Three 4-monosubstituted butyrolactones from a regulatory gene mutant of Streptomyces rochei 7434AN4.

Extensive metabolite analysis of Streptomyces rochei 7434AN4 was performed to discover uncharacterized secondary metabolites. A mutant strain of S. rochei, in which two regulatory genes srrC (a tetR-type repressor) and srrY (SARP-type activator) were inactivated, accumulated three 4-monosubstituted γ-butyrolactones YT02-A, YT02-B, and KH01-A, which were not detected in the parent strain. Their structures were identified as 4,10-dihydroxy-10-methyldodecan-4-olide, 4,10-dihydroxy-10-methylundecan-4-olide, and 4-hydroxy-11-oxo-10-methyldodecan-4-olide. A structural comparison indicated that the three butanolides and the signaling molecules, termed S. rochei butenolides (SRBs), could share common C12 or C13 fatty acids for their biosynthesis intermediates, however, these three butanolides did not induce antibiotic production even at 50 µM concentration (1000-folds of the minimum antibiotic-inducing concentration of SRBs) in S. rochei.

Cleavage of α-1,4-glycosidic linkages by the glycosylphosphatidylinositol-anchored α-amylase AgtA decreases the molecular weight of cell wall α-1,3-glucan in Aspergillus oryzae.

Aspergillus fungi contain α-1,3-glucan with a low proportion of α-1,4-glucan as a major cell wall polysaccharide. Glycosylphosphatidylinositol (GPI)-anchored α-amylases are conserved in Aspergillus fungi. The GPI-anchored α-amylase AmyD in Aspergillus nidulans has been reported to directly suppress the biosynthesis of cell wall α-1,3-glucan but not to degrade it in vivo. However, the detailed mechanism of cell wall α-1,3-glucan biosynthesis regulation by AmyD remains unclear. Here we focused on AoAgtA, which is encoded by the Aspergillus oryzae agtA gene, an ortholog of the A. nidulans amyD gene. Similar to findings in A. nidulans, agtA overexpression in A. oryzae grown in submerged culture decreased the amount of cell wall α-1,3-glucan and led to the formation of smaller hyphal pellets in comparison with the wild-type strain. We analyzed the enzymatic properties of recombinant (r)AoAgtA produced in Pichia pastoris and found that it degraded soluble starch, but not linear bacterial α-1,3-glucan. Furthermore, rAoAgtA cleaved 3-α-maltotetraosylglucose with a structure similar to the predicted boundary structure between the α-1,3-glucan main chain and a short spacer composed of α-1,4-linked glucose residues in cell wall α-1,3-glucan. Interestingly, rAoAgtA randomly cleaved only the α-1,4-glycosidic bonds of 3-α-maltotetraosylglucose, indicating that AoAgtA may cleave the spacer in cell wall α-1,3-glucan. Consistent with this hypothesis, heterologous overexpression of agtA in A. nidulans decreased the molecular weight of cell wall α-1,3-glucan. These in vitro and in vivo properties of AoAgtA suggest that GPI-anchored α-amylases can degrade the spacer α-1,4-glycosidic linkages in cell wall α-1,3-glucan before its insolubilization, and this spacer cleavage decreases the molecular weight of cell wall α-1,3-glucan in vivo.

SrrB, a Pseudo-Receptor Protein, Acts as a Negative Regulator for Lankacidin and Lankamycin Production in Streptomyces rochei.

Streptomyces rochei 7434AN4, a producer of lankacidin (LC) and lankamycin (LM), carries many regulatory genes including a biosynthesis gene for signaling molecules SRBs (srrX), an SRB receptor gene (srrA), and a SARP (Streptomyces antibiotic regulatory protein) family activator gene (srrY). Our previous study revealed that the main regulatory cascade goes from srrX through srrA to srrY, leading to LC production, whereas srrY further regulates a second SARP gene srrZ to synthesize LM. In this study we extensively investigated the function of srrB, a pseudo-receptor gene, by analyzing antibiotic production and transcription. Metabolite analysis showed that the srrB mutation increased both LC and LM production over four-folds. Transcription, gel shift, and DNase I footprinting experiments revealed that srrB and srrY are expressed under the SRB/SrrA regulatory system, and at the later stage, SrrB represses srrY expression by binding to the promoter region of srrY. These findings confirmed that SrrB acts as a negative regulator of the activator gene srrY to control LC and LM production at the later stage of fermentation in S. rochei.

