Expression of calcitonin gene-related peptide in anterior and posterior horns of the spinal cord after brachial plexus injury

This study shows the expression pattern of calcitonin gene-related peptide (CGRP) in the anterior and posterior horns of the spinal cord after brachial plexus injury. The animals were divided into three injury groups: group 1, right C₇ anterior root avulsion; group 2, right C₇ anterior root avulsion and cut right C₅–T₁ posterior roots; and group 3, right C₇ anterior root avulsion plus right hemitransection between the C₅ and C₆ segments of the spinal cord. These animals were killed at 1, 3, 7 and 14 days after injury. In the anterior horn of all three injured groups, the expression of CGRP increased progressively from day 1 to day 7 (p < 0.05), peaked on day 7, and then began to decrease slowly. In the posterior horn of all three injured groups, the expression of CGRP decreased gradually from day 1 to day 14 after the operation and was significantly lower on day 14 compared to day 1. At each time point (days 1, 3, 7 and 14), the expression of CGRP was the highest in group 1 and the lowest in group 2, with significant differences among the three groups. The CGRP in the anterior horn of the spinal cord was derived from the cell bodies of motor neurons and was possibly involved in repair mechanisms and regeneration after nerve injury. However, the CGRP in the posterior horn was mainly derived from the posterior root ganglion and was possibly associated with the conduction of noxious stimulation.     

Changes of chemoarchitectural organization of the rat spinal cord following ventral and dorsal root transection.

Time-related changes in the distribution of chemical messengers in the rat spinal cord following the transection of dorsal and ventral roots were observed by using immunohistochemistry for the following antigens: microtubule-associated protein 2 (MAP2), calcitonin gene-related peptide (CGRP), substance P (SP), galanin (Gal), Met-enkephalin (Enk), neuropeptide Y (NPY), and serotonin (5-HT). To investigate dendrocytoarchitectonic organizational changes, morphometric analyses were performed on both the gray and the white matter of tissue samples stained with MAP2 antiserum. A significant reduction in the area of gray matter on the lesioned side was seen from 1 to 24 weeks postoperation, and progressive changes in the shape of the gray matter were also observed. CGRP-immunoreactive fibers were reduced in number in the posterior horn after root transection, except in the lateral part of lamina I. In contrast, CGRP immunoreactivity in the anterior horn cells of the ipsilateral side was increased early after transection, but later it progressively decreased. Root transection also caused significant reduction in the number of SP-immunoreactive fibers in the posterior horn, but no changes were seen in the anterior horn. Gal immunoreactivity was also affected by root transection, and it changed in a similar way to CGRP immunoreactivity. 5-HT-immunoreactive fibers were increased in the posterior horn after transection, and later decreased. In the anterior horn, there were no changes in the intensity or distribution pattern of 5-HT-immunoreactive nerve fibers following root transection. Enk and NPY immunoreactivity in the anterior and posterior horns was not affected by root transection up to 24 weeks postoperative. These results show that spinal root transection caused significant changes in the chemoarchitectural organization of nerve fibers containing certain types of chemical messengers, such as CGRP, SP, Gal, and 5-HT, in addition to altering dendritic geometry in the spinal cord.     

Neuroprotective effect of estrogen after chronic spinal cord injury in ovariectomized rats

