L3-L5 Segments Research Articles

Technical aspects of rod-insertion forceps (persuader) application in reducing construct failure after lumbar spine fusion surgery: a biomechanical cadaveric study in Germany.

A prospective experimental study. This biomechanical in vitro study aimed to examine the extent to which the use of a rod persuader (RP) leads to additional mechanical stress on the screw-rod system and determine its influence on the bony anchoring of primary pedicle screws. Degenerative spine diseases and deformities are the most common indications for the stabilization and fusion of spinal segments. The pedicle screw-rod system is considered the gold standard for dorsal stabilization, and an RP is also increasingly being considered to fit the spondylodesis material. Ten lumbar spines from body donors were examined. Bisegmental dorsal spinal lumbar interbody fusion of the L3-L5 segments was performed using a pedicle screw-rod system (ROCCIA Multi-LIF Cage; Silony Medical, Germany). In group 1, the titanium rod was inserted without tension, whereas in group 2, the rod was attached to the pedicle screws at the L4 and L5 levels, creating a 5-mm gap. To attach the rod, the RP was used to press the rod into the pedicle screw. The rod was left in place for 30 minutes and then removed. The rod reduction technique significantly increased the mechanical load on the overall construct measured by strain gauges (p<0.05) and resulted in outright implant failure with pedicle screw pullout in 88.9%. In cases where the spondylodesis material is not fully attached within the pedicle screw, an RP can be used with extreme caution, particularly in osteoporotic bones, to avoid pedicle screw avulsion and screw anchor failure.

Application of polyetheretherketone rod semi-rigid pedicle screw internal fixation in lumbar non-fusion surgery

To investigate the effect of Polyetheretherketone (PEEK) rod semi-rigid pedicle screw fixation system in lumbar spine non-fusion surgery. A total of 74 patients with tow-level lumbar degenerative diseases who underwent surgery from March 2017 to December 2019 were divided into PEEK rod group and titanium rod group. In the PEEK rod group, there were 34 patients, including 13 males and 21 females, aged from 51 to 79 years old with an average of (62.4±6.8) years old;There were 1 patient of L1-L3 segments, 7 patients of L2-L4 segments, 20 patients of L3-L5 segments and 6 patients of L4-S1 segments. In the titanium rod group, there were 40 patients, including 17 males and 23 females, aged from 52 to 81 years old with an average of (65.2±7.3) years old;There were 3 patient of L1-L3 segments, 11 patients of L2-L4 segments, 19 patients of L3-L5 segments and 7 patients of L4-S1 segments. The general conditions of operation, such as operation time, intraoperative blood loss, postoperative drainage was recorded. The visual analogue scale (VAS) for low back pain and Oswestry disability index (ODI) were compared in preoperatively and postoperatively(3 months, 12 months and last follow-up) between two groups. The change of range of motion (ROM) was observed by flexion and extension x-ray of lumbar. All patients successfully completed the operation. The follow-up time ranged from 22 to 34 months with an average of(26.8±5.6) months. The operative time (142.2±44.7) min and intraoperative blood loss(166.5±67.4)ml in PEEK group were lower than those in titanium group [(160.7±57.3) min、(212.8±85.4) ml](P<0.05). There was no significant differences in postoperative drainage between the two groups (P>0.05). At the final follow-up visit, in PEEK group and titanium group VAS of low back pain[(0.8±0.4) points vs (1.0±0.5) points], VAS for leg pain [ (0.7±0.4) points vs (0.8±0.5) points] and ODI [(9.8±1.6)% vs (12.1±1.5)%] were compared with preoperative [ (5.8±1.1) points vs (6.0±1.1)points], [ (7.2±1.7) points vs (7.0±1.6) points], [(68.5±8.9)% vs(66.3±8.2)%] were significantly different(P<0.05). There was no significant difference in VAS scores between the two groups at each postoperative time point (P>0.05). At 3 months after surgery, there was no difference in ODI between the two groups (P>0.05). There were significant differences in ODI between PEEK group and titanium rod group at 12 months [(15.5±2.1)% vs (18.4±2.4)%] and at the last follow-up [(9.8±1.6)% vs (12.1±1.5)%] (P<0.05). The total range of motion (ROM) of lumbar decreased in both groups after surgery. At 12 months after surgery and the last follow-up, the PEEK group compared with the titanium rod group, the total range of motion of lumbar was statistically significant (P<0.05). The range of motion (ROM) of the fixed segments decreased in both groups after surgery. The ROM of the fixed segments in PEEK group decreased from (9.5±4.6)° to (4.1±1.9)° at the last follow-up (P<0.05), which in the titanium rod group was decreased from (9.8±4.3)°to (0.9±0.5)° at the last follow-up (P<0.05). The range of motion (ROM) of upper adjacent segment increased in both groups, there was statistical significance in the ROM of upper adjacent segment between the two groups at 12 months after surgery and the last follow-up, (P<0.05). There was no screw loosening and broken rods in both groups during the follow-up period. The PEEK rod semi-rigid pedicle screw internal fixation system used in lumbar non-fusion surgery can retain part of the mobility of the fixed segment, showing comparable short-term clinical efficacy to titanium rod fusion. PEEK rod semi-rigid pedicle screw internal fixation system is a feasible choice for the treatment of lumbar spine degenerative diseases, and its long-term efficacy needs further follow-up observation.

