in the wave II–III loss group showed favorable results. Histological data and electrophysiological properties allowed reasonable assumptions of wave origin: wave I from extrapyramidal tracts, wave II from the ventral corticospinal tract, and wave III from the dorsal corticospinal tract. Complete destruction of pyramidal tracts in both dorsal and ventral fibers was essential for long-term impairment of locomotion.

Impact of different drugs and different volumes injected intraspinally on the nerve function: experiment with rats

To investigate the impact of different drugs and different volumes injected intraspinally on the nerve function and find out the safe intraspinally injection volume. Ninety-six adult Sprague-Dawley rats were randomly divided into 12 equal groups to be injected intraspinally with normal saline (NS) 2 microl, 4 microl, 6 microl, or 10 microl, viral buffer 2 microl, 4 microl, 6 microl, or 10 microl, or Ad-LacZ (1 x 10(9) pfu/ml) 2 microl, 4 microl, 6 microl, or 10 microl at the level of T13 respectively immediately after the laminectomy was performed. Another 6 adult SD rats only underwent T13 luminal decompression and used as controls. Basso-Beattie-Bresnahan locomotor rating scale was used to evaluate the motor function of their hind limbs after 1, 2, 3, 4, 5, and 6 days, and 1, 2, 3, and 4 weeks. One and 2 weeks after the injection 2 rats from each group were killed and the remaining 4 rats in each group were killed 4 weeks after the injection. Histological examination was done with HE staining and toluidine blue staining. X-gal staining was used to observe the expression of adenovirus in the spinal cord. The extent of motor deficits and tissue damage increased following the increase of intraspinal injection volume (P = 0.000). The severity of damage of the Ad-LacZ was significantly greater than those of the NS and virus buffer groups (P = 0.044), however, there were no significant differences in the severity of damage among NS groups and virus buffer groups (P = 1.000). X-Gal staining showed that the reporter gene LacZ was expressed in all Ad-LacZ groups. The great the dose the more severe the damage. Four weeks later the effective residual rates f the grey and white matters of the Ad-LacZ groups were all significantly lower than those of the other 2 groups, however, there were no significant differences in the effective residual rates f the grey and white matters among the NS and virus buffer groups. Intraspinal injection results in functional deficit and tissue damage in the spinal cord. The extent of tissue damage increases following the increase of intraspinal injection volume. Different drugs cause different tissue damage. The intraspinal injection of Ad-LacZ results in more tissue damage. The volume of 4 microl is the safe volume for injection, 6 microl is a critical volume.

Evaluation of the anatomic effect of physical therapy exercises for mobilization of lumbar spinal nerves and the dura mater in dogs

To adapt and standardize neural tissue mobilization exercises, quantify nerve root movement, and assess the anatomic effects of lumbar spinal nerve and dural mobilization in dogs. 15 canine cadavers. 5 cadavers were used in the preliminary part of the study to adapt 3 neural tissue mobilization physical therapy exercises to canine anatomy. In the other 10 cadavers, the L4 to L7 nerve roots and the dura at the level of T13 and L1 were isolated and marked. Movements during the physical therapy exercises were standardized by means of goniometric control. Movement of the nerve roots in response to each exercise was digitally measured. The effects of body weight and crownrump length on the distance of nerve root movement achieved during each exercise were also assessed. Each exercise was divided into 4 steps, and the overall distance of neural movement achieved was compared with distances achieved between steps. Neural tissue mobilization exercises elicited visible and measurable movement of nerve roots L4 to L7 and of the dura at T13 and L1 in all cadavers. The physical therapy exercises evaluated had measurable effects on nerve roots L4 to L7 and the dura mater in the T13 and L1 segments. These exercises should be evaluated in clinical trials to validate their efficacy as primary treatments or ancillary postsurgical therapy in dogs with disorders of the thoracolumbar and lumbosacral segments of the vertebral column.

