Opioids evoke postural asymmetry in rats: the side of the flexed paw depends on the type of opioid agonist

Opioid kappa-agonists bremazocine and dynorphin (1-13), sigma-agonist SKF 10.047 and delta-agonist D-Ala2, D-Leu5-enkephalin (DADL) induce postural asymmetry of rats hind limbs under subarachnoidal administration below the level of spinal cord section (T3-T4). The side of the flexed leg depends on the opioid agonist type: bremazocine and dynorphin (1-13) induce predominantly right flexion. SKF 10.047--the left flexion, but not in all doses, DADL--in small doses (1 and 100 pg per animal)--of the right one, in larger doses (up to 10 ng per animal)--of the left one. Saline and opiate mu-agonist morphine do not induce postural asymmetry. Opiate antagonist naloxone prevents asymmetry development when injected prior opioid agonists, and also decreases the number of asymmetries induced by these agonists. Naloxone alone does not influence the per cent of animals with pose asymmetry. The opioid receptors are involved in asymmetry development. The revealed ability of opioid kappa-, delta- and sigma-agonists may be based on lateralization of opioid receptors in the rat spinal cord.     

Met-enkephalin-induced release into the blood of a factor causing postural asymmetry

Met- and Leu-enkephalin applied subarachnoidally into the rostral portion of a transected spinal cord (at the T6-T7 level) induce postural asymmetry of the hind limbs in rats, Met-enkephalin being predominantly responsible for the flexion of the right, and Leu-enkephalin of the left, hind leg. The blood serum of rats injected with Met-enkephalin contains a factor which, when administered subarachnoidally into the caudal portion of the transected spinal cord, is capable of inducing the hind limb postural asymmetry--predominantly, with the right leg flexion. This factor is inactivated by papain and differs from Met- and Leu-enkephalin in chromatographic properties. Apparently, Met-enkephalin induces the release of a peptide factor into the blood, from the brain or organs innervated by the neurons lying above the cut. It is then carried with the blood to the hind limbs and effects the hind limb postural asymmetry.     

The activity of postural asymmetry factors in symmetrical sections of the rat spinal cord

The distribution of the low-molecular weight and high-molecular weight postural asymmetry factors (FPA) activity in the left and right parts of the lumbal region of the rat spinal cord was studied. Low-molecular weight FPA induces flexion of the hind limb ipsilateral to the half of the spinal cord from which FPA was isolated, while high-molecular weight FPA induces contralateral flexion. The activities of the low- and high-molecular weight FPAs in each half of the spinal cord are comparable in normal rat. After the suction lesion of the motor areas in the left hemisphere the increase of the low-molecular weight FPA activity in the right half of the lumbal region of the spinal cord was observed.     

Role of the hypophysis in fixing postural asymmetry at the segmental level in hemisection of the spinal cord

The role of the pituitary in the mechanisms of posttraumatic reorganizations of the segmental apparatus was studied. Hypophysectomized rats failed to demonstrate postural asymmetry after spinal cord hemisection. The lack of asymmetry is connected with a considerable decrease in postural asymmetry factor activity in the cerebrospinal fluid and cerebral tissue. It was established that pituitary tissue is characterized by the maximal level of postural asymmetry factor activity after hemisection.     

Formation of postural asymmetry in rat hindlimbs during colchicine-induced unilateral blockade of axonal transport in cortico-lumbar projections

The possibility of the formation of spinal cord functional asymmetry by the blockade of axonal transport in corticolumbar projections with colchicine was investigated. To identify the blockade of axonal transport, the method of retrograde transport of horseradish peroxidase was used. The blockade of axonal transport led to the formation of the asymmetric functional status of the spinal cord, manifesting in postural asymmetry of the hind limbs and characteristic changes in the pattern of bioelectrical activity of the flexor muscles. An endogenous factor inducing postural asymmetry in intact recipients was detected in the cerebrospinal fluid of colchicine treated animals. Based on the experimental data the conclusion is drawn that interruption of normal axonal transport attests to the destruction of central neurons.     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

Mechanism of expiratory muscle activation during lower thoracic spinal cord stimulation.

