Mechanical thresholds for initiation and persistence of pain following nerve root injury: mechanical and chemical contributions at injury

There is much evidence supporting the hypothesis that magnitude of nerve root mechanical injury affects the nature of the physiological responses which can contribute to pain in lumbar radiculopathy. Specifically, injury magnitude has been shown to modulate behavioral hypersensitivity responses in animal models of radiculopathy. However, no study has determined the mechanical deformation thresholds for initiation and maintenance of the behavioral sensitivity in these models. Therefore, it was the purpose of this study to quantify the effects of mechanical and chemical contributions at injury on behavioral outcomes and to determine mechanical thresholds for pain onset and persistence. Male Holtzman rats received either a silk or chromic gut ligation of the L5 nerve roots, a sham exposure of the nerve roots, or a chromic exposure in which no mechanical deformation was applied but chromic gut material was placed on the roots. Using image analysis, nerve root radial strains were estimated at the time of injury. Behavioral hypersensitivity was assessed by measuring mechanical allodynia continuously throughout the study. Chromic gut ligations produced allodynia responses for nerve root strains at two-thirds of the magnitudes of those strains which produced the corresponding behaviors for silk ligation. Thresholds for nerve root compression producing the onset (8.4%) and persistence of pain (17.4%-22.2%) were determined for silk ligation in this lumbar radiculopathy model. Such mechanical thresholds for behavioral sensitivity in a painful radiculopathy model begin to provide biomechanical data which may have utility in broader experimental and computational models for relating injury biomechanics and physiologic responses of pain.     

Chronic Compression of the Dorsal Root Ganglion Enhances Mechanically Evoked Pain Behavior and the Activity of Cutaneous Nociceptors in Mice

Radicular pain in humans is usually caused by intraforaminal stenosis and other diseases affecting the spinal nerve, root, or dorsal root ganglion (DRG). Previous studies discovered that a chronic compression of the DRG (CCD) induced mechanical allodynia in rats and mice, with enhanced excitability of DRG neurons. We investigated whether CCD altered the pain-like behavior and also the responses of cutaneous nociceptors with unmyelinated axons (C-fibers) to a normally aversive punctate mechanical stimulus delivered to the hairy skin of the hind limb of the mouse. The incidence of a foot shaking evoked by indentation of the dorsum of foot with an aversive von Frey filament (tip diameter 200 μm, bending force 20 mN) was significantly higher in the foot ipsilateral to the CCD surgery as compared to the contralateral side on post-operative days 2 to 8. Mechanically-evoked action potentials were electrophysiologically recorded from the L3 DRG, in vivo, from cell bodies visually identified as expressing a transgenically labeled fluorescent marker (neurons expressing either the receptor MrgprA3 or MrgprD). After CCD, 26.7% of MrgprA3⁺ and 32.1% MrgprD⁺ neurons exhibited spontaneous activity (SA), while none of the unoperated control neurons had SA. MrgprA3⁺ and MrgprD⁺ neurons in the compressed DRG exhibited, in comparison with neurons from unoperated control mice, an increased response to the punctate mechanical stimuli for each force applied (6, 20, 40, and 80 mN). We conclude that CCD produced both a behavioral hyperalgesia and an enhanced response of cutaneous C-nociceptors to aversive punctate mechanical stimuli.     

Regional Distribution of Calcitonin Gene-Related Peptide-, Substance P-, Cholecystokinin-, Met5-Enkephalin-, and Dynorphin A (1–8)-Like Materials in the Spinal Cord and Dorsal Root Ganglia of Adult Rats: Effects of Dorsal Rhizotomy and Neonatal Capsaicin