Dorsal column lesion reduces mechanical allodynia in the induction, but not the maintenance, phase in spinal hemisected rats

The dorsal column–medial lemniscal (DC–ML) system is known to be a route of ascending input signals for mechanical allodynia following peripheral nerve injury. We examined whether the pain signals after spinal hemisection were transmitted via the DC–ML system in the induction and maintenance phases of the neuropathic pain. Under enflurane anesthesia, rats were subjected to spinal hemisection at T13 level and bilateral DC lesion was made at T8 level 1 day or 3 weeks after the hemisection. The DC lesion 1 day after the hemisection significantly reduced the mechanical, but not cold, allodynia, whereas the DC lesion 3 weeks after the hemisection did not change both mechanical and cold allodynia. These results suggest that the signals for mechanical allodynia following spinal hemisection should be transmitted via the DC–ML system in the induction, but not maintenance, phase.

Effects of antithrombin III on spinal cord-evoked potentials and functional recovery after spinal cord injury in rats.

From the view of motor function, spinal cord-evoked potentials, and histology, we evaluated the effects of antithrombin III on a spinal cord injury resulting in incomplete paraplegia. To investigate the effect of antithrombin III on the recovery process after acute incomplete rat spinal cord injury. Antithrombin III is used for treating disseminated intravascular coagulation by its anticoagulant effect and is also reported to reduce organ damage by the release of prostaglandin I2 from endothelial cells, induced by antithrombin III. Therefore, antithrombin III has potential as a therapeutic agent for spinal cord damage. The spinal cord injury was induced by placing a 25 g weight on the dorsal surface of the spinal cord at the 10th thoracic spine level for 20 minutes. Antithrombin III (250 U/kg) was administered intravenously 5 minutes before the compression, whereas 2 mL of physiologic saline solution was administered to the control group. We recorded the spinal cord-evoked potentials at the T13 level after stimulation of the brain and C7 level. For 12 weeks after the injury, we observed the recovery course of waveform and motor function. The recovery of motor function was evaluated by using inclined table and modified Tarlov scores. We also examined the histology of the compressed site in the spinal cord. There were statistically significant differences in the motor recovery process between the two groups. Evoked potentials of the antithrombin III group recovered earlier than those of the control group. Histologically, hemorrhage and tissue defects in the spinal cord were less in the antithrombin III group. Because antithrombin III facilitated the recovery of behavior and evoked potentials, these findings suggest that antithrombin III may have a positive effect on the recovery of incomplete spinal cord injury.

An Optimal Regimen of 0.2% Ropivacaine-Sufentanil as a Continuous Thoracic Epidural Infusion for Pain Control after Thoracotomy

Background: Thoracotomy is considered one of the most painful surgical procedures. Thus optimal pain control is essential in the postoperative care of thoracotomy patients. In this randomized double-blinded study, we sought to determine an optimal dose-combination of sufentanil and ropivacaine 0.2% for postoperative epidural analgesia. Methods: Forty-five of fifty-two patients that underwent thoracotomy were assigned to three groups to receive continuous high thoracic epidural analgesia with ropivacaine 0.2% + sufentanil 0.5g/ml (group I), ropivacaine 0.2% + sufentanil 0.75g/ml (group II), or ropivacaine 0.2% + sufentanil 1.0g/ml (group III). Upon completion of surgery, a thoracic epidural catheter was inserted at the T1-3 level, and moved downward by 5-7 cm. Epidural continuous infusion was administered at 5 ml/hr. Resting VAS (visual analogue scale), dynamic VAS, forced vital capacity, and side effects were recorded at three times: immediate after extubation; 12 hours after arrival at the ICU; and 24 hours after arrival at the ICU. Results: Resting VAS was significantly lower in group III immediately after extubation (P g/ml provides effective analgesia for post-thoracotomy pain with fewer side effects than the other two combinations tested.