MRI-Derived Dural Sac and Lumbar Vertebrae 3D Volumetry Has Potential for Detection of Marfan Syndrome

Purpose: To assess the feasibility and diagnostic accuracy of MRI-derived 3D volumetry of lower lumbar vertebrae and dural sac segments using shape-based machine learning for the detection of Marfan syndrome (MFS) compared with dural sac diameter ratios (the current clinical standard). Materials and methods: The final study sample was 144 patients being evaluated for MFS from 01/2012 to 12/2016, of whom 81 were non-MFS patients (46 [67%] female, 36 ± 16 years) and 63 were MFS patients (36 [57%] female, 35 ± 11 years) according to the 2010 Revised Ghent Nosology. All patients underwent 1.5T MRI with isotropic 1 × 1 × 1 mm3 3D T2-weighted acquisition of the lumbosacral spine. Segmentation and quantification of vertebral bodies L3-L5 and dural sac segments L3-S1 were performed using a shape-based machine learning algorithm. For comparison with the current clinical standard, anteroposterior diameters of vertebral bodies and dural sac were measured. Ratios between dural sac volume/diameter at the respective level and vertebral body volume/diameter were calculated. Results: Three-dimensional volumetry revealed larger dural sac volumes (p &lt; 0.001) and volume ratios (p &lt; 0.001) at L3-S1 levels in MFS patients compared with non-MFS patients. For the detection of MFS, 3D volumetry achieved higher AUCs at L3-S1 levels (0.743, 0.752, 0.808, and 0.824) compared with dural sac diameter ratios (0.673, 0.707, 0.791, and 0.848); a significant difference was observed only for L3 (p &lt; 0.001). Conclusion: MRI-derived 3D volumetry of the lumbosacral dural sac and vertebral bodies is a feasible method for quantifying dural ectasia using shape-based machine learning. Non-inferior diagnostic accuracy was observed compared with dural sac diameter ratio (the current clinical standard for MFS detection).