Spin textures and Larmor precession of holes modulated by the spin-orbit coupling confined in the two-dimensional Si x Ge1-x mixed-alloy quantum well system.

We theoretically study the spin textures of holes confined in the two-dimensional (2D) [Formula: see text] quantum well (QW) system. We particularly focus on the spin-orbit interaction (SOI) caused by the bulk-inversion-asymmetry (BIA) and explore the effective magnetic field (EMF) generated by the combination of the SOI couplings. For the study of the semiconductor mixed-alloy (MA) system, we propose the extended [Formula: see text] perturbation approach including possible perturbation terms crossing with the SOI couplings up to the second order ones. We then study the distribution and orientation of the EMF and investigate how the EMF changes the spin textures of the heavy-mass holes (HHs), light-mass holes (LHs) and separated holes (SHs), respectively. Finally, we study the Larmor spin precession of these holes having the characteristic spin textures driven by the SOI couplings.

Recovery from acute pediatric complex regional pain syndrome type I after ankle sprain by early pharmacological and physical therapies in primary care: a case report.

BACKGROUND: Complex regional pain syndrome type I (CRPS I) in children is a serious condition disrupting the family and school life of patients with the condition after it fully develops. It has been emphasized that early diagnosis is closely associated with earlier reduction of pain leading to preferable outcomes. OBJECTIVES: To report a case of acute CRPS I in a boy who was found to develop this condition by a routine visual analog scale (VAS) pain monitoring and who recovered from CRPS I at an early phase by prompt pharmacological, physical, and educational therapies. STUDY DESIGN: Case report. CASE REPORT: A 12-year-old boy sprained his left ankle while playing soccer and was referred to our clinic 4 days after the injury. At the first visit, he could walk, reporting motion pain with a VAS scale of 80 mm. On day 5, pain intensity increased to 100 mm, and a diagnosis of acute CRPS I was made. On day 7, he could not move the injured ankle; therefore celecoxib and pregabalin were administered, and physical and educational therapies started. On day 35, pain intensity was 0 mm and he could walk and run normally. CONCLUSION: Routine monitoring of VAS for every patient in pain is useful to discover an abnormal transition of VAS, enabling the early diagnosis of CRPS I. Inflammation and peripheral or central sensitization are postulated for early development of CRPS I. The present case suggested a combination of physical therapy and pharmacological intervention with celecoxib and pregabalin reduced peripheral and central sensitization.

Initial performance evaluation of iterative model reconstruction in abdominal computed tomography.

OBJECTIVE: The aim of this study was to present initial evaluation of the performance of the iterative model reconstruction algorithm (IMR) in abdominal computed tomography (CT). METHODS: Computed tomographic examinations were performed for clinical study of 36 patients and for phantom study. We reconstructed the raw data with 1.0- and 5.0-mm slice thicknesses using filtered back projection (FBP), iDose4, and IMR and evaluated image quality objectively and subjectively. RESULTS: For almost all subjective characteristics, the image quality was better using IMR than iDose4. Objective image noise was significantly less using IMR than iDose4 (P < 0.0001). The contrast-noise ratio of both slice thicknesses increased in order from FBP to iDose4 to IMR. The spatial resolution of reconstructed images was almost identical using IMR, FBP, and iDose4. CONCLUSIONS: The IMR can significantly improve image noise and low-contrast resolution and maintain edge sharpness in abdominal CT images compared with iDose4 or FBP.

Effectiveness of L2 spinal nerve infiltration for selective discogenic low back pain patients.