BACKGROUND: At present, there is still lack of effective drugs for chronic spinal cord injury, whereas it is found recently that estrogen has a neuroprotective effect on brain and spinal cord injuries. OBJECTIVE: To observe the effect of estrogen on the apoptosis of nerve cells after gradual chronic spinal cord injury in ovariectomized rats. DESIGN: A randomized controlled animal trial. SETTING: Institute of Orthopaedics, the Second Hospital of Lanzhou University. MATERIALS: Sixty-five female Wistar rats of common degree, weighing 220–250 g, were provided by the experimental animal center of Lanzhou University. The rats were randomly divided into sham-operated group (n =5), estrogen-treated group (n =30) and saline control group (n =30), and the latter two groups were observed at 1, 3, 7, 14, 28 and 60 days respectively, and 5 rats for each time point. METHODS: All the rats were treated with bilateral oophorectomy 2 weeks before the experiment. T10 vertebral lamina was revolved into using plastic screw. The spinal canal impingement was not induced initially. After that, the original incision was opened to expose the screw every 7–10 days. MAIN OUTCOME MEASURES: The apoptosis and Caspase-3 positive cells in the damaged spinal cord were detected using terminal deoxynucleotidal transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and Caspase-3 immunohistochemical staining at 1, 3, 7, 14, 28 and 60 days after chronic spinal cord injury respectively. RESULTS: Totally 65 rats were used, and the deleted ones during the experiment were supplemented by others. Changes of Caspase-3 expression after spinal cord injury: In the sham-operated group, only a small amount of Caspase-3 proteins were observed in the rat spinal cord, mainly located in motor neurons of spinal cord anterior horn. In the estrogen-treated group and saline control group, positive cells expressed occasionally at 1 day postoperatively, began to increase obviously at 7 days after injury, strongly expressed at 14 and 28 days, but decreased at 60 days, mainly located in the neurons of spinal cord gray matter anterior horn, and they expressed fewer in the motor neurons and white matter of ventral horn, and there were obvious differences between the estrogen-treated group and saline control group at 7, 14, 28 and 60 days (P < 0.05). CONCLUSION: Estrogen can reduce the apoptosis of nerve cells and promote the recovery of neurological function following gradual chronic spinal cord injury.     

Effects of continuous peripheral nerve block by tetrodotoxin on growth associated protein-43 expression during neuropathic pain development

BACKGROUND: Peripheral nerve injury may lead to neuropathic pain and cause a markedly increase expression of growth associated protein-43 (GAP-43) in the spinal cord and dorsal root ganglion, local anesthetics blocking electrical impulse propagation of nerve fibers may also affect the expression of GAP-43 in the spinal cord and dorsal root ganglion. OBJECTIVE: To determine the effects of continuous peripheral nerve block by tetrodotoxin before and after nerve injury on GAP-43 expression in the dorsal root ganglion during the development of neuropathic pain. DESIGN: A randomized controlled animal experiment. SETTINGS: Department of Anesthesiology, the Second Hospital of Xiamen City; Department of Anesthesiology, the Second Affiliated Hospital of Shantou University Medical College. MATERIALS: Thirty-five Sprague Dawley (SD) rats, weighing 200–250 g, were randomly divided into four groups: control group (n =5), simple sciatic nerve transection group (n =10), peripheral nerve block before and after sciatic nerve transection groups (n =10). All the sciatic nerve transection groups were divided into two subgroups according to the different postoperative survival periods: 3 and 7 days (n =5) respectively. Mouse anti-GAP-43 monoclonal antibody (Sigma Co., Ltd.), supervision TM anti-mouse reagent (HRP, Changdao antibody diagnosis reagent Co., Ltd., Shanghai), and HMIAS-100 image analysis system (Qianping Image Engineering Company, Tongji Medical University) were employed in this study. METHODS: This experiment was carried out in the Department of Surgery and Pathological Laboratory, the Second Affiliated Hospital of Shantou University Medical College from April 2005 to April 2006. ①The animals were anesthetized and the right sciatic nerve was exposed and transected at 1 cm distal to sciatic notch. ② Tetrodotoxin 10 μg/kg was injected percutaneously between the greater trochanter and the posterior superior iliac spine of right hind limb to block the sciatic nerve proximally at 1 hour before or 4 hours after nerve injury respectively, the injection was repeated in all the rats every 12 hours. ③ At 3 or 7 days after nerve injury, immunohistochemistry and image analysis were used to evaluate the expression of GAP-43 in the dorsal root ganglions of L5 to the transected sciatic nerve, and quantitative analysis was also performed. ④ Statistical analysis was performed using one way analysis of variance followed by t test. MAIN OUTCOME MEASURE: Expression of GAP-43 in the right dorsal root ganglions of L5. RESULTS: All the 35 SD rats were involved in the final analysis of results. In normal rats, there were very low expressions of GAP-43 in the dorsal root ganglions. In simple sciatic nerve transection rats 3 and 7 days after sciatic nerve transection, the average absorbance value of GAP-43 immunopositive neurons were significantly different from that in normal rats (t =8.806, 6.771, P < 0.01). Whereas 3 and 7 days after sciatic nerve transection in rats with peripheral nerve block before and after nerve injury, the average absorbance value of GAP-43 immunopositive neurons were not significantly different from that in normal rats (P > 0.05). CONCLUSION: Local anesthetic continuous peripheral nerve block before or after nerve injury can suppress nerve injury induced high expression of GAP-43 during the development of neuropathic pain.     