Lower thoracic spinal cord stimulation (SCS) may be a useful method to restore an effective cough mechanism. In dogs, two groups of studies were performed to evaluate the mechanism of the expiratory muscle activation during stimulation at the T(9)-T(10) level, which results in the greatest changes in airway pressure. In one group, expiratory muscle activation was monitored by evoked muscle compound action potentials (CAPs) from the internal intercostal muscles in the 10th, 11th, and 12th interspaces and from portions of the external oblique innervated by the L(1) and L(2) motor roots. SCS, applied with single shocks, resulted in short-latency CAPs at T(10) but not at more caudal levels. SCS resulted in long-latency CAPs at each of the more caudal caudal recording sites. Bilateral dorsal column sectioning, just below the T(11) spinal cord level, did not affect the short-latency CAPs but abolished the long-latency CAPs and also resulted in a fall in airway pressure generation. In the second group, sequential spinal root sectioning was performed to assess their individual mechanical contribution to pressure generation. Section of the ventral roots from T(8) through T(10) resulted in negligible changes, whereas section of more caudal roots resulted in a progressive reduction in pressure generation. We conclude that 1) SCS at the T(9)-T(10) level results in direct activation of spinal cord roots within two to three segments of the stimulating electrode and activation of more distal roots via spinal cord pathways, and 2) pathway activation of motor roots makes a substantial contribution to pressure generation.     

Postural asymmetry in rats induced by stress and pain stimuli

A significant number of rats (31–68%) subjected to pain stimulation (intraperitoneal injection of 1% or 5% acetic acid, 5 ml/kg), immobilization stress (6 hrs in a supine position) or cold stress (3 hrs at 4–6°C) exhibited postural asymmetry of hind limbs after spinal cord transection at T7 level. The number of rats with right and left limb flexions was approximately equal. Naloxone (2 mg/kg intraperitoneally) prevented the development of postural asymmetry induced by pain and stress stimuli. Postural asymmetry of hind limbs appears to be due to an asymmetric CNS response to stress and pain stimuli. Our data imply that endogenous opioid peptides and opiate receptors may be involved in the regulation of this response.     

应用cDNA微阵列技术筛选大鼠脊髓损伤修复相关基因

脊髓损伤是一类常见的、高致残率的中枢神经系统疾病,由于多种复杂因素影响其损伤后的修复过程,损伤脊髓的再生能力非常有限。本研究采用cDNA微阵列技术筛选大鼠脊髓损伤后出现的差异表达基因。实验组动物在T8-T9进行脊髓全横断手术,对照组动物只打开椎板;4．5d后取脊髓进行RNA提取并在反转录过程中进行Cy3／Cy5标记,然后与预制的、带有4041条特异性探针的芯片进行杂交。Cy5／Cy3信号比值≥2．0视为脊髓损伤后出现差异表达的基因。通过筛选,我们得到了65个上调表达基因（21个已知基因,30个已知EST和14个未知基因）和79个下调基因（20个已知基因,42个已知EST和17个未知基因）。进一步通过半定量RT-PCR对其中的5个上调已知基因（Timpl,Tagln,Vim,Fc gamma receptor,Ctss）和三个下调已知基因（stearyl-CoA desaturase,F2,Ensa）的表达情况进行了验证,结果显示与芯片结果一致。这些基因可能在脊髓损伤后的修复过程中起一定的作用,对其深入研究将有助于揭示脊髓损伤修复的分子机制。     

Translational constraint influences dynamic spinal canal occlusion of the thoracic spine: an in vitro experimental study