Increased nociceptive behaviors and spinal c-Fos expression in the formalin test in a rat repeated cold stress model

Repeated cold stress (RCS) can trigger the development of fibromyalgia (FM)-like symptoms, including persistent deep-tissue pain, although nociceptive changes to the skin have not been fully characterized. Using a rat RCS model, we investigated nociceptive behaviors induced by noxious mechanical, thermal, and chemical stimuli applied to plantar skin. Neuronal activation in the spinal dorsal horn was examined using the formalin pain test. In rats exposed to RCS, nociceptive behavioral hypersensitivity was observed in all modalities of cutaneous noxious stimuli: the mechanical withdrawal threshold was decreased, and the heat withdrawal latency was shortened one day after the cessation of stress. The duration of nocifensive behaviors in the formalin test was prolonged in phase II but not in phase I. The number of c-Fos-positive neurons increased in the entire dorsal horn laminae I-VI, ipsilateral, but not contralateral, to formalin injection at the L3-L5 segments. The duration of nocifensive behavior in phase II was significantly and positively correlated with the number of c-Fos-positive neurons in laminae I-II. These results demonstrate that cutaneous nociception is facilitated in rats exposed to RCS for a short time and that the spinal dorsal horn neurons are hyperactivated by cutaneous formalin in the RCS model.

Differential dopamine modulation of spinal reflex amplitudes is associated with the presence or absence of the autonomic nervous system

The spinal cord contains a highly collateralized network of descending dopamine (DA) fibers that stem from the dorso-posterior hypothalamic A11 region in the brain, however, the modulatory actions of DA have generally only been assessed in lumbar segments L2-L5. In contrast to these exclusively sensorimotor segments, spinal cords segments T1-L2 and, in mouse, L6-S2, additionally contain the intermediolateral (IML) nucleus, the origin of autonomic nervous system (ANS).Here, we tested if the different spinal circuits in sensorimotor and IML-containing segments react differently to the modulation of the monosynaptic reflex (MSR) by DA. Bath-application of DA (1 μM) led to a decrease of MSR amplitude in L3-L5 segments; however, in IML-containing segments (T10-L2, and S1/2) the MSR response was facilitated. We did not observe any difference in the response between thoracic (sympathetic) and lumbosacral (parasympathetic) segments. Application of the D2-receptor agonists bromocriptine or quinpirole mimicked the effects of DA, while blocking D2 receptor pathways with raclopride or application with the D1-receptor agonist SKF 38393 led to an increase of the MSR in L3-L5 segments and a decrease of the MSR in IML-containing segments. In contrast, in the presence of the gap-junction blockers, carbenoloxone and quinine, DA modulatory actions in IML-containing segments were similar to those of sensorimotor L3-L5 segments.We suggest that DA modulates MSR amplitudes in the spinal cord in a segment-specific manner, and that the differential outcome observed in ANS segments may be a result of gap junctions in the IML.

Transcriptome Changes In Dorsal Spinal Cord Of Rats With Neuropathic Pain.

BackgroundMechanisms of neuropathic pain are not fully understood. Molecular changes in spinal dorsal horn take part in the initiation and development of neuropathic pain.MethodsTo detect the transcriptome changes in the dorsal spinal cord of neuropathic pain rat, sciatic nerve chronic constriction injury (CCI) rats were used. Then, the CCI ipsilateral dorsal spinal cords of lumbar L3-L5 segments were collected at 14th day post-CCI and subjected to microRNA and long non-coding RNA (lncRNA)/mRNA microarray. To evaluate functions of differential mRNAs, bioinformatics methods including gene ontology (GO) and KEGG pathway analysis were conducted for significantly up- and downregulated mRNAs.ResultsMicroRNA microarrays showed that 13 microRNAs were differently expressed between CCI and sham-operated rats (fold change ≥ 2.0). Six of them were upregulated, and the other seven were downregulated in CCI group. MicroRNA-1b overexpressed 18.7 times after CCI. LncRNA/mRNA microarray detected 876 lncRNAs with significant differential expression (fold change ≥ 2.0). Among them, 339 were significantly upregulated, and 537 were downregulated in CCI group. Sixteen of them differentially expressed more than 10 times and the lncRNA XR_356687 overexpressed as high as 53 times. In addition, 950 mRNAs were differentially expressed (fold change ≥ 2.0), including 405 upregulated and 545 downregulated in CCI group. Ten of these mRNAs with changed expressions of more than 10 times. The Hspa1b (encodes heat shock protein 70) overexpressed 24 times in CCI rats. Gene ontology analysis revealed that hundreds of differentially expressed mRNAs involved in the biological processes, cellular component, and molecular function. In addition, these genes significantly enriched into 32 KEGG pathways, including the TNF, FoxO, cytokine–cytokine receptor interaction, and calcium signaling pathways.ConclusionNeuropathic pain induced comprehensive changes of transcription profile in the dorsal spinal cord. These differentially expressed transcripts in spinal cord could be potential targets in defeating neuropathic pain.