Panax notoginseng saponins improve recovery after spinal cord transection by upregulating neurotrophic factors

Saponins extracted from Panax notoginseng are neuroprotective, but the mechanisms underlying this effect remain unclear. In the present study, we established a rat model of thoracic(T10) spinal cord transection, and injected Panax notoginseng saponins(100 mg/kg) or saline 30 minutes after injury. Locomotor functions were assessed using the Basso, Beattie, and Bresnahan(BBB) scale from 1 to 30 days after injury, and immunohistochemistry was carried out in the ventral horn of the spinal cord at 1 and 7 days to determine expression of nerve growth factor(NGF) and brain-derived neurotrophic factor(BDNF). Our results show that at 7–30 days post injury, the BBB score was higher in rats treated with Panax notoginseng saponins than in those that received saline. Furthermore, at 7 days, more NGF- and BDNF-immunoreactive neurons were observed in the ventral horn of the spinal cord of rats that had received Panax notoginseng saponins than in those that received saline. These results indicate that Panax notoginseng saponins caused an upregulation of NGF and BDNF in rats with spinal cord transection, and improved hindlimb motor function.     

Effects of C8 ventral root avulsion or transection on spinal alpha motoneurons in adult rats★○ A qualitative light and electron microscopic study

BACKGROUND： Nerve root avulsion is a frequent finding in patients with brachial plexus injury following road traffic accidents or as a result of severe arm traction during complicated deliveries. This injury constitutes a challenging clinical and surgical problem. The morphological characteristics of motoneurons after nerve root avulsion deserve further analysis.
OBJECTIVE： To study the different morphological changes of α-motoneurons under light and electron microscopy after C8 spinal ventral rootlets avulsion and transection at various stages. DESIGN： Controlled animal study.
SETTING： Department of Anatomy, King Faisal University.
MATERIALS： The experiment was carried out at the Department of Anatomy, College of Medicine, King Faisal University between January 2005 and March 2006. Six adult Sprague Dawley rats weighing 200-350 g, irrespective of gender, were used for this study. The animals were bred at the animal house, College of Medicine, King Faisal University, and fed on rat maintenance diet. Water and standard diet were supplied ad libitum. Animal interventions were carried out according to animal ethical standards.
METHODS： Three animals were randomly chosen for avulsion of the right ventral rootlets of C8 spinal nerves. The other three received transection of the right ventral rootlets of C8 spinal nerves. ①Avulsion experiment： After rats were anesthetized, the right ventral rootlets of C8 spinal nerves were identified. The ventral rootlets were avulsed from the spinal cord by traction with a fine hook （Fine Science Tools Inc., No. 10031-13, Germany）. Traction was exerted in a direction parallel to the course of the spinal root. Under the operating microscope, the C8 segment was exactly located. After checking the successfulness of the surgical procedure, the C8 segment was separated from the spinal cord. The outcome of the avulsion procedure was as follows： two animals had true avulsion, i.e., no remaining stump was attached to the spinal cord surface. One rat had a stump     