Biochemical mapping of five different peptide-like materials--calcitonin gene-related peptide (CGRP), substance P (SP), Met5-enkephalin (ME), cholecystokinin (CCK), and dynorphin A (1-8) (DYN)--was conducted in the dorsal and ventral zones of the spinal cord at the cervical, thoracic, and lumbar levels in 3-month-old rats 10 days after unilateral dorsal rhizotomy at the cervical level (C4-T2) or after neonatal administration of capsaicin (50 mg/kg s.c.). In control rats, all peptide-like materials were more abundant in the dorsal than in the ventral zone all along the spinal cord. However, in both zones, absolute concentrations of CGRP, SP, ME, and CCK were significantly higher at the lumbar than at the cervical level. Rhizotomy-induced CGRP depletion (-85%) within the ipsilateral dorsal zone of the cervical cord was more pronounced than that due to neonatal capsaicin (-60%), a finding suggesting that this peptide is contained in both capsaicin-sensitive (mostly unmyelinated) and -insensitive (myelinated) primary afferent fibers. In contrast, similar depletions of SP (-50%) were observed after dorsal rhizotomy and neonatal capsaicin treatment, as expected from the presence of SP only in the capsaicin-sensitive small-diameter primary afferent fibers. Although the other three peptides remained unaffected all along the cord by either intervention, evidence for the existence of capsaicin-insensitive CCKergic primary afferent fibers could be inferred from the increased accumulation of CCK (together with SP and CGRP) in dorsal root ganglia ipsilateral to dorsal root sections.     

Sensory perturbations using suture and sutureless repair of transected median nerve in rats

The effects of changes to cold, mechanical, and heat thresholds following median nerve transection with repair by sutures (Su) or Rose Bengal adhesion (RA) were compared to sham-operated animals. Both nerve-injured groups showed a transient, ipsilateral hyposensitivity to mechanical and heat stimuli followed by a robust and long-lasting hypersensitivity (6–7 weeks) with gradual recovery towards pre-injury levels by 90 days post-repair. Both tactile and thermal hypersensitivity were seen in the contralateral limb that was similar in onset but differed in magnitude and resolved more rapidly compared to the injured limb. Prior to injury, no animals showed any signs of aversion to cold plate temperatures of 4–16?°C. After injury, animals showed cold allodynia, lasting for 7 weeks in RA-repaired rats before recovering towards pre-injury levels, but were still present at 12 weeks in Su-repaired rats. Additionally, sensory recovery in the RA group was faster compared to the Su group in all behavioural tests. Surprisingly, sham-operated rats showed similar bilateral behavioural changes to all sensory stimuli that were comparable in onset and magnitude to the nerve-injured groups but resolved more quickly compared to nerve-injured rats. These results suggest that nerve repair using a sutureless approach produces an accelerated recovery with reduced sensorimotor disturbances compared to direct suturing. They also describe, for the first time, that unilateral forelimb nerve injury produces mirror-image-like sensory perturbations in the contralateral limb, suggesting that the contralateral side is not a true control for sensory testing. The potential mechanisms involved in this altered behaviour are discussed.     

Anti-allodynic effects of intrathecally and intracerebroventricularly administered 26RFa, an intrinsic agonist for GRP103, in the rat partial sciatic nerve ligation model

26RFa and QRFP are endogenous ligands of GPR103. 26RFa binding sites are widely distributed in the brain and the spinal cord where they are involved in processing pain. In the present study, the effects of intrathecal and intracerebroventricular applications of 26RFa on the level of mechanical allodynia induced by partial sciatic nerve ligation were examined in rats. The level of mechanical allodynia was measured using von Frey filaments. Intrathecal and intracerebroventricular injection of 26RFa attenuated the level of mechanical allodynia. 26RFa has been reported to activate not only GPR103 but also neuropeptide FF2 receptor and the effect of intrathecally and intracerebroventricularly administered 26RFa was not antagonized by BIBP3226, an antagonist of neuropeptide FF receptor. Immunohistochemical examination revealed that QRFP-like immunoreactivity (QRFP-LI) was expressed mainly in the small to medium sized neurons in the L5 dorsal root ganglion (DRG) and that partial sciatic nerve injury increased the percentage of QRFP-LI positive neurons. 7 days after the nerve injury, QRFP-LI positive neurons in the L5 DRG ipsilateral to the partial sciatic nerve injury were larger than those in the L5 DRG ipsilateral to the sham operation. These data suggest that (1) exogenously applied 26RFa modulates nociceptive transmission at the spinal and the supraspinal brain in the neuropathic pain model, (2) the mechanism 26RFa uses to produce an anti-allodynic effect may be mediated by the activation of GPR103, and (3) partial sciatic nerve ligation affects the expression of QRFP-LI in the dorsal root ganglion.     