BACKGROUND: It has been reported that rat L5/6 lumbar discs are innervated mainly by L2 dorsal root ganglion neurons. We previously reported that L2 spinal nerve infiltration was effective for discogenic low back pain (DLBP) patients, although the diagnosis was based only on the results of physical examination, plain films, and magnetic resonance imaging (MRI). The purpose of the current study was to evaluate L2 spinal nerve block for DLBP patients retrospectively based on MRI findings and surgical results. METHODS: A total of 62 patients with only LBP and no accompanying radicular pain were investigated. Patients had only one level of disc degeneration on MRI. When pain was provoked during discography, we performed surgery at the next stage (40 patients). In all, 22 patients were excluded owing to negative discography results. Of the 40 patients, we evaluated 25 strictly selected patients suffering from DLBP. DLBP was diagnosed when the patient experienced pain relief at least 2 years after anterior lumbar interbody fusion. Fifteen patients who did not show pain relief after surgery were used for the non-DLBP group. L2 spinal nerve infiltration using 1.5 ml of lidocaine was performed in all 40 patients before surgery. The visual analogue scale (VAS) score after L2 spinal nerve infiltration was recorded, and an association of L2 spinal nerve infiltration and DLBP was explored. RESULTS: Low back pain scores assessed using the VAS score, the Japanese Orthopedic Association score, and the Oswestry Disability Index score in the two groups were not significantly different. L 2 spinal nerve infiltration was effective for 27 patients but not effective for 13 patients; the VAS score after 15 min and 2 h improved in the DLBP group compared with that of the non-DLBP group (P < 0. 05). L2 spinal nerve infiltration was more effective in DLBP patients (21 patients, 84%) than in the non-DLBP group (6 patients, 40%) (P < 0.05). CONCLUSIONS: In the current study, L2 spinal nerve infiltration was effective in 84% of selected DLBP patients and is thought to be a useful tool for diagnosing DLBP. However, we should take into consideration that the L2 spinal nerve infiltration was effective in 40% of non-DLBP patients as well.

Dorsoventral organization of sensory nerves in the lumbar spine as indicated by double labeling of dorsal root ganglion neurons.

BACKGROUND: Referred pain due to lumbar disc disorders can be analyzed using the stereoscopic structure of the peripheral sensory nervous system. The rostrocaudal structure has been clarified. The dorsoventral structure of the lumbar spine would be useful for mapping areas of pain perception in spinal disorders. METHODS: The neurotracer 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (DiI) was applied to the lateral portion of the L5/6 intervertebral disc in rats to examine the dorsoventral organization of the sensory nervous system in the lumbar spine and related tissues. Fluorogold (FG) was applied to reference sites located at the spinous process of the L5 vertebra, the L5/6 facet joint, the psoas muscle at the L5 level, or the rectus abdominis muscle at the pubic symphysis. FG was also applied to the lateral portion of the disc (DiI application site) at L5 or at the L5 level as controls for the double labeling. Labeled neurons were counted in dorsal root ganglia (DRGs) from L1 through L4. RESULTS: The proportion of neurons double-labeled with DiI and FG in the total number of DiI-labeled and FG-labeled neurons was 32.9% in the control group; 0% in the spinous process, 0.6% in the facet joint, 2.3% in the psoas muscle, and 0.1% in the rectus abdominis muscle. DRG neurons with dichotomizing afferent fibers were most prevalent (2.3%) between the lateral disc and the psoas muscle at the groin; they were rare or absent between the disc and other reference sites. CONCLUSIONS: Dorsoventral organization of the primary sensory system in the lumbar body trunk was suggested from the proportion of DRG neurons with dichotomizing afferent fibers innervating the lumbar disc and other tissues. The present findings provide a pathomechanism of groin referred pain in lumbar disc disorders.

Sclerotomes in the thoracic and lumbar spine, pelvis, and hindlimb bones of rats.

UNLABELLED: Pain in lumbar radiculopathy shows a segmental distribution in muscles and bones, requiring knowledge of myotomes and sclerotomes for diagnosis of the involved nerve roots. The rostrocaudal coordinate in sensory space was examined for 49 reference sites placed on the periostium in the spine and hindlimb bones of rats to clarify the sclerotomes. Neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to reference sites. DiI-labeled neurons were counted from the T9 through S3 dorsal root ganglia. The coordinate was calculated for each reference site as the location of the "median" neuron of all DiI-labeled neurons. The coordinate was between T13 and L3 for the lumbar spine, L2 and S1 in the coxal bone, L3 and L4 in the hip joint, femur, knee joint, tibia, and first digit, and L5 in the fibula and fifth digit. The routes of sclerotome boundary lines (SBLs) were determined based on the coordinates of the reference sites. SBLs obliquely demarcated the lumbar spine. SBLs were aligned parallel rostrodorsally to caudoventrally in the coxal bone, with medially-oriented convergence. The SBL between L3 and L4, which corresponded to the level of the furcal nerve, passed from the femur, tibia, and toward the first digit in the hindlimb bones. PERSPECTIVE: The present study is the first report of the detailed sclerotome chart of rats. The sclerotome chart is not only useful for basic research of lumbar radiculopathy using rats, but would also facilitate an understanding of the spatial distribution of pain in patients with lumbar radiculopathy.