Labeling of central projections of primary afferents in adult rats: a comparison between biotinylated dextran amine, neurobiotin™ and Phaseolus vulgaris-leucoagglutinin

The efficacy of anterograde labeling of the central projections of primary afferent fibers were compared between biotinylated dextran amine (BDA), neurobiotin™ (NB) and Phaseolus vulgaris-leucoagglutinin (PHA-L) after injections into the L5 or T13 dorsal root ganglia (DRGs) of adult rats. Excellent labeling was obtained with BDA, which visualized fibers with fine terminal boutons in the L5 and T13 spinal cord segments, Clarke's nucleus and the gracile nucleus. Rarely observed crossed projections to the gracile nucleus and L5 ventral horn of the contralateral side could also be distinguished. Even in the most successful experiments, however, BDA labeled only about one-third of the axons originating from the injected dorsal root ganglion. BDA was also efficient as transganglionic tracer after application to the transected sciatic nerve. NB produced no significant labeling of the L5 primary afferents, and was only moderately effective on the T13 level. PHA injections resulted in sparse terminal labeling of the T13 and L5 afferents. Thus, BDA is an effective tracer for long-range labeling of primary afferent projections in the spinal cord and brain stem. Since not all stem fibers become labeled, however, the method does not allow quantification of all axon branches and terminals arising from the injected DRGs.

Neurones in the dorsal root ganglia of T13, L1 and L2 innervate the dorsal portion of lower lumbar discs in rats

Based on a study using a retrograde neurotracer, we have previously found that the dorsal portion of the L5/6 disc in the rat is multisegmentally innervated by dorsal root ganglia (DRG) from the level of T13 to L6, and that sensory nerve fibres from DRG of T13, L1 and L2 pass through the paravertebral sympathetic trunks. In this study in newborn rats, we injected crystals of 1,1'-dioctadecyl-3,3,3',3'-tetramethylinedocarbocyanine perchlorate (DiI) into the DRG of T13, L1 and L2 and showed DiI-labelled sensory nerve fibres in the dorsal portion of the discs from the level of T13/L1 to L5/6. Our results show that the dorsal portion of the lumbar discs is innervated by the DRG from levels T13 to L2.

Neurones in the dorsal root ganglia of T13, L1 and L2 innervate the dorsal portion of lower lumbar discs in rats

Based on a study using a retrograde neurotracer, we have previously found that the dorsal portion of the L5/6 disc in the rat is multisegmentally innervated by dorsal root ganglia (DRG) from the level of T13 to L6, and that sensory nerve fibres from DRG of T13, L1 and L2 pass through the paravertebral sympathetic trunks. In this study in newborn rats, we injected crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylinedocarbocyanine perchlorate (DiI) into the DRG of T13, L1 and L2 and showed DiI-labelled sensory nerve fibres in the dorsal portion of the discs from the level of T13/L1 to L5/6. Our results show that the dorsal portion of the lumbar discs is innervated by the DRG from levels T13 to L2.

C-Fos induction in spinal cord neurons after renal arterial or venous occlusion.

Experiments were done in the anesthetized rat to identify the dorsal root ganglia (DRG) and the spinal cord segments that contain neurons activated by either renal venous occlusion (RVO) or by renal arterial occlusion (RAO). Fos induction, detected immunohistochemically in DRG and the spinal cord neurons, was used as a marker for neuronal activation. RVO induced Fos immunoreactivity in neurons in the DRG of spinal segments T8-L2 on the side ipsilateral to that of occlusion. The largest number of Fos-labeled neurons was found in the T11 DRG. In the spinal cord the largest number of Fos-labeled neurons was found in the ipsilateral dorsal horn of spinal segments T11-T12, predominantly in a cluster near the dorsomedial edge of laminae I-II. A few additional Fos-labeled neurons were observed in laminae IV and V. After RAO Fos-labeled neurons were found in the ipsilateral DRG of spinal segments similar to those observed to contain neurons after RVO. However, most of the Fos-labeled neurons were observed within the T12-L1 DRG. In the spinal cord Fos-labeled neurons were scattered throughout lamina I-II of the ipsilateral dorsal horn of spinal segments T8-L2, although the largest number was observed at the T13 level. Additionally, a distinct cluster of Fos-labeled neurons was observed predominantly in the region of the ipsilateral intermediolateral cell column, although a few neurons were found scattered throughout the nucleus intercalatus, central autonomic areas, and laminae IV and V of the cord bilaterally. No Fos labeling was observed in the complementary contralateral DRG or dorsal horns after either RVO or RAO. In addition, renal nerve transection prevented Fos labeling in the ipsilateral DRG and dorsal horns after RVO or RAO. Taken together, these data suggest that functionally different renal afferent fibers activate DRG neurons that may have distinct projections in the spinal cord.