Spinal CCL2 pronociceptive action is no longer effective in CCR2 receptor antagonist-treated rats

A better understanding of the mechanisms linked to chemokine pronociceptive effects is essential for the development of new strategies to better prevent and treat chronic pain. Among chemokines, MCP-1/CCL2 involvement in neuropathic pain processing is now established. However, the mechanisms by which MCP-1/CCL2 exerts its pronociceptive effects are still poorly understood. In the present study, we demonstrate that MCP-1/CCL2 can alter pain neurotransmission in healthy rats. Using immunohistochemical studies, we first show that CCL2 is constitutively expressed by primary afferent neurons and their processes in the dorsal horn of the spinal cord. We also observe that CCL2 is co-localized with pain-related peptides (SP and CGRP) and capsaicin receptor (VR1). Accordingly, using in vitro superfusion system of lumbar dorsal root ganglion and spinal cord explants of healthy rats, we show that potassium or capsaicin evoke calcium-dependent release of CCL2. In vivo, we demonstrate that intrathecal administration of CCL2 to healthy rats produces both thermal hyperalgesia and sustained mechanical allodynia (up to four consecutive days). These pronociceptive effects of CCL2 are completely prevented by the selective CCR2 antagonist (INCB3344), indicating that CCL2-induced pain facilitation is elicited via direct spinal activation of CCR2 receptor. Therefore, preventing the activation of CCR2 might provide a fruitful strategy for treating pain.     

Distribution,immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.     

Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles.

We hypothesized that a 10-s rest interval (at zero load) inserted between each load cycle would increase the osteogenic effects of mechanical loading near previously identified thresholds for strain magnitude and cycle numbers. We tested our hypothesis by subjecting the right tibiae of female C57BL/6J mice (16 wk, n = 70) to exogenous mechanical loading within a peri-threshold physiological range of strain magnitudes and load cycle numbers using a noninvasive murine tibia loading device. Bone responses to mechanical loading were determined via dynamic histomorphometry. More specifically, we contrasted bone formation induced by cyclic vs. rest-inserted loading (10-s rest at zero load inserted between each load cycle) by first varying peak strains (1,000, 1,250, or 1,600 micro epsilon) at fixed cycle numbers (50 cycles/day, 3 days/wk for 3 wk) and then varying cycle numbers (10, 50, or 250 cycles/day) at a fixed strain magnitude (1,250 micro epsilon). Within the range of strain magnitudes tested, the slope of periosteal bone formation rate (p.BFR/BS) with increasing strain magnitudes was significantly increased by rest-inserted compared with cyclical loading. Within the range of load cycles tested, the slope of p.BFR/BS with increasing load cycles of rest-inserted loading was also significantly increased by rest-inserted compared with cyclical loading. In sum, the data of this study indicate that inserting a 10-s rest interval between each load cycle amplifies bone's response to mechanical loading, even within a peri-threshold range of strain magnitudes and cycle numbers.     

The peptide content of colonic afferents decreases following colonic inflammation.

Peripheral injury produces long term changes in peptide content in dorsal root ganglion (DRG) cells that contribute to the inflammatory process in the periphery and neuronal plasticity in the spinal cord. We report here the proportion of colonic afferents labeled for calcitonin gene-related peptide (CGRP), substance P (SP) or somatostatin (Som) in the T13-L2 and L6-S2 DRG and changes in the percentage of SP or CGRP labeled afferents 6, 24, and 72 h following induction of experimental colitis. Following injection of fluorogold (FG) into the descending colon, significantly more FG labeled DRG cells were observed in the T13-L2 than L6-S2 DRG. In noninflamed rats, in both spinal regions, 60-70% of the colonic afferents that were labeled with FG were double labeled for SP. Similar results were obtained when double labeling for CGRP. Only 20-30% of the FG labeled afferents were double labeled for Som. Following experimental colitis induced by intracolonic zymosan, there was a significant decrease in the percentage of cells double labeled for SP in the T13-L2 and L6-S2 DRG at 6, 24, and 72 h. The percentage of CGRP double labeled cells was decreased in the T13-L2 DRG at all time points, but only at 24 h in the L6-S2 DRG. The cell bodies of CGRP labeled colonic afferents were significantly larger than SP or Som in control rats. Inflammation did not affect the mean size of the double labeled cells. These results suggest that colonic inflammation increases SP and CGRP release in the spinal cord and the colon that is manifest as a decrease in peptide content in the cell bodies of the colonic afferents during the first 72 h following injury.     