Increased calcitonin gene-related peptide (CGRP), substance P,and enkephalin immunoreactivities in dorsal spinal cord and loss of CGRP-immunoreactive motoneurons in arthritic rats depend on intact peripheral nerve supply

The distribution of peptides thought to be involved in pain modulation—substance P, calcitonin gene-related peptide (CGRP), and enkephalin—were studied in the spinal cord and dorsal root ganglia of polyarthritic rats and in rats with one sciatic nerve sectioned prior to induction of arthritis. In arthritic rats there was a bilateral increase of CGRP- and substance P-immunoreactive fibers and appearance of enkephalin-immunoreactive cell bodies in the dorsal horn of the lumbar (L₄) spinal cord when compared to controls. In the corresponding dorsal root ganglia there were significant increases of CGRP- (P<0.02) and substance P- (P<0.001) immunoreactive cell bodies compared to controls. In the ventral horn of the control rats CGRP-immunoreactive motoneurons were abundant but were significantly (P<0.001) reduced in the arthritic spinal cord. Less pronounced changes were seen in the contralateral L₄ spinal cord of arthritic rats with unilateral sciatic nerve section. In the ipsilateral dorsal horn, however, CGRP- and substance P-immunoreactive fibers were markedly depleted, and no enkephalin cell bodies were present. Furthermore, a number of CGRP-immunoreactive motoneurons were observed. In the ipsilateral L₄ ganglia CGRP- (P<0.02) and substance P- (P<0.02) immunoreactive cells were significantly decreased compared to the contralateral side. The data suggest that pain perception is linked to complex interactions between CGRP, substance P, and enkephalin in sensory pathways and an intact peripheral input. The loss of CGRP-immunoreactive motoneurons may reflect muscular dysfunction associated with the arthritic condition.     

Spinal cord compression injury in adult rats initiates changes in dorsal horn remodeling that may correlate with development of neuropathic pain

Spinal cord injury commonly causes chronic, neuropathic pain. The mechanisms are poorly understood but may include structural plasticity within spinal and supraspinal circuits. Our aim was to determine whether structural remodeling within the dorsal horn rostral to an incomplete injury differs from a complete spinal cord transection. Four immunohistochemical populations of primary afferent C‐fibers, and descending catecholamine and serotonergic projections, were examined in segments T9–T12 at 2 and 12 weeks after a T13 clip‐compression injury in adult male rats. Dorsal root ganglia were also examined. Two weeks after injury, fibers immunoreactive for calcitonin gene‐related peptide (CGRP) or GDNF‐family receptors (GFRα1, GFRα2, GFRα3) showed distinct injury responses within the superficial dorsal horn. CGRP fibers decreased, but GFRα1, GFRα2 and GFRα3 fibers did not change. In contrast, all groups were decreased by 12 weeks after injury. Catecholamine fibers showed a decrease at 2 weeks followed by an increase in density at 12 weeks, whereas serotonergic fibers showed a decrease (restricted to deep dorsal horn) at 12 weeks. These results show that the dorsal horn of the spinal cord undergoes substantial structural plasticity rostral to a compression injury, with the most profound effect being a prolonged and possibly permanent loss of primary afferent fibers. This loss was more extensive and more prolonged than the loss that follows spinal cord transection. Our results provide further evidence that anatomical reorganization of sensory and nociceptive dorsal horn circuits rostral to an injury could factor in the development or maintenance of spinal cord injury pain. J. Comp. Neurol. 513:668–684, 2009. © 2009 Wiley‐Liss, Inc.     