Somatotopic organization of lumbar muscle-innervating neurons in the ventral horn of the rat spinal cord.

The ventral horn of the rat spinal cord was investigated with respect to the somatotopic organization of the motor neurons that innervate the lumbar muscles. Neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was applied to specific sites in lumbar muscles. Spinal cord segments at L1 through L4 levels were cut into 40-mum serial transverse sections. Labeled neurons were located in the ventromedial nucleus (VM) and lateromedial nucleus (LM) nuclei of Rexed's lamina IX. Motor neurons innervating the m. interspinales lumborum and m. multifidus were without exception present in the VM, whereas all motor neurons innervating the m. rectus abdominis were present in the LM. Forty percent of motor neurons innervating the m. quadratus lumborum were present in the VM and the other 60% were in the LM. Although most of the motor neurons innervating the m. psoas major were present in the LM, a few labeled neurons existed in the VM. These results suggest that the border zone demarcating the areas of innervation of the dorsal and ventral rami of spinal nerves crosses the m. quadratus lumborum.

Peripheral nerve pathways of afferent fibers innervating the lumbar spine in rats.

UNLABELLED: We investigated the pathways of afferent fibers innervating the lumbar spine. The neurotracer DiI was applied to reference sites at the L5 level in rats. One of 4 surgeries was performed before DiI application: (1) transaction of the dorsal ramus of the L2 spinal nerve, (2) transaction of the ventral ramus of the L2 spinal nerve, (3) transaction of the psoas major muscle at L3-L4, or (4) removal of the paravertebral sympathetic trunks from L3-L5. The number of DiI-labeled neurons in the dorsal root ganglia after surgery was compared with neuron numbers in surgery-naïve rats. The number of DiI-labeled neurons decreased drastically with transection of the L2 ventral ramus or psoas major muscle for the ventral and lateral portions of the disc and vertebral body and after transection of the L2 dorsal ramus for the facet joint and spinous process. Removal of the sympathetic trunks did not reduce the number of DiI-labeled neurons significantly in the extra-spinal canal sites. In contrast, significant reductions occurred after the removal of the paravertebral sympathetic trunks in the intra-spinal canal sites. Extra-spinal canal sites received afferent fibers primarily through somatic routes, but intra-spinal canal sites received afferent fibers via the sympathetic trunks. PERSPECTIVE: Extra-spinal canal sites of the lumbar spine received afferent fibers from muscles originating in the site. Intra-spinal canal sites received a considerable number of afferent fibers via the paravertebral sympathetic trunks. These results may provide new insights for nerve block treatment of low back pain.

Existence of nerve growth factor receptors, tyrosine kinase a and p75 neurotrophin receptors in intervertebral discs and on dorsal root ganglion neurons innervating intervertebral discs in rats.

STUDY DESIGN: We evaluated 2 types of nerve growth factor (NGF) receptors on dorsal root ganglion (DRG) cells and nerve fibers innervating rat lumbar intervertebral discs. OBJECTIVE: To examine the NGF receptors, tyrosine kinase A (TrkA) and p75 neurotrophin receptor (p75NTR) on DRG cells and nerve fibers innervating rat lumbar intervertebral discs using immunohistochemistry and a retrograde neurotracing method. SUMMARY OF BACKGROUND DATA: Nerve innervation of intervertebral discs is thought to be a pathology of discogenic low back pain. NGF is also important for mediating inflammatory pain from intervertebral discs via the high affinity receptor, TrkA. Recent research has also revealed that the low affinity NGF receptor, p75NTR plays an important role in inflammatory pain. However, the presence of TrkA and p75NTR-immunoreactive (NTR-IR) DRG neurons innervating the rat L5/6 intervertebral disc, and p75NTR-IR nerve fibers in rat intervertebral discs, has not been explored. METHODS: The Fluoro-gold neurotracer was applied to rat L5/6 intervertebral discs to determine the DRG neurons innervating the discs (n = 20). Fourteen days after surgery, bilateral DRG from the L1-L6 levels were harvested, sectioned, and immunostained for TrkA and p75NTR. The percentages of TrkA and p75NTR-IR DRG neurons were counted, and p75NTR-IR nerve fibers in L5/6 discs evaluated. RESULTS: p75NTR-IR nerve fibers were found in superficial layers in the annulus fibrosus in L5/6 intervertebral discs. Fluoro-gold-labeled neurons innervating the L5/6 discs were distributed throughout DRG from the L1-L6 levels. The percentage of TrkA-immunoreactive (TrkA-IR) neurons was 75.1% +/- 3.9% (mean +/- SE) and that of p75NTR-IR neurons was 75.8% +/- 5.1%. These percentages were similar for each level. CONCLUSION: Rat L5/6 intervertebral discs were innervated by multisegmental levels of DRG. Most DRG neurons innervating the discs were positive for 2 types of NGF receptors.