Mechanical constraints to spine motion can arise in a variety of real-world situations such as when shoulder belts prevent anterior translation of the thorax during automotive collisions. The effect of such constraint on spinal column-spinal cord interaction during injury remains unknown. The purpose of the present study was to compare maximal dynamic spinal canal occlusion, measured via a specialized transducer, in cadaveric upper thoracic spine specimens under a variety of anterior-posterior constraint conditions. Four injury models were produced using 24 cadaveric spine specimens (T1-T4). Incremental compressive trauma was applied under constrained (i.e. blocked anterior-posterior translation) flexion-compression, pure-compression and extension-compression, and under unconstrained (i.e. free anterior-posterior translation) flexion-compression. All displacements were applied at 500 mm/s. For all three constrained trauma groups, complete transducer occlusion occurred between 20 and 30 mm of compressive displacement. The extension-compression caused transducer occlusion significantly less than the other constrained models (p < 0.022) at 20 mm compression. For unconstrained flexion-compression, a compression of up to 50 mm resulted in a mean of 26% transducer occlusion. The constrained pure-compression tests led to burst fracture with significant body height loss at T2. The constrained flexion-compression and extension-compression tests caused fracture-dislocation injury at the T2-T3 level. Constrained trauma clearly led to more spinal canal occlusion than the unconstrained in these models, and more severe injury to the spinal column. The results add to our understanding of the effect of column injury pattern on spinal cord injury. This information has clear implications for the design of injury prevention devices.     

The effect of follower load on the intersegmental coupled motion characteristics of the human thoracic spine: An in vitro study using entire rib cage specimens

The mechanical coupling behaviour of the thoracic spine is still not fully understood. For the validation of numerical models of the thoracic spine, however, the coupled motions within the single spinal segments are of importance to achieve high model accuracy. In the present study, eight fresh frozen human thoracic spinal specimens (C7-L1, mean age 54 ± 6 years) including the intact rib cage were loaded with pure bending moments of 5 Nm in flexion/extension (FE), lateral bending (LB), and axial rotation (AR) with and without a follower load of 400 N. During loading, the relative motions of each vertebra were monitored. Follower load decreased the overall ROM (T1-T12) significantly (p < 0.01) in all primary motion directions (extension: −46%, left LB: −72%, right LB: −72%, left AR: −26%, right AR: −26%) except flexion (−36%). Substantial coupled motion was found in lateral bending with ipsilateral axial rotation, which increased after a follower load was applied, leading to a dominant axial rotation during primary lateral bending, while all other coupled motions in the different motion directions were reduced under follower load. On the monosegmental level, the follower load especially reduced the ROM of the upper thoracic spine from T1-T2 to T4-T5 in all motion directions and the ROM of the lower thoracic spine from T9-T10 to T11-T12 in primary lateral bending. The facet joints, intervertebral disc morphologies, and the sagittal curvature presumably affect the thoracic spinal coupled motions depending on axial compressive preloading. Using these results, the validation of numerical models can be performed more accurately.     

Modification of radiation myelopathy by the transplantation of neural stem cells in the rat

In a novel approach, neural stem cells were transplanted to ameliorate radiation-induced myelopathy in the spinal cords of rats. A 12-mm section of the cervical spinal cord (T2-C2) of 5-week-old female Sprague-Dawley rats was locally irradiated with a single dose of 22 Gy of (60)Co gamma rays. This dose is known to produce myelopathy in all animals within 6 months of irradiation. After irradiation, the animals were subdivided into three groups, and at 90 days after irradiation, neural stem cells or saline (for controls) were injected into the spinal cord, intramedullary, at two sites positioned 6 mm apart on either side of the center of the irradiated length of spinal cord. The injection volume was 2 microl. Group I received a suspension of MHP36 cells, Group II MHP15 cells, and Group III (controls) two injections of 2 microl saline. All rats received 10 mg/kg cyclosporin (10 mg/ml) daily i.p. to produce immunosuppression. All animals that received saline (Group III) developed paralysis within 167 days of irradiation. The paralysis-free survival rates of rats that received transplanted MHP36 and MHP15 cells (Groups I and II) were 36.4% and 32% at 183 days, respectively. It was concluded that transplantation of neural stem cells 90 days after irradiation significantly (P = 0.03) ameliorated the expression of radiation-induced myelopathy in the spinal cords of rats.     