Morphometric Analysis of the Lumbar Vertebrae Concerning the Optimal Screw Selection for Transpedicular Stabilization.

Transpedicular stabilization is a frequently used spinal surgery for fractures, degenerative changes, or neoplastic processes. Improper screw fixation may cause substantial vascular or neurological complications. This study seeks to define detailed morphometric measurements of the pedicle (height, width, and surface area) in the aspects of screw length and girth selection and the trajectory of its implantation, i.e., sagittal and transverse angle of placement. The study was based on CT examinations of 100 Caucasian patients (51 women and 49 men) aged 27-75 with no anatomical, degenerative, or post-traumatic spine changes. The results were stratified by gender and body side, and they were counter compared with the available literature database. Pedicle height decreased from L1 to L4, ranging from 15.9 to 13.3mm. Pedicle width increased from L1 to L5, extending from 6.1 to 13.2mm. Pedicle surface area increased from L1 to L5, ranging from 63 to 140mm2. Distance from the point of entry into the pedicle to the anterior surface of the vertebral body, defining the maximum length of a transpedicular screw, varied from 54.0 to 50.2mm. Variations concerning body sides were inappreciable. A transverse angle of screw trajectory extended from 20° to 32°, shifting caudally from L1 to L5, with statistical differences in the L3-L5 segments. A sagittal angle varied from 10° to 12°, without such definite relations. We conclude that the L1 and L2 segments display the most distinct morphometric similarities, while the greatest differences, in both genders, are noted for L3, L4, and L5. The findings enable the recommendation of the following screw diameters: 4mm for L1-L2, 5mm for L3, 6mm for L4-L5, and the length of 50mm. We believe the study has extended clinical knowledge on lumbar spine morphometry, essential in the training physicians engaged in transpedicular stabilization.

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord.

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Human spinal ejaculation generator.

A spinal ejaculation generator (SEG) has been identified in the rat with lumbar galaninergic interneurons playing a pivotal role (Science 2002;297:1566-1569). The aim was to evidence a SEG in humans. Spatial distribution of galaninergic neurons was studied in postmortem spinal cord segments of 6 men and compared with that of 6 women for evidencing sexual dimorphism. Based on the identified segmental distribution of galaninergic neurons, the ability for penile vibratory stimulation (PVS) to elicit ejaculation when the concerned spinal segments were injured was studied in 384 patients with clinically complete spinal cord injury (SCI) and consequent anejaculation. Such patients represent a unique model to investigate the role of defined spinal segments in the control of ejaculation. Galaninergic neurons were mostly located between L2 and L5 segments in medial lamina VII, with a maximal density within L4. Three-dimensional 3D reconstruction showed that these neurons were grouped into single columns bilaterally to the central canal. In addition, galaninergic neuron density was found higher in L3 and L4 segments in men as compared to women supporting sexual dimorphism. In the patients' cohort, injury of L3-L5 segments was the sole independent predictor for failure of PVS to induce ejaculation. Although evidence from clinical observations was indirect, there is close correspondence to neuroanatomical data. Organization and sexual dimorphism of human spinal galaninergic neurons were similar to the rat's SEG. Neurohistological data, together with clinical results, corroborate the existence of an SEG in humans in L3-L5 segments. Such a generator could be targeted to treat neurogenic and non-neurogenic ejaculatory disorders. ANN NEUROL 2017;81:35-45.