Selective plasticity of primary afferent innervation to the dorsal horn and autonomic nuclei following lumbosacral ventral root avulsion and reimplantation in long term studies

Previous studies involving injuries to the nerves of the cauda equina and the conus medullaris have shown that lumbosacral ventral root avulsion in rat models results in denervation and dysfunction of the lower urinary tract, retrograde and progressive cell death of the axotomized motor and parasympathetic neurons, as well as the emergence of neuropathic pain. Root reimplantation has also been shown to ameliorate several of these responses, but experiments thus far have been limited to studying the effects of lesion and reimplantation local to the lumbosacral region. Here, we have expanded the region of investigation after lumbosacral ventral root avulsion and reimplantation to include the thoracolumbar sympathetic region of the spinal cord. Using a retrograde tracer injected into the major pelvic ganglion, we were able to define the levels of the spinal cord that contain sympathetic preganglionic neurons innervating the lower urinary tract. We have conducted studies on the effects of the lumbosacral ventral root avulsion and reimplantation models on the afferent innervation of the dorsal horn and autonomic nuclei at both thoracolumbar and lumbosacral levels through immunohistochemistry for the markers calcitonin gene-related peptide (CGRP) and vesicular glutamate transporter 1 (VGLUT1). Surprisingly, our experiments reveal a selective and significant decrease of CGRP-positive innervation in the dorsal horn at thoracolumbar levels that is partially restored with root reimplantation. However, no similar changes were detected at the lumbosacral levels despite the injury and repair targeting efferent neurons, and being performed at the lumbosacral levels. Despite the changes evident in the thoracolumbar dorsal horn, we find no changes in afferent innervation of the autonomic nuclei at either sympathetic or parasympathetic segmental levels by CGRP or VGLUT1. We conclude that even remote, efferent root injuries and repair procedures can have an effect on remote and non-lesioned sensory systems sharing common peripheral ganglia.     

Glucose utilization and reflex activity of the transected rat spinal cord

Spinal cord glucose utilization (SCGU) of gray and white matter was studied with the quantitative autoradiography [¹⁴C]2-deoxyglucose methodology, below and above a complete low thoracic transection. One day after transection, a generalized decrease in SCGU was observed in gray matter, particularly marked in the dorsal horn of the lumbar cord. A progressive increase in SCGU was observed thereafter. Values reached levels greater than those of non-transected controls by 2 and 4 weeks after the intervention in ventral horn of the lumbar cord, and ventral and dorsal horn of the cervical cord. A similar behavior of SCGU was observed in white matter of transected animals. The development of 17 lumbar reflex modalities was quantified between days 1 and 28 after spinal transection. The delay in emergence of these reflexes was related to their complexity. Correlation of reflex scores with SCGU was significant for all lumbar cord regions but linearity of this relationship was only observed in white matter. These results uncover a close relationship between SCGU and reflex activity of the spinal cord below a complete transection, particularly striking in white matter and suggests a role of the fasciculi proprii of the spinal cord in this phenomenon.     

Ventral root avulsion versus transection at the cervical 7 level of the cat spinal cord

The results of unilateral ventral root avulsions at the cervical 7 (C7) level were compared using light microscopy with unilateral transactions of the ventral root C7 at the surface of the cat spinal cord after survival times ranging from one to 90 days. Sections were stained for Nissl, acetylcholinesterase, neurofilaments, glial fibrillary acidic protein, and B-50/GA P43. A distinct difference in neuronal reaction was found between the effects of avulsion and transection of ventral root C7. After avulsion, changes in the Nissl-and acetylcholinesterase-stained sections and the sections stained with antibodies against neurofilaments remained present up to 90 days, although the number of neurons in the ventral horn on the avulsed side had significantly decreased at 60 and 90 days. This was in contrast to the transection where the changes in the stainings tended to return to the normal situation between 30 and 60 days and showed the normal pattern 90 days after transection. No decrease in the number of neurons was found. Both lesions were situated in the transitional region of the nerve roots. After avulsion axons break in the white matter of the CNS, while the axons were transected in the PNS compartment of the CNS-PNS transitional region. After avulsion, not only were the axons disconnected, as in transection, but the connections of recurrent motor axon collaterals probably were damaged. This may explain the different reaction after avulsion and transection.     