Neuropeptide Y, peptide YY, and pancreatic polypeptide modulate duodenal and colonic motility at a thoracic spinal site in rats.

Neuropeptide Y, PYY, and PP (200 pmol) alter intraluminal pressure in the duodenum and colon of rats following their administration into the thoracic (T8-T10) region of the spinal cord. Neuropeptide Y decreases the tone of the duodenum and the colon following intrathecal (T8-T10) administration prior to an increase in tone to baseline or greater. There is no effect on intraluminal pressure of either the duodenum or the colon following intrathecal administration of NPY or PP into the lumbar (L4-L5) region of the spinal cord. Following intrathecal (T8-T10) administration of PYY and PP, increases in intraduodenal pressures are observed (+2.1 and +3.0 mmHg from saline baseline). Phasic contractions of the duodenum are increased following intrathecal administration of PYY into the thoracic spinal cord of rats. Neuropeptide Y, PYY, and PP increase intracolonic pressure +2.2, +3.3, and +3.7 mmHg from saline baseline, respectively. Phasic contractions of the colon are increased following PP intrathecal thoracic administration. Responsiveness of the duodenum or colon to the different ligands of the PP-fold peptide family in the absence of alpha-adrenergic blockade did not vary. The increases in intraluminal pressure of the duodenum and colon following intrathecal administration of the PP-fold peptides are attenuated by both alpha-1 adrenergic (prazosin) and alpha-2 adrenergic (yohimbine) blockade. There is a difference in responsiveness of the colon between the ligands of the PP-fold family in the presence of the alpha-2 adrenergic blockade. The findings of this study indicate that duodenal and colonic motility are modulated by the PP-fold peptides at thoracic spinal sites via alteration of sympathetic outflow.     

Left vagal stimulation induces dynorphin release and suppresses substance P release from the rat thoracic spinal cord during cardiac ischemia

Electrostimulatory forms of therapy can reduce angina that arises from activation of cardiac nociceptive afferent fibers during transient ischemia. This study sought to determine the effects of electrical stimulation of left thoracic vagal afferents (C(8)-T(1) level) on the release of putative nociceptive [substance P (SP)] and analgesic [dynorphin (Dyn)] peptides in the dorsal horn at the T(4) spinal level during coronary artery occlusion in urethane-anesthetized Sprague-Dawley rats. Release of Dyn and SP was measured by using antibody-coated microprobes. While Dyn and SP had a basal release, occlusion of the left anterior descending coronary artery only affected SP release, causing an increase from lamina I-VII. Left vagal stimulation increased Dyn release, inhibited basal SP release, and blunted the coronary artery occlusion-induced release of SP. Dyn release reflected activation of descending pathways in the thoracic spinal cord, because vagal afferent stimulation still increased the release of Dyn after bilateral dorsal rhizotomy of T(2)-T(5). These results indicate that electrostimulatory therapy, using vagal afferent excitation, may induce analgesia, in part, via inhibition of the release of SP in the spinal cord, possibly through a Dyn-mediated neuronal interaction.     

Effect of spinal level and loading conditions on the production of vertebral burst fractures in a porcine model

Vertebral burst fractures are commonly studied with experimental animal models. There is however a lack of consensus as to what parameters are important to create an unstable burst fracture with a significant canal encroachment on such model. This study aims to assess the effect of the loading rate, flexion angle, spinal level, and their interactions on the production of a vertebral thoracolumbar burst fracture on a porcine model. Sixteen functional spinal units composed of three vertebrae were harvested from mature Yucatan minipigs. Two loading rates (0.01 and 500 mm/s), two flexion angles (0° and 15°), and two spinal levels (T11-T13 and T14-L2) were studied, following a full factorial experimental plan with one repetition. Compression was applied to each functional unit to create a vertebral fracture. The load-to-failure, loss of compressive stiffness, final canal encroachment, and fracture type were used as criteria to evaluate the resulting fracture. All specimens compressed without flexion resulted in burst fractures. Half of the specimens compressed with the 15° flexion angle resulted in compression fractures. Specimens positioned without flexion lost more of their compressive stiffness and had more significant canal encroachment. Fractured units compressed with a higher loading rate resulted in a greater loss of compressive stiffness. The spinal level had no significant effect on the resulting fractures. The main parameters which affect the resulting fracture are the loading rate and the flexion angle. A higher loading rate and the absence of flexion favors the production of burst fractures with a greater canal encroachment.     