Sensory innervation of lumbar vertebral bodies in rats.

STUDY DESIGN: Using a retrograde neurotracing method with Fluoro-Gold (FG), the level at which dorsal root ganglions (DRGs) innervate the L2 and L5 vertebral bodies and the innervation pathways were investigated in rats. OBJECTIVE: To clarify the levels at which DRGs innervate the lumbar vertebral bodies and to determine the pathways from the L2 and L5 vertebral bodies to DRGs. SUMMARY OF BACKGROUND DATA: Elderly patients with osteoporosis sometimes experience lumbar vertebral fracture and may also feel diffuse nonlocalized pain in the back, lateral portion of the trunk, and area surrounding the iliac crest. However, the pattern of sensory innervation of vertebral bodies remains unclear. METHODS: Forty female Sprague-Dawley rats were used. FG crystals were applied to the L2 (L2 vertebra group) or L5 (L5 vertebra group) vertebral bodies via an anterior approach, and numbers of labeled neurons in DRGs from T10 to L6 were counted. To determine sensory pathways, bilateral sympathectomy was performed. RESULTS: In nonsympathectomy animals, FG-labeled neurons were present in DRGs from T11 through L3 in the L2 vertebra group and from T13 through L6 in the L5 vertebra group. The number of labeled neurons following sympathectomy was not significantly different in L1, L2, and L3 DRGs in the L2 vertebra group or in L3, L4, L5, and L6 DRGs in the L5 vertebra group from those in nonsympathectomy animals. In contrast, fewer labeled DRG neurons were present in sympathectomy animals at T11, T12, and T13 in the L2 vertebra group, and at T13, L1, and L2 in the L5 vertebra group than in nonsympathectomy animals (P < 0.01). CONCLUSION: Sensory nerve fibers in the L2 and L5 vertebral bodies are derived from the T11-L3 and T13-L6 DRGs, respectively. Some sensory nerves from the L2 and L5 vertebral bodies enter the paravertebral sympathetic trunks and reach the DRGs at multisegmental levels. The present findings regarding multisegmental innervation to vertebral bodies may explain the diffuse pain that originates within osteoporotic vertebral fractures in elderly patients.

Segmental somatotopic organization of cutaneous afferent fibers in the lumbar spinal cord dorsal horn in rats.

In the present study, we investigated the central representation of segmental cutaneous afferent fiber projection fields in the horizontal plane of the spinal cord dorsal horn in adult rats. The neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) was applied to spinal nerves T12-S2 and cutaneous ventrodorsal axial lines T13-S1. The Dil fluorescent zones in transverse sections of the dorsal horn were observed microscopically. Mediolateral locations of Dil fluorescent zones were measured, followed by reorganization on the horizontal plane through lamina I-I111. Rostral and caudal boundary lines of the central projection fields of spinal nerves T12-S2 formed 'waves' in the horizontal plane of the dorsal horn, pitching rostrocaudally about one spinal cord segment. The rostral and caudal apexes of the waves could be linked with those of adjacent segments, suggesting that the wave pattern is continuous rostrocaudally in the dorsal horn. The waves were markedly transformed in the central projection fields of the hindlimb and genital regions, in the L5 and L6 spinal cord segments.

Characteristics of sensory dorsal root ganglia neurons innervating the lumbar vertebral body in rats.