Penile erectile dysfunction after brachial plexus root avulsion injury in rats

Our previous studies have demonstrated that some male patients suffering from brachial plexus injury, particularly brachial plexus root avulsion, show erectile dysfunction to varying degrees. However, the underlying mechanism remains poorly understood. In this study, we evaluated the erectile function after establishing brachial plexus root avulsion models with or without spinal cord injury in rats. After these models were established, we administered apomorphine （via a sub- cutaneous injection in the neck） to observe changes in erectile function. Rats subjected to simple brachial plexus root avulsion or those subjected to brachial plexus root avulsion combined with spinal cord injury had significantly fewer erections than those subjected to the sham operation. Expression of neuronal nitric oxide synthase did not change in brachial plexus root avulsion rats. However, neuronal nitric oxide synthase expression was significantly decreased in brachial plexus root avulsion ＋ spinal cord injury rats. These findings suggest that a decrease in neuronal nitric oxide synthase expression in the penis may play a role in erectile dysfunction caused by the combi- nation of brachial plexus root avulsion and spinal cord injury.     

Pathology of spinal cord lesions caused by ossification of the posterior longitudinal ligament,with special reference to reversibility of the spinal cord lesion

Abstract

This report describes pathological findings of the spinal cord damage, with ossification of the posterior longitudinal ligament (OPLL), with special reference to reversibility of such lesions. Twenty-five autopsy cases associated with OPLL were examined!, and the spinal cord damage was pathologically classified into four categories based on degree of destruction (stage 0-3). In stage 0 and stage 1major pathological changes in the gray matter and the degree of compression on the spinal cord were well correlated to deformity of the anterior horn. In stage 2 and stage 3, neurons were almost completely obliterated and necrosis with cavitation were frequently observed. Destruction of the spinal cord in stage 2 and stage 3 is considered to be irreversible; therefore, surgical treatment is recommended at stage 0 or stage 1. [Neurol Res 1992; 14: 312-314]     

A monoclonal antibody distinguishes anterior horn cells of human embryonic spinal cord during a transient period of development.

Monoclonal antibodies were prepared by using the anterior horn region of human embryonic spinal cord as immunogen. To increase the specificity of the immune response towards the anterior horn cells, mice were first injected with antigens from the posterior horn and then immunosuppressed with cyclophosphamide; subsequently antigens from the anterior horn were injected. One of the monoclonal antibodies recognizes a small population of anterior horn cells of human embryonic spinal cord during a transient period of development (9-10th embryonic week); these cells are probably motoneurons according to their location in the spinal cord, their positive staining for acetylcholinesterase and their large nuclei. The staining pattern has a special axial distribution as it is limited to the cervical and thoracic regions of the spinal cord. The antibody is species-specific and shows a high degree of tissue specificity. Since this antibody distinguishes a small group of anterior horn cells in the spinal cord during a specific developmental stage, it opens stimulating perspectives for further investigation on the nature of the antigen and its putative role during the development of the human embryonic spinal cord.     

Neuroprotection and Axonal Regeneration After Lumbar Ventral Root Avulsion by Re-implantation and Mesenchymal Stem Cells Transplant Combined Therapy

Ventral spinal root avulsion causes complete denervation of muscles in the limb and also progressive death of segmental motoneurons (MN) leading to permanent paralysis. The chances for functional recovery after ventral root avulsion are very poor owing to the loss of avulsed neurons and the long distance that surviving neurons have to re-grow axons from the spinal cord to the corresponding targets. Following unilateral avulsion of L4, L5 and L6 spinal roots in adult rats, we performed an intraspinal transplant of mesenchymal stem cells (MSC) and surgical re-implantation of the avulsed roots. Four weeks after avulsion the survival of MN in the MSC-treated animals was significantly higher than in vehicle-injected rats (45 % vs 28 %). Re-implantation of the avulsed roots in the injured spinal cord allowed the regeneration of motor axons. By combining root re-implantation and MSC transplant the number of surviving MN at 28 days post-injury was higher (60 %) than in re-implantation alone animals (46 %). Electromyographic tests showed evidence of functional re-innervation of anterior tibialis and gastrocnemius muscles by the regenerated motor axons only in rats with the combined treatment. These results indicate that MSC are helpful in enhancing neuronal survival and increased the regenerative growth of injured axons. Surgical re-implantation and MSC grafting combined had a synergic neuroprotective effect on MN and on axonal regeneration and muscle re-innervation after spinal root avulsion.     