Efferent sympathetic preganglionic and afferent spinal transit pathways of the cat stellate ganglion

Using a technique of retrograde axonal transport of horseradish peroxidase, labeled neurons were detected in the intermedialateral nucleus (pars principalis and pars funicularis), intercalatous spinal nucleus, and in the ventral horns of the spinal cord in cats. Afferent spinal transit pathways pass in all the above branches as well as the vertebral nerve. Bodies of the labeled neurons with branches passing in the vertebral nerve are located in the T2-T7 spinal ganglia, whereas those with branches passing in other nerves--are located in the C8-T8.     

Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury

The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MP-treated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.     

Dynamics of the activity of the postural asymmetry factor after unilateral damage to the motor area of the cortex

The authors studied the time-course of functional rearrangements of the segmental apparatus after unilateral injury of the rat motor cortex. It was found that one day after injury the postural asymmetry of the hind limbs was fixed by the lumbal region of the spinal cord. This functional state of the segmental apparatus lasted 10 days after injury in the presence of the maximal activity of postural asymmetry factor (PAF) in the CSF and increasing activity of the factor in the brain tissue. Recovery of the segmental apparatus to symmetrical function by the end of the third week following injury was accompanied by PAF inactivation.     

First transplantation of embryonic serotonergic neurons in primate spinal cord

Three adult monkeys (Macaca fascicularis) underwent a total section of the spinal cord at thoracic level (T6). 1 week later, two of them received at T8 level an injection of a cellular suspension prepared from the raphe region of a foetal macaque 39 days old. The third animal received one injection of buffer solution. 1 month later, the animals were sacrificed, and their spinal cord was processed for the immunocytochemical detection of serotonin with light and electron microscopy. Serotonergic neurons had survived after transplantation, and had grown axons and dendrites. Afferent and efferent synapses to serotonergic neurons were readily identified.     

Successful treatment of paralytic relapses in adoptive experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance.

We have previously demonstrated that Ag-specific tolerance induced by the i.v. administration of splenocytes coupled with neuroantigens, such as mouse spinal cord homogenate, myelin basic protein (MBP), and proteolipid protein, and their encephalitogenic peptides, results in dramatic inhibition of clinical and histologic signs of both actively induced and adoptively transferred relapsing experimental autoimmune encephalomyelitis (R-EAE). We report here that the administration of splenocytes coupled with mouse spinal cord homogenate (i.e., a mixture of neuroantigens), after the first paralytic episode of adoptive R-EAE triggered by MBP-specific T cells but before the appearance of the first relapse, effectively reduced the onset and severity of all subsequent relapses, as determined by both clinical and pathologic criteria. In contrast, the i.v. administration of splenocytes coupled with MBP (i.e., the specificity of the initiating T cell response), under similar conditions, effectively inhibited the initial clinical relapse, but subsequent relapses occurred with the same incidence rate and severity as those in control animals. Collectively, these results demonstrate that neuroantigen-specific tolerance is effective at specifically down-regulating an ongoing autoimmune response. This may have potential clinical applicability for treatment of autoimmune diseases. The results also support the hypothesis that the neuroantigen specificity of later relapses of R-EAE may be due to effector T cells with specificities different from those that triggered the initial clinical episode. Thus, potential therapy for the advanced stages of R-EAE, and perhaps other autoimmune diseases, may have to be directed not simply against the effector cells initiating the disease but also against effector cells with differing specificities recruited as a result of tissue damage occurring in the initial acute disease.