UNLABELLED: Characteristics of sensory dorsal root ganglia (DRG) neurons innervating the L5 vertebral body were investigated in rats by using a retrograde neurotransport method, lectin affinity- and immuno-histochemistry to further elucidate the causes of diffuse pain suffered by some elderly patients in their back, lateral trunk, and iliac crest, after lumbar osteoporotic vertebral fracture. We used calcitonin gene-related peptide (CGRP) as a marker of small peptide-containing neurons and the glycoprotein binding the isolectin from Griffonia simplicifolia (IB4) as a marker of small non-peptide-containing neurons. Neurons innervating the L5 vertebral bodies, retrogradely labeled with fluoro-gold (FG), were distributed throughout DRGs from T13 to L6. The proportion of CGRP-immunoreactive (IR) FG-labeled neurons was 32%. The proportion of IB4-binding FG-labeled neurons was significantly smaller, at 4%. Other neurons that were non-CGRP-IR and non-IB4-binding were mostly large neurons, and they may transmit proprioception from vertebral bodies. Most neurons transmitting pain are CGRP-IR peptide-containing neurons. They may have a more significant role in pain sensation in the vertebral bodies as peptidergic DRG neurons. PERSPECTIVE: This article shows that vertebral bodies are innervated by CGRP-IR neurons. CGRP-IR neurons may play a role in pain sensation through peptidergic DRG neurons. These findings contribute to an understanding of pain associated with the vertebral body such as tumor, infection, or osteoporotic fracture.

Projection field of primary afferent fibers innervating the ventral portion of the lumbar intervertebral disc in the spinal cord dorsal horn.

In the present study, we investigated the central projection of afferent fibers innervating the lumbar intervertebral disc using the fluorescent neurotracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil). The tracer Dil was applied to the ventrolateral portion of the L5-L6 intervertebral disc in 11 adult rats. Fluorescent sites were observed microscopically on spinal cord transverse sections. Fluorescent spots in laminae I-III were plotted on the central projection map of cutaneous afferents. In six of 11 rats, Dil was restricted to the application site. Of these six rats, three showed no evident fluorescent sites. In the remaining three rats, small fluorescent spots were scattered in the dorsal horn. Fluorescent spots in dorsal horn lamina I were located in the central projection fields of the low back and groin skin. Fluorescent spots were observed, also sporadically, in Clarke's column in T12-L1 segments. The central projection of afferent fibers innervating the rat lumbar intervertebral disc was indistinct with Dil labeling. We presumed this was due to the scarcity of central terminal arbors of disc afferent fibers. Spotty projections in laminae I-IllIIere present near the central projection fields of the loin and groin, indicating that pain would be perceived in the groin.

Extracellular signal-regulated kinase mitogen-activated protein kinase activation in the dorsal root ganglion (DRG) and spinal cord after DRG injury in rats.

STUDY DESIGN: We investigated the extracellular signal-regulated kinase (ERK) activation by immunohistochemically detecting phosphorylated ERK (pERK) in the dorsal root ganglion (DRG) and spinal cord. OBJECTIVE: To clarify the ERK activation in the rat nervous system following DRG injury. SUMMARY OF BACKGROUND DATA: Radicular pain is known to be associated with DRG injury caused by intervertebral disc herniation. ERK is activated by phosphorylation in the DRG and spinal cord by noxious stimuli, which are related to pain hypersensitivity. METHODS: From 2 minutes to 24 hours after the left L4 DRG crush injury, L4 DRGs and spinal cords were resected to prepare serial sections, which were investigated immunohistochemically. RESULTS: In the DRG, ERK activation was detected in neurons and satellite cells at 2 minutes; the former was maintained at increased levels for 20 minutes, and the latter for 4 hours. At 30 minutes, pERK immunoreactivity was observed in Schwann cells, which continued for up to 24 hours. In the spinal cord, pERK-positive neurons were detected at 2 minutes, and the pERK levels were maintained at increased levels for 20 minutes. CONCLUSIONS: Profiles of pERK induction in neurons after DRG injury were similar between the DRG and spinal cord, whereas pERK induction in the satellite cells was more long lasting. The pERK induction in Schwann cells in the DRG was late onset and the most long lasting.

Innervation of the lumbar intervertebral disc by nerve growth factor-dependent neurons related to inflammatory pain.