Stimulation-induced expression of immediate early gene proteins in the dorsal horn is increased in neuropathy

Background and aimsPeripheral neuropathic pain is described as a pain state caused by an injury or dysfunction of the nervous system, and could have clinical manifestations such as hyperalgesia, allodynia and spontaneous pain. The development of neuropathic pain may depend on long-term forms of neuronal plasticity in the spinal cord (SC). Expression of the immediate early gene proteins (IEGPs) Arc, Zif268, and c-Fos are implicated in establishment of long-term potentiation (LTP) induced by conditioning stimulation (CS) of primary afferent fibres. However, the impact of the neuropathic state (Bennett's model) on CS-induced expression of IEGPs has not been studied. The aim of this study was to compare the levels of Arc, c-Fos and Zif268 immunoreactivity prior to and after conditioning stimulation in animals with developed neuropathic pain, with sham operated, non-ligated controls. MethodsTwenty-four animals were divided equally into the neuropathic and non-neuropathic groups. Neuropathic pain was induced in all animals by conducting a loose ligation of the sciatic nerve with Chromic Catgut 4.0 sutures 7 days prior to conditioning stimulation or sham operation. The loose ligation was performed by placing sutures around the sciatic nerve compressing the nerve slightly just enough to reduce but not completely diminish the perineural circulation. A state of neuropathy was confirmed by a significant decrease in mechanical withdrawal threshold measured by von Frey's fibres. Immunohistochemical analysis was performed on transverse sections obtained from the L3–L5 segments of the SC at 2 and 6h post-CS and IEGP positive cells were counted in lamina I and II of the dorsal horn. During statistical analyses, the groups were compared by means of analysis of variance (univariate general linear model). If significant differences were found, each set of animals was compared with the sham group with post hoc Tukey's multiple comparison test. ResultsStrikingly, all IEGPs exhibited a significant increase in immunoreactivity at both time points compared to time-matched, sham operated controls. Maximal IEGP expression was found 2h after CS in neuropathic rats, and there was a smaller but still significant increase 6h after CS. The unstimulated side of the dorsal horn in stimulated animals did not show any significant change of the number of IEGP positive cells and was approximately at the same level as sham operated animals. The number of IEGP positive cells in sham operated controls (non-neuropathic and non-stimulated animals) showed same immunoreactivity in 2 and 6h post sham operation. Conclusions and implicationsThe neurophysiological process of neuropathic pain development is complex and needs to be studied further in order to clarify its nature and components. This present study is meant to reveal a step towards further understanding the role of Arc, c-Fos and Zif268 in neuropathic pain. Moreover, this study might contribute to the knowledge base for further research on better therapeutic possibilities for neuropathic pain.

The expression of calcitonin gene-related peptide on the neurons associated Zusanli (ST 36) in rats.

To investigate the biochemical characteristic of the neurons associated Zusanli (ST 36) in the rat by using Alexa Fluor 594 conjugated cholera toxin subunit B (AF594-CTB) neural tracing and calcitonin gene-related peptide (CGRP) fluorescent immunohistochemical techniques. Four male Sprague Dawley rats were injected with AF594-CTB into the corresponding area of the Zusanli in the human body. After 3 surviving days, the rat's spinal cord and dorsal root ganglia (DRGs) at lumbar segments were dissected following perfusion with 4% paraformaldehyde, cut into sections, and then stained with CGRPfluorescent immunohistochemical method. AF594-CTB labeled sensory neurons were detected in the L3-L6 DRGs with high concentration in L4 DRG, and the labeled motor neurons located in the dorsolateral and intermediate regions of lamina IX from L3-L5 segments with high concentration at L4. Meanwhile, CGRPpositive neural labeling distributed symmetrically on both sides of DRGs, anterior and dorsal horns of spinal cord. In the AF594-CTB labeled neurons, 37% sensory neurons and 100% motor neurons expressed CGRPpositive. These findings present the morphological evidence to demonstrate that the sensory and motor neurons associated Zusanli in the rat distributed with segmental and regional patterns, and contained CGRP-expression.