Vertebral hemangiosarcoma as a cause of spinal cord compression in a horse

A case compression of the spinal cord in a horse by a tumor located in the vertebra is described. A 10-year-old gelding was euthanized after being found recumbent with hind limb paresis of unknown duration. At necropsy an hemangiosarcoma was detected in the vertebral body of T3. The tumoral mass invaded upwards through the bone tissue of the vertebral body into the vertebral canal, compressing the spinal cord and causing Wallerian degeneration at T1-3 levels of the cord.

Mechanisms within the spinal cord are involved in the movement-induced attenuation of an H reflex in the dog.

1. H reflexes were elicited in the second interosseous muscle of the hindpaw of the anesthetized dog during passive rotation of the shank about the ipsilateral or contralateral knee. Reflexes sampled at four points in the cycle of movement were compared with stationary controls. For both the ipsilateral and contralateral limb manipulations, reflexes were significantly reduced (P < 0.05) across the cycle of movement. Position-related modulation of the reflex amplitude was not detected (P > 0.05) in either instance. 2. The experiments were then repeated after the spinal transection of each animal at the level of T13. Passive rotation about either the ipsilateral or contralateral knee significantly attenuated (P < 0.05) the H reflex across a cycle of movement in the spinal dog. There was little difference in the amount of inhibition produced by the movement between the intact and spinal animals. On average, the reflex was attenuated 29 +/- 2.4% (mean +/- SE) in the intact animals and 32 +/- 2.1% in the spinal animals. 3. It is concluded that passive rotation about the knee of either leg leads to suppression of the H reflex of the second interosseous muscle both in the ipsilateral, moving leg and the contralateral, stationary one. This reflex suppression occurs across the cycle of movement. The mediating circuitry lies within the spinal cord, caudal to T13.

The use of local anaesthetic microinjections to identify central pathways: a quantitative evaluation of the time course and extent of the neuronal block.

The time course and extent of local anaesthetic blocks within the spinal cord of cats were evaluated. A monopolar stimulation electrode with the tip lowered into the dorsal columns (DC) 1000 microns below cord surface was used to activate antidromically DC fibers at the T13 level and evoke cord dorsum potentials at the level of the lumbar spinal cord. The amplitude of the negative deflection, the N-wave, was determined for various stimulation intensities (stimulation-response-function, SRF). Lidocaine (1%) was microinjected in volumes of 0.5 or 1.0 microliter into the DC from a glass micropipette 1 mm caudal to the stimulation site. Conduction block was characterized by a reversible shift of the SRFs to higher stimulation intensities. The diameter of the blocked area in the transverse plane was evaluated from threshold intensities and was found to be 0.9 +/- 0.1 mm 4 to 30 min after the injection of 0.5 microliter lidocaine and 1.6 +/- 0.36 mm 10 to 45 min after the injection of 1.0 microliter lidocaine. In the sagittal plane, the diameter of the blocked area following 1.0 microliter lidocaine was found to be up to 2.8 mm. The DC-block was reversible within 92 min following injection of 1.0 microliter and 69 min after the injection of 0.5 microliter lidocaine. The application of the present findings for blocks in other CNS structures is discussed.

Neuropeptides in sensory perikarya projecting to the rat ovary

Afferent perikarya in dorsal root ganglia (DRG) at the T13 and L1 segmental levels projecting to the rat ovary were identified by utilizing the fluorescent retrograde tracer true blue (TB). Subsequently, TB-labeled ovarian afferent perikarya in DRG were examined for vasoactive intestinal polypeptide (VIP), substance P (SP), cholecystokinin-8 (CCK-8), neuropeptide Y (NPY), and somatostatin (SOM) immunoreactivity and for the presence of fluoride-resistant acid phosphatase (FRAP) enzyme activity. Of the ovarian afferent perikarya at the T13 and L1 segmental levels, 20.5% displayed VIP immunoreactivity, 23.8% displayed SP immunoreactivity, and 43.1% were immunoreactive for CCK-8. No ovarian afferent perikarya contained SOM or NYP immunoreactivity or FRAP activity. It is suggested that different neuropeptides may participate in modulation of specific ovarian functions.