STUDY DESIGN: We used anatomic tracers and immunoreactivity in rats to define dorsal root ganglion neuron populations innervating the lumbar discs in physiologic and inflammatory states. OBJECTIVES: To investigate the percentages of calcitonin gene-related peptide-immunoreactive (CGRP-ir) and isolectin B4 (IB4)-binding neurons innervating lumbar discs. SUMMARY OF BACKGROUND DATA: Small neurons are classified into two types. One contains CGRP and expresses the nerve growth factor receptor. The other binds IB4 and expresses the glial cell line-derived neurotrophic factor receptor. METHODS: A neurotracer, Fluoro-Gold, was applied to the L5-L6 disc in rats. Five days later, 50-microL saline (control group: n = 8) or Complete Freund's adjuvant (inflammatory group: n = 8) was applied to the disc. Seven days after the second operation, T13-L5 dorsal root ganglions were processed for double staining of CGRP and IB4. RESULTS: Of the Fluoro-Gold-labeled neurons, 50.1 +/- 4.6% (mean +/- SEM) were positive for CGRP and 0.7 +/- 0.6% positive for IB4 in the control group, while 65.6 +/- 4.7% were positive for CGRP and 1.0 +/- 1.0% positive for IB4 in the inflammatory group. The percentage of CGRP-ir neurons was significantly higher than that of IB4-binding neurons in both groups (P < 0.001, each). The percentage of CGRP-ir neurons in the inflammatory group was significantly higher than in the control group (P < 0.05). CONCLUSIONS: We found that most small neurons innervating the disc were CGRP-ir. Furthermore, disc inflammation caused an increase in CGRP-ir neurons but not IB4-binding neurons, suggesting that CGRP-ir, nerve growth factor-dependent neurons are more responsible for discogenic pain.

Sensory innervation of the lateral portion of the lumbar intervertebral disc in rats.

BACKGROUND CONTEXT: An annular tear extending to the outer one-third of the annulus is thought to be one of the causes of low back pain. However, some patients have bilateral low back symptoms, even if the annular tear is localized in the lateral disc. Because nociceptive information from the lateral disc is transmitted by the dorsal root ganglion (DRG) neurons innervating the lateral disc, we investigated the distribution of the DRG neurons innervating the lateral portion of the disc. PURPOSE: To clarify the distribution and pathway of the DRG neurons innervating the lateral portion of the L5-L6 disc in rats. STUDY DESIGN/SETTING: Using the retrograde tracing method, we studied the innervation pattern of the lateral portion of the L5-L6 intervertebral disc in rats. METHODS: The retrograde transport of Fluoro-Gold (F-G; Fluorochrome, Denver, CO) was used in 22 rats. Subjects included a nontreated group (n=16) and a sympathectomized group (n=6). Seven days after the application of F-G crystals to the left lateral portion of the L5-L6 disc, bilateral T12-L6 DRGs were observed by fluorescent microscopy. RESULTS: In the nontreated group, of all the F-G-labeled neurons, 93.1% were present in the left DRGs and 6.9% were in the right DRGs. The number of labeled neurons was largest in the left L2 DRGs. In the sympathectomized group, the numbers of labeled neurons in the T13, L1 and L2 DRGs were significantly lower than the numbers in the nontreated group. CONCLUSION: Results of this study indicate that DRG neurons innervating the lateral portion of the disc are distributed mainly in the ipsilateral side but also in the contralateral side. The DRG neurons in T13, L1 and L2 innervate the lateral portion of the L5-L6 disc through the paravertebral sympathetic trunks.

Distribution and immunocytochemical characterization of dorsal root ganglion neurons innervating the lumbar intervertebral disc in rats: a review.

Previously, it was believed that the lumbar intervertebral disc was innervated segmentally by dorsal root ganglion (DRG) neurons via the sinuvertebral nerves. Recently, it was demonstrated using retrograde tracing methods that the lower disc (L5-L6) is innervated predominantly by upper (L1 and L2) DRG neurons via the sympathetic trunks. Furthermore, we investigated the expression of various pain-related molecules such as calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), P2X(3) receptor and vanniloid receptor 1 (VR1) in DRG neurons innervating the disc using a combination of immunostaining with the retrograde tracing method. This review outlines the distribution and immunocytochemical characterization of DRG neurons innervating the disc. Small nociceptive DRG neurons are classified into nerve growth factor (NGF)-dependent neurons and glial cell line-derived neurotrophic factor (GDNF)-dependent neurons and they can be distinguished by their reactivity for CGRP and IB4, respectively. We found that about half of the neurons innervating the disc were CGRP-immunoreactive (-ir), whilst, only 0.6% of the DRG neurons were IB4-positive, thereby indicating that NGF-dependent neurons are the main subpopulation which transmits and modulates nociceptive information from the disc. In addition, we also demonstrated P2X(3)- and VR1-immunoreactivity in DRG neurons innervating the disc and noted that they were mainly localized in NGF-dependent neurons. It is well known that NGF has sensitizing effects on DRG neurons, with a recent study demonstratng the presence of NGF in the painful intervertebral disc. Therefore, it is suggested that NGF is involved in the generation of discogenic low back pain.