Simultaneous identification of unmyelinated and myelinated primary somatic afferents by co-injection of isolectin B4 and Cholera toxin subunit B into the sciatic nerve of the rat

Several studies have used the transganglionic tracers cholera toxin subunit B (CTb) and either Bandeiraea simplicifolia isolectin B4 (IB4) or wheat-germ agglutinin (WGA) to label myelinated and unmyelinated afferent fibres respectively. In this study, we aim to determine whether co-injection of CTb and either IB4 or WGA into the sciatic nerve of rat will selectively label myelinated and unmyelinated simultaneously. A double immunofluorescence approach was used to detect these tracers in dorsal root ganglia (DRGs) and afferent fibre terminals in the spinal cord. CTb- and IB4-labelled neurons were seen mainly in L4 and L5 DRGs, with CTb labelling detected primarily in large sized neurons and IB4 staining seen mainly in smaller cells. Only a minority of CTb labelled DRG neuron profiles (5.1%) were also labelled with IB4. In the spinal cord, IB4-labelling was largely confined to lamina II of spinal segments L3–L5, whereas CTb-labelled terminals were seen in all laminae but sparse in lamina II. Confocal microscopy showed no evidence for colocalisation of CTb and IB4 labelling in any terminals in laminae I–III. Although the central distribution of CTb labelling in laminae I and II inner-IV had the same rostro-caudal and medio-lateral coverage as IB4 labelling in spinal segments L3–L5, CTb labelling in ventral laminae (of putative proprioceptor afferents) extended between T12 and S1. Similar patterns of central labelling were found when CTb and WGA were injected together. We therefore concluded that this co-injection approach provides a reliable method to identify both myelinated and unmyelinated somatic primary afferents simultaneously.

Nociceptive input from the rat thoracolumbar fascia to lumbar dorsal horn neurones

In anaesthetised rats, systematic electrophysiological recordings from dorsal horn neurones in spinal segments Th13–L5 were made to obtain information about the spinal nociceptive processing from the lumbar thoracolumbar fascia. Six to fourteen percent of the neurones in the spinal segments Th13–L2 had nociceptive input from the thoracolumbar fascia in naïve animals, no neurones responsive to input from the lumbar fascia were found in segments L3–L5. The segmental location of the receptive fields in the fascia was shifted 2–4 segments caudally relative to the spinal segment recorded from. Most neurones were convergent in that they received additional input from other deep somatic tissues in the low back (87%) and from the skin in the abdominal wall or the proximal leg (50%). The proportion of neurones responsive to input from the thoracolumbar fascia rose significantly from 4% to 15% ( P < 0.05) in animals with an experimentally-induced inflammation of a low back muscle (multifidus). Moreover, neurones in spinal segment L3 – that did not receive input from the fascia in normal animals – responded to fascia input in animals with inflamed muscle. The data suggest that the nociceptive input from the thoracolumbar fascia contributes to the pain in low back pain patients.

Collateral Actions of Premotor Interneurons on Ventral Spinocerebellar Tract Neurons in the Cat

Strong evidence that premotor interneurons provide ventral spinocerebellar tract (VSCT) neurons with feedback information on their actions on motoneurons was previously found for Ia inhibitory interneurons and Renshaw cells, while indications for similar actions of other premotor interneurons were weaker and indirect. Therefore the aim of the present study was to reexamine this possibility with respect to interneurons relaying actions of group Ib afferents from tendon organs and group II afferents from muscle spindles. In all, 133 VSCT neurons in the L3-L5 segments (including 41 spinal border neurons) were recorded from intracellularly in deeply anesthetized cats to verify that stimuli applied in motor nuclei evoked monosynaptic inhibitory postsynaptic potentials (IPSPs) attributable to stimulation of axon collaterals of premotor interneurons. IPSPs were found in over two thirds of the investigated neurons. When intraspinal stimuli were preceded by stimuli applied to a muscle nerve at critical intervals, IPSPs evoked from motor nuclei were considerably reduced, indicating a collision of nerve volleys in axons of interneurons activated by group I and group II afferents. In individual VSCT neurons monosynaptic IPSPs were evoked from both biceps-semitendinosus and gastrocnemius-soleus motor nuclei, in parallel with disynaptic IPSPs from group Ib and group II as well as group Ia afferents. These observations indicate that individual VSCT neurons may monitor the degree of inhibition of both flexor and extensor motoneurons by premotor interneurons in inhibitory pathways from group Ib and group II afferents to motoneurons. They may thus be providing the cerebellum with feedback information on actions of these premotor interneurons on motoneurons.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 microg in 50 microL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed's lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Distribution and Localization of 5-HT1AReceptors in the Rat Lumbar Spinal Cord after Transection and Deafferentation