Primary afferent projections of the major splanchnic nerve to the spinal cord and gracile nucleus of the cat.

Splanchnic afferent projections to the spinal cord and gracile nucleus were labeled following the application of HRP to the central cut end of the major splanchnic nerve. Labeled afferent fibers were detected in the ipsilateral dorsal column, in Lissauer's tract (LT), in laminae 1, 5, 7, and 10, and in the dorsal gray commissure at T1-T13 levels of the spinal cord. Afferent projections were not identified in laminae 2-4. Collaterals from LT projected ventrally along the lateral and medial margins of the dorsal horn (called lateral and medial pathways, respectively). Afferents in the lateral pathway formed small bundles, spaced rostrocaudally at intervals of 300-1,000 microns, which passed medially at the base of the dorsal horn into laminae 5, 7, and 10 and to the contralateral spinal cord. Some afferents in the lateral pathway projected to the intermediolateral nucleus where labeled sympathetic preganglionic neurons were located. Afferents in the medial pathway entered the lateral aspect of the dorsal column and projected as a group near the midline rostrally to the medulla. The dorsal column pathway terminated in the ventral gracile nucleus in four or five clusters, each occupying a region ranging in size from 0.01-0.1 mm3 and separated in the rostrocaudal axis by distances of 400-800 microns. These clusters were concentrated in the middle and caudal portions of the nucleus below the obex. A comparison of the present results with those from earlier experiments on the central projections of afferent fibers from the heart, kidney, and pelvic organs demonstrates a consistent pattern of visceral afferent termination in the thoracolumbar and sacral segments of the spinal cord. This is not unexpected, since visceral afferent pathways to different organs perform similar functions, such as the transmission of nociceptive information and the initiation of autonomic reflexes.

Retrograde changes in the nerve fibers of the dorsal spinocerebellar and spinocervical tracts in the cat.

The potential retrograde changes that occur in the proximal segment of transected nerve fibers found in the region of the spinocervical tract (SCT) and in the region of the dorsal spinocerebellar tract (DSCT) after unilateral dorsolateral column (DLC) lesions were studied in kittens and adult cats. Nerve fibers in the region of the SCT and DSCT in the spinal segments caudal to the lesion were studied in transverse sections of Epon embedded material that had been fixed by vascular perfusion with a 4% buffered paraformaldehyde solution, post-fixed in osmium tetroxide, and stained with Azur II-methylene blue. T8 level DLC lesions in the 10-week old kitten resulted in a 14 to 25% decrease in mean fiber diameter in the SCT fibers (larger than or equal to 5 mu) of the T13 level. Retrograde fiber degeneration occurred in the small and large caliber fibers of the DSCT. A 15 to 30% decrease in mean fiber diameter occurred in the DSCT fibers (greater than or equal to 5 mu). The fiber density of the DSCT decreased by 20 to 30% and this could be correlated with a 20% loss of class C neurons (Loewy, '70) and a severe atrophy affecting the remaining 80% of the surviving class C neurons in the L3 segment. Mid-cervical DLC lesions in the 10-week old kitten resulted in a 10% decrease in mean fiber diameter in the SCT fibers (less than or equal to 5 mu), a 15% decrease in mean fiber diameter in the DSCT fibers (less than or equal to 5 mu), and no change in fiber density could be determined despite the finding that these lesions produced about a 10% loss of class C neurons. A T1 level lesion in a 20-week old kitten produced no change in the SCT fibers suggesting that the changes seen in the 10-week old kitten after midcervical lesions were probably age-dependent changes. A 20% decrease in mean fiber diameter (less than 5 mu) and a 6% decrease in fiber density was detected in the DSCT. T13-L1 level DLC lesions in the adult cats resulted in degeneration of some of the large and medium size fibers in the DSCT which was most noticeable at 20 to 25 days after surgery. DLC lesions did not cause degenerative changes in the SCT fibers 5 to 25 days after surgery.