Changes of the neuropeptides content and gene expression in spinal cord and dorsal root ganglion after noxious colorectal distension

Visceral pain/hypersensitivity is a cardinal symptom of functional gastrointestinal disorders. With their peripheral and central (spinal) projections, sensory neurons in the dorsal root ganglia (DRG) are the "gateway" for painful signals emanating from both somatic and visceral structures. In contrast to somatic pain, the neurochemical pathways involved in visceral pain/hypersensitivity have not been well studied. We hypothesized the neuropeptide changes in spinal cord and DRG during visceral pain would mirror similar changes in somatic nociception. Noxious (painful) colorectal distension (CRD) was done by distending a rectal balloon up to 60 mm Hg phasically for 1 h in Sprague-Dawley rats. The spinal content of calcitonin gene-related peptide (CGRP), substance P (SP), galanin and vasoactive intestinal peptide (VIP) as well as their mRNAs in DRG were measured at 0, 4 and 24 h after the CRD. Visceromotor reflex (VMR) was measured by recording the electromyogram at the abdominal muscle in response to CRD. Distal colorectum was removed for evaluating the presence of inflammation. No significant evidence of histological inflammation was seen in the colonic mucosa/submucosa after repeated CRD, which is confirmed by myeloperoxidase assay. The spinal content of CGRP and SP decreased significantly 4 h after CRD, while galanin and VIP levels increased gradually and reached highest level at 24 h (p<0.05). The mRNAs in DRG of the neuropeptides were significantly upregulated after CRD (p<0.05). VMR recording showed the rat's colon became hypersensitive 4 h after CRD, a sequence parallel to the spinal changes of CGRP and SP in timeframe. Noxious mechanical distension of the colorectum causes an acute change in the spinal levels of excitatory neurotransmitters (CGRP and SP), probably reflecting central release of these peptides from sensory neurons and contributing to the hypersensitivity following the noxious CRD. This is followed by a slower change in the levels of the inhibitory neurotransmitter galanin and VIP. Such stimulation results in significant alternation of the gene expression in DRG, reflecting the plasticity of the neuronal response. In the absence of visceral inflammation, the aforementioned neuropeptides are important mediators in the processing of visceral pain/hypersensitivity.     

诱发性耳声发射的掩蔽

３４例听觉正常受试者（共４８耳）进行疏波短声诱发性耳声发射（ＥＯＡＥ）掩蔽实验,项目包括同侧同时掩蔽、同侧后掩蔽和对倒后掩蔽。同时掩蔽的掩蔽声是稳态白噪声,后掩蔽的掩蔽声是宽带噪声。同侧同时掩蔽强度达３０ｄＢＳＬ时,未观察到对ＥＯＡＥ的掩蔽效应,但对主观听觉感受有掩蔽作用,表明ＥＯＡＥ的客观属性反映听觉行为有其局限性、同侧及对侧后掩蔽出现掩蔽效应时的掩蔽强度分别为３０和５０ｄＢＳＬ,掩蔽阈约分别为５９和６８ｄＢＳＬ。耳蜗的机械特性－非线性或耳蜗内存在的某种功能性的反馈调节系统可能是同侧后掩蔽的作用机理。下行的对侧橄榄耳蜗内侧束对外毛细胞主动收缩的抑制性作用,可有效解释对倒后掩蔽的ＥＯＡＥ变化。     

Nuclear Localization of SP, CGRP, and NK 1 R in a Subpopulation of Dorsal Root Ganglia Subpopulation Cells in Rats

Signals generated by renal pelvic afferent nerves in response to stimulation are transmitted from peripheral processes of dorsal root ganglia neurons to their central terminals in the dorsal horn of the spinal cord to cause the release of neuropeptides, including SP and CGRP. All of the cellular activities of SP are considered to be mediated through interaction with NK₁R located on the cell surface. We have investigated the colocalization and subcellular distribution of NK₁R, SP, and CGRP in different subpopulations of neurons that innervate renal tissue. Our findings therefore provide the first evidence for the presence of NK₁R, SP, and CGRP in the nuclei of DGR neural cells. The physiological significance of this localization remains unknown. One possibility is that pelvic sensory neurons may regulate their responses to different stimuli by modulating the ratio of CGRP and SP release and/or nuclear NK₁R expression.     

Can periods of static loading be used to enhance the resistance of the spine to cumulative compression?