The serotonergic system is highly plastic, capable of adapting to changing afferent information in diverse mammalian systems. We hypothesized that removing supraspinal and/or peripheral input would play an important role in defining the distribution of one of the most prevalent serotonergic receptors, the 5-HT(1A) receptor (R), in the spinal cord. We investigated the distribution of this receptor in response to a complete thoracic (T7-T8) spinal cord transection (eliminating supraspinal input), or to spinal cord isolation (eliminating both supraspinal and peripheral input) in adult rats. Using two antibodies raised against either the second extracellular region (ECL(2)) or the third intracellular region (ICL(3)) of the 5-HT(1A)R, we compared the 5-HT(1A)R levels and distributions in specific laminae of the L3-L5 segments among the control, spinal cord-transected, and spinal cord-isolated groups. Each antibody labeled different populations of 5-HT(1A)R: ECL(2) labeled receptors in the axon hillock, whereas ICL(3) labeled receptors predominantly throughout the soma and proximal dendrites. Spinal cord transection increased the number of ECL(2)-positive cells in the medial region of laminae III-IV and lamina VII, and the mean length of the labeled axon hillocks in lamina IX. The number of ICL(3)-labeled cells was higher in lamina VII and in both the medial and lateral regions of lamina IX in the spinal cord-transected compared to the control group. In contrast, the length and number of ECL(2)-immunolabeled processes and ICL(3)-immunolabeled cells were similar in the spinal cord-isolated and control groups. Combined, these data demonstrate that the upregulation in 5-HT(1A)R that occurs with spinal cord transection alone is dependent on the presence of sensory input.

Different Forms of Glycine- and GABAA-Receptor Mediated Inhibitory Synaptic Transmission in Mouse Superficial and Deep Dorsal Horn Neurons

BackgroundNeurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood.Methods and ResultsHere we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23°C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 ± 3.9 vs. 64.7 ± 5.0 pA; n = 25 each), decay time was slower (8.5 ± 0.8 vs. 5.5 ± 0.3 ms), and frequency was lower (0.15 ± 0.03 vs. 0.72 ± 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 ± 2.4, n = 14 vs. 25. ± 2.0 pA, n = 18) and frequencies (0.21 ± 0.08 vs. 0.18 ± 0.04 Hz) in both regions; however, decay times were slower (23.0 ± 3.2 vs. 18.9 ± 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 ± 1.6 pS, n = 11 vs. 55.7 ± 1.8, n = 8) and GABAARs (22.7 ± 1.7 pS, n = 10 vs. 22.4 ± 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 μM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons.ConclusionTogether these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.