Meta analysis of olfactory ensheathing cell transplantation promoting functional recovery of motor nerves in rats with complete spinal cord transection

OBJECTIVE:To evaluate the effects of olfactory ensheathing cell transplantation on functional recovery of rats with complete spinal cord transection.DATA SOURCES: A computer-based online search of Medline(1989–2013),Embase(1989–2013),Cochrane library(1989–2013),Chinese Biomedical Literature Database(1989–2013),China National Knowledge Infrastructure(1989–2013),VIP(1989–2013),Wanfang databases(1989–2013) and Chinese Clinical Trial Register was conducted to collect randomized controlled trial data regarding olfactory ensheathing cell transplantation for the treatment of complete spinal cord transection in rats.SELECTION CRITERIA: Randomized controlled trials investigating olfactory ensheathing cell transplantation and other transplantation methods for promoting neurological functional recovery of rats with complete spinal cord transection were included in the analysis.Meta analysis was conducted using Rev Man 4.2.2 software.MAIN OUTCOME MEASURES: Basso,Beattie and Bresnahan scores of rats with complete spinal cord transection were evaluated in this study.RESULTS: Six randomized controlled trials with high quality methodology were included.Meta analysis showed that Basso,Beattie and Bresnahan scores were significantly higher in the olfactory ensheathing cell transplantation group compared with the control group(WMD = 3.16,95% CI(1.68,4.65); P 0.00001).CONCLUSION: Experimental studies have shown that olfactory ensheathing cell transplantation can promote the functional recovery of motor nerves in rats with complete spinal cord transection.     

Cholecystokinin in Mammalian Primary Sensory Neurons and Spinal Cord: In Situ Hybridization Studies in Rat and Monkey

The peptide cholecystokinin (CCK) has been suggested to be involved in nociception, but its exact localization at the level of the spinal cord and in spinal ganglia has been a controversial issue. Therefore the distribution of messenger RNA (mRNA) for CCK was studied by in situ hybridization using oligonucleotide probes on sections of adult rat lumbar dorsal root ganglia following unilateral section of the sciatic nerve and on sections of untreated monkey trigeminal ganglia, spinal cord and spinal ganglia from all levels. For comparison, calcitonin gene-related peptide (CGRP) mRNA was also studied in the monkey tissue using the same techniques. Peripheral sectioning of the sciatic nerve in the rat resulted in the appearance of detectable CCK mRNA in up to 30% of remaining ipsilateral L4 and L5 dorsal root ganglion neurons 3 weeks after surgery, with a distinct but more limited appearance also in the contralateral ganglia. No cells, or only single cells, could be seen in normal control rat ganglia. In contrast, in the normal monkey, ∼20% of dorsal root ganglion neurons, regardless of spinal level, and 10% of trigeminal ganglia neurons expressed mRNA for CCK. CGRP mRNA was expressed at detectable levels in ∼80% of these monkey dorsal root ganglion neurons. In the monkey spinal cord, CCK mRNA was detected in the dorsal horn and in motoneurons, whereas CGRP mRNA was only seen in motoneurons. The present results suggest that CCK peptides can be involved in sensory processing in the dorsal horn of the spinal cord in normal monkeys and in rats after peripheral nerve injury, adding one more possible excitatory peptide to the group of mediators in the dorsal horn.     

Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species

Calcitonin gene-related peptide (CGRP) immunoreactivity was found throughout the entire spinal cord of man, marmoset, horse, pig, cat, guinea pig, mouse, rat, and frog. CGRP-immunoreactive fibers were most concentrated in the dorsal horn. In the ventral horn of some species large immunoreactive cells, tentatively characterized as motoneurons, were present. Pretreatment of rats with colchicine enhanced staining of these large cells but did not reveal CGRP-immunoreactive cell bodies in the dorsal horn. In the dorsal root ganglia, CGRP immunoreactivity was observed in most of the small and some of the intermediate sized cells. Substance P immunoreactivity, where present, was co-localized with CGRP to a proportion of the small cells. In the cat the ratio of substance P-immunoreactive to CGRP-immunoreactive ganglion cells was 1:2.7 (p less than 0.001). The concentration of CGRP-immunoreactive material in tissue extracts was determined by radioimmunoassay. In the dorsal horn of the rat spinal cord the levels of peptide were found to range from 225.7 +/- 30.0 pmol/gm of wet weight in the cervical region to 340.6 +/- 74.6 pmol/gm in the sacral spinal cord. In the rat ventral spinal cord, levels of 15.7 +/- 2.7 to 35.1 +/- 10.6 pmol/gm were found. The concentration in dorsal root ganglia of the lumbar region was 225.4 +/- 46.9 pmol/gm. Gel permeation chromatography of this extractable CGRP-like immunoreactivity revealed three distinct immunoreactive peaks, one eluting at the position of synthetic CGRP and the others, of smaller size, eluting later. In cats and rats, rhizotomy induced a marked loss of CGRP-immunoreactive fibers from the dorsal horn of the spinal cord. In the cat, unilateral lumbosacral dorsal rhizotomy resulted in a significant (p less than 0.05) reduction of extractable CGRP from the ipsilateral lumbar dorsal horn (5.6 +/- 1.2 pmol/gm of wet weight) compared to the contralateral side (105.0 +/- 36.0 pmol/gm of wet weight). We conclude that the major origin of CGRP in the dorsal spinal cord is extrinsic, from afferent fibers which are probably derived from cells in the dorsal root ganglia. The selective distribution of CGRP throughout sensory, motor, and autonomic areas of the spinal cord suggests many putative roles for this novel peptide.     

Colitis induces calcitonin gene-related peptide expression and Akt activation in rat primary afferent pathways

Previous study has shown that colitis-induced increases in calcitonin gene-related peptide (CGRP) immunoreactivity in bladder afferent neurons result in sensory cross-sensitization. To further determine the effects of colitis on CGRP expression in neurons other than bladder afferents, we examined and compared the levels of CGRP mRNA and immunoreactivity in the lumbosacral dorsal root ganglia (DRG) and spinal cord before and during colitis in rats. We also examined the changes in CGRP immunoreactivity in colonic afferent neurons during colitis. Results showed increases in CGRP mRNA levels in L1 (2.5-fold, p < 0.05) and S1 DRG (1.9–2.4-fold, p < 0.05). However, there were no changes in CGRP mRNA levels in L1 and S1 spinal cord during colitis. CGRP protein was significantly increased in L1 (2.5-fold increase, p < 0.05) but decreased in S1 (50% decrease, p < 0.05) colonic afferent neurons, which may reflect CGRP release from these neurons during colitis. In L1 spinal cord, colitis caused increases in the number of CGRP nerve fibers in the deep lamina region extending to the gray commissure where the number of phospho-Akt neurons was also increased. In S1 spinal cord, colitis caused the increases in the intensity of CGRP fibers in the regions of dorso-lateral tract, and caused the increases in the level of phospho-Akt in the superficial dorsal horn of the spinal cord. In spinal cord slice culture, exogenous CGRP increased the phosphorylation level of Akt but not the phosphorylation level of extracellular-signal regulated kinase ERK1/2 even though our previous studies showed that colitis increased the phosphorylation level of ERK1/2 in L1 and S1 spinal cord. These results suggest that CGRP is synthesized in the DRG and may transport to the spinal cord where it initiates signal transduction during colitis.