Results of in vitro studies conducted on isolated bone specimens have indicated a higher tolerance to static load than exists when exposed to cyclic loading, when controlled for creep rate. If this difference in load tolerance exists, it may be exploited to extend the life of vertebral bone exposed to repetitive compression, and potentially alter the development of spinal injury. However, little work has been conducted on functional spinal units to determine if bone displays this characteristic within an intact joint. Additionally, static loading may result in load redistribution within the intervertebral disc forcing more of the compressive load towards the periphery of the endplate away from the nucleus. In order to examine these potential mechanisms, 218 osteoligamentous porcine functional spinal units were assigned to one of 15 loading scenarios. This involved one of three normalized peak load magnitudes (50%, 70% and 90% of estimated compressive tolerance) and one of five normalized static load applications (0%, 50%, 100%, 200% and 1000% of the total dynamic work duration). Load magnitude significantly altered the resistance to cumulative compression with decreased peak magnitudes corresponding to both increased cumulative load tolerance and increased height loss. Static load periods did not alter the resistance of the spinal unit to cumulative compression or impact the number of cycles tolerated to failure. The insertion of static load periods impacted the total survival time to failure, but only for the 1000% static load group, an exposure unlikely to occur for most in vivo exposures. The insertion of static load periods decreased the amount of height loss during testing which may play a protective role by allowing load redistribution within the vertebral bone and intervertebral disc.     

Search for critical loading condition of the spine--a meta analysis of a nonlinear viscoelastic finite element model

The relative vulnerability of spinal motion segments to different loading combinations remains unknown. The meta-analysis described here using the results of a validated L2-L3 nonlinear viscoelastic finite element model was designed to investigate the critical loading and its effect on the internal mechanics of the human lumbar spine. A Box-Behnken experimental design was used to design the magnitude of seven independent variables associated with loads, rotations and velocity of motion. Subsequently, an optimization method was used to find the primary and secondary variables that influence spine mechanical output related to facet forces, disc pressure, ligament forces, annulus matrix compressive/shear stresses and anulus fibers strain. The mechanical responses with respect to the two most-relevant variables were then regressed linearly using the response surface quadratic model. Axial force and sagittal rotation were identified as the most-relevant variables for mechanical responses. The procedure developed can be used to find the critical loading for finite element models with multi input variables. The derived meta-models can be used to predict the risk associated with various loading parameters and in setting safer load limits.     

Direct evidence for the involvement of brain-derived neurotrophic factor in the development of a neuropathic pain-like state in mice

Thermal hyperalgesia and tactile allodynia induced by sciatic nerve ligation were completely suppressed by repeated intrathecal (i.t.) injection of a TrkB/Fc chimera protein, which sequesters endogenous brain-derived neurotrophic factor (BDNF). In addition, BDNF heterozygous (+/-) knockout mice exhibited a significant suppression of nerve ligation-induced thermal hyperalgesia and tactile allodynia compared with wild-type mice. After nerve ligation, BDNF-like immunoreactivity on the superficial laminae of the ipsilateral side of the spinal dorsal horn was clearly increased compared with that of the contralateral side. It should be noted that a single i.t. injection of BDNF produced a long-lasting thermal hyperalgesia and tactile allodynia in normal mice, and these responses were abolished by i.t. pre-treatment with either a Trk-dependent tyrosine kinase inhibitor K-252a or a selective protein kinase C (PKC) inhibitor Ro-32-0432. Supporting these findings, we demonstrated here for the first time that the increase in intracellular Ca2+ concentration by application of BDNF in cultured mouse spinal neurons was abolished by pre-treatment with either K-252a or Ro-32-0432. Taken together, these findings suggest that the binding of spinally released BDNF to TrkB by nerve ligation may activate PKC within the spinal cord, resulting in the development of a neuropathic pain-like state in mice.     

Search for critical loading condition of the spine–A meta analysis of a nonlinear viscoelastic finite element model

The relative vulnerability of spinal motion segments to different loading combinations remains unknown. The meta-analysis described here using the results of a validated L2–L3 nonlinear viscoelastic finite element model was designed to investigate the critical loading and its effect on the internal mechanics of the human lumbar spine. A Box-Behnken experimental design was used to design the magnitude of seven independent variables associated with loads, rotations and velocity of motion. Subsequently, an optimization method was used to find the primary and secondary variables that influence spine mechanical output related to facet forces, disc pressure, ligament forces, annulus matrix compressive/shear stresses and anulus fibers strain. The mechanical responses with respect to the two most-relevant variables were then regressed linearly using the response surface quadratic model. Axial force and sagittal rotation were identified as the most-relevant variables for mechanical responses. The procedure developed can be used to find the critical loading for finite element models with multi input variables. The derived meta-models can be used to predict the risk associated with various loading parameters and in setting safer load limits.     