Recording Temperature Affects the Excitability of Mouse Superficial Dorsal Horn Neurons, In Vitro

Superficial dorsal horn (SDH) neurons in laminae I-II of the spinal cord play an important role in processing noxious stimuli. These neurons represent a heterogeneous population and are divided into various categories according to their action potential (AP) discharge during depolarizing current injection. We recently developed an in vivo mouse preparation to examine functional aspects of nociceptive processing and AP discharge in SDH neurons and to extend investigation of pain mechanisms to the genetic level of analysis. Not surprisingly, some in vivo data obtained at body temperature (37 degrees C) differed from those generated at room temperature (22 degrees C) in spinal cord slices. In the current study we examine how temperature influences SDH neuron properties by making recordings at 22 and 32 degrees C in transverse spinal cord slices prepared from L3-L5 segments of adult mice (C57Bl/6). Patch-clamp recordings (KCH(3)SO(4) internal) were made from visualized SDH neurons. At elevated temperature all SDH neurons had reduced input resistance and smaller, briefer APs. Resting membrane potential and AP afterhyperpolarization amplitude were temperature sensitive only in subsets of the SDH population. Notably, elevated temperature increased the prevalence of neurons that did not discharge APs during current injection. These reluctant firing neurons expressed a rapid A-type potassium current, which is enhanced at higher temperatures and thus restrains AP discharge. When compared with previously published whole cell recordings obtained in vivo (37 degrees C) our results suggest that, on balance, in vitro data collected at elevated temperature more closely resemble data collected under in vivo conditions.

Lateral spinal nucleus of the rat: NADPH-diaphorase activity and Fos expression after noxious peripheral stimulation

Results of electrophysiological studies suggest a significant role of the lateral spinal nucleus (LSpN) in the transmission of nociceptive signals. In our study, the presence of Fos immunoreactivity and NADPH-diaphorase positivity was observed in the rat LSpN following noxious peripheral subcutaneous stimulation. Formalin-induced unilateral hindpaw stimulation in the rat caused bilateral NADPH-d reactivity and ipsilateral Fos expression in this nucleus. In the LSpN of the L3–L5 segments of stimulated rats, on average, 4.1 ± 1.2 NADPH-d-positive, NADPH-d(+), 5.1+1.8 Fos-immunoreactive, Fos(+), and 3.0 ± 1.1 double-labeled neurons per 25-µm-thick section were found unilaterally. A close anatomical relationship between NADPH-d(+) processes and Fos(+) cell nuclei in the LSpN was also observed following noxious peripheral stimulation. These neuroanatomical findings support the hypothesis that the LSpN is involved in pain processing and suggest an important role of nitric oxide-mediated signal transduction in this nucleus.

Altered potassium channel function in the superficial dorsal horn of the spastic mouse

The spastic mouse has a naturally occurring glycine receptor (GlyR) mutation that disrupts synaptic input in both motor and sensory pathways. Here we use the spastic mouse to examine how this altered inhibitory drive affects neuronal intrinsic membrane properties and signal processing in the superficial dorsal horn (SDH), where GlyRs contribute to pain processing mechanisms. We first used in vitro patch clamp recording in spinal cord slices (L3-L5 segments) to examine intrinsic membrane properties of SDH neurones in spastic and age-matched wildtype controls ( approximately P23). Apart from a modest reduction ( approximately 3 mV) in resting membrane potential (RMP), neurones in spastic mice have membrane and action potential (AP) properties identical to wildtype controls. There was, however, a substantial reorganization of AP discharge properties in neurones from spastic mice, with a significant increase (14%) in the proportion of delayed firing neurones. This was accompanied by a change in the voltage sensitivity of rapid A-currents, a possible mechanism for increased delayed firing. To assess the functional consequences of these changes, we made in vivo patch-clamp recordings from SDH neurones in urethane anaesthetized (2.2 g kg(-1), i.p.) spastic and wildtype mice ( approximately P37), and examined responses to innocuous and noxious mechanical stimulation of the hindpaw. Overall, responses recorded in wildtype and spastic mice were similar; however, in spastic mice a small population of spontaneously active neurones ( approximately 10%) exhibited elevated spontaneous discharge frequency and post-pinch discharge rates. Together, these results are consistent with the altered intrinsic membrane properties of SDH neurones observed in vitro having functional consequences for pain processing mechanisms in the spastic mouse in vivo. We propose that alterations in potassium channel function in the spastic mouse compensate, in part, for reduced glycinergic inhibition and thus maintain normal signal processing in the SDH.