Vascular changes associated with spinal root avulsion injury

Abstract

This paper has investigated the hypothesis that spinal root avulsion (SRA) injury produces alterations in blood flow that contribute to avulsion injury induced pain-like behaviour in rodents. Photoplethysmography (PPG) is an established way of assessing blood flow in the central nervous system (CNS) and laser Doppler flowmetry (LDF) is the most widely used technique for measuring tissue perfusion. Using an established model of SRA injury that produces mechanical hypersensitivity, the PPG and LDF signals were recorded in animals 2 weeks post-injury and compared to naive recordings. PPG and LDF measurements were assessed on the ipsilateral and contralateral sides of the spinal cord rostral and caudal to the avulsion injury and at the level of the injury. Two weeks after injury, a time when vascular blood vessel endothelial markers are known to be decreased, no significant changes were seen in the spinal cord blood flow (SCBF) above, at, or below the injury site or when comparing the ipsilateral vs. contralateral side. Assessment of oxygenation levels again revealed no significant differences between naive and spinal root injured animals along the rostrocaudal axis (i.e., above, at, and below the site of injury or its equivalent on the contralateral side). From these experiments it is concluded that SRA does not significantly alter blood flow or tissue oxygen levels and so ischemia may play a less prominent role in avulsion injury induced pain.     

Effect of multiple dorsal rhizotomies on calcitonin gene-related peptide-like immunoreactivity in the lumbosacral dorsal spinal cord of the cat: a radioimmunoassay analysis

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) was measured by radioimmunoassay in the cat lumbosacral dorsal spinal cord following unilateral dorsal rhizotomy of 5 consecutive dorsal roots. The dorsal rhizotomies greatly reduced but did not eliminate the CGRP-LI from the ipsilateral rhizotomized segments. The amount of CGRP-LI remaining in the rhizotomized segments was greatest in the most caudal segment (846 +/- 311 pmoles/g tissue) and decreased below 300 pmoles/g tissue in the remaining segments. When these values were compared to the intact contralateral side, the percent CGRP remaining ranged from 65% in the sacral segments to less than 20% in the lumbar segments. Rostral to the rhizotomized segments there was a gradual return of CGRP-LI to control levels within 3 segments. Small diameter primary afferent fibers are the only known source of CGRP within the dorsal spinal cord. These results suggest that the most likely origin of the CGRP that remained in the rhizotomized lumbar segments was the rostrally and caudally projecting branches of ipsilateral primary afferents that entered the spinal cord through intact dorsal roots caudal and rostral to the transected roots. These results support the hypothesis that small diameter primary afferents project several segments in the cat spinal cord.     

Bone and joint neuropathy in rats with type-2 diabetes

We have previously demonstrated that Goto-Kakizaki (GK) rats with spontaneous type-2 diabetes and peripheral neuropathy exhibit regional osteopathic changes. In the present study on 18 GK rats and 21 control Wistar rats, the occurrence of the sensory neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP), and the autonomic neuropeptide Y (NPY) was analysed in bone and joints, dorsal root ganglia and lumbar spinal cord by immunohistochemistry and radioimmunoassay (RIA). Immunohistochemistry disclosed a predominance of immunoreactivities in vessel-related nerve fibers, although some were also seen in free terminals. While SP, CGRP and NPY in periosteum, cortical bone and synovium was confined to neuronal tissue, the bone marrow in addition exhibited an abundance of NPY-positive megakaryocytes. Apart from this cellular source of NPY, the observations suggest that the three neuropeptides analysed in bone and joints are of neuronal origin. Quantification by RIA showed a significant decrease of NPY in cortical bone (-36%), bone marrow (-66%) and ankle (-29%) of GK rats. CGRP was decreased in the spinal cord (-19%) and dorsal root ganglia (-26%) but was unchanged in bone and joints, as with SP. Given the suggested anabolic role of NPY and CGRP on bone, neuropeptidergic deficit in diabetes may prove to be an important factor underlying the development of regional osteopenia.