Neurochemical characterization of neuronal populations expressing protein kinase C gamma isoform in the spinal cord and gracile nucleus of the rat.

Protein kinase C gamma (PKCgamma) is widely distributed throughout the CNS and is thought to play a role in long term hyper-excitability in nociceptive neurones. Here, we provide the first report of PKCgamma cells in the dorsal column nuclei of the adult rat. Retrograde labeling of PKCgamma cells from the thalamus with choleragenoid revealed that 25% of the PKCgamma positive gracile cells projected to the thalamus. Further, we have characterized the distribution of PKCgamma within gracile nucleus in terms of colocalization with various neurotransmitter receptors or enzymes and calcium binding proteins, and compared this with PKCgamma colocalization in cells of laminae I-III of the spinal cord. We show that approximately 90% of the PKCgamma cells in the gracile nucleus and 60% in the dorsal horn were immuno-positive for the AMPA receptor subunit glutamate 2/3 (GluR2/3). Little coexpression was seen with neurokinin 1 receptor, nitric oxide synthase (NOS) and the AMPA receptor subunit GluR1, markers of distinct neuronal subpopulations. In the spinal cord, a quarter of PKCgamma cells expressed calbindin, but very few cells did so in the gracile nucleus. Electrical stimulation at c-fiber strength of the normal or injured sciatic nerve was used to induce c-fos as a marker of postsynaptic activation in the spinal cord and gracile nucleus. Quantitative analysis of the number of PKCgamma positive gracile cells that expressed also c-fos increased from none to 24% after injury, indicating an alteration in the sensory activation pattern in these neurones after injury. C-fos was not induced in inner lamina II following c-fiber electrical stimulation of the intact or axotomized sciatic nerve, indicating no such plasticity at the spinal cord level. As dorsal column nuclei cells may contribute to allodynia after peripheral nerve injury, pharmacological modulation of PKCgamma activity may therefore be a possible way to ameliorate neuropathic pain after peripheral nerve injury.

The prevalence of neuropathic pain after non-traumatic spinal cord lesion.

STUDY DESIGN: Retrospective register study. OBJECTIVE: To investigate the predictive value of the following parameters for the development of neuropathic pain after non-traumatic spinal cord lesion: that is age at onset of spinal cord disease, gender, completeness of lesion, level of lesion, and aetiology. SETTING: A unit for patients with post-acute traumatic and non-traumatic spinal cord lesions in the greater area of Stockholm, Sweden. METHOD: All patients with non-traumatic spinal cord lesions visiting the unit between 1995 and 2000 were classified according to the following: that is neuropathic pain at or below lesion level according to IASP criteria, age at time of the onset of the spinal cord symptoms, injury level, complete/incomplete injury, and aetiology. Results were analysed with chi(2) - analysis and logistic regression. RESULTS: In total, 38% had neuropathic pain, 15% had pain predominantly at the level of lesion, and 23% predominantly below the level of lesion. Of those with pain, 67% reported that the pain affected daily life. Women reported neuropathic pain below the level of lesion more often (40%) than men (13%). The prevalence was particularly high (64%) for patients with malignant spinal cord diseases. Neither age at onset of the spinal cord symptoms, nor complete/incomplete injury nor injury level had significant influence on the prevalence. CONCLUSION: Neuropathic pain is common among patients with acquired non-traumatic spinal cord lesions regardless of aetiology, often causing severe problems in daily life.

Distribution of antinociceptive adenosine A1 receptors in the spinal cord dorsal horn, and relationship to primary afferents and neuronal subpopulations.

Adenosine can reduce pain and allodynia in animals and man, probably via spinal adenosine A1 receptors. In the present study, we investigate the distribution of the adenosine A1 receptor in the rat spinal cord dorsal horn using immunohistochemistry, in situ hybridization, radioligand binding, and confocal microscopy. In the lumbar cord dorsal horn, dense immunoreactivity was seen in the inner part of lamina II. This was unaltered by dorsal root section or thoracic cord hemisection. Confocal microscopy of the dorsal horn revealed close anatomical relationships but no or only minor overlap between A1 receptors and immunoreactivity for markers associated with primary afferent central endings: calcitonin gene-related peptide, or isolectin B4, or with neuronal subpopulations: mu-opioid receptor, neuronal nitric oxide synthase, met-enkephalin, parvalbumin, or protein kinase Cgamma, or with glial cells: glial fibrillary acidic protein. A few adenosine A1 receptor positive structures were double-labeled with alpha-amino-3-hydroxy-5-methyl-4-isoaxolepropionic acid glutamate receptor subunits 1 and 2/3. The results indicate that most of the adenosine A1 receptors in the dorsal horn are located in inner lamina II postsynaptic neuronal cell bodies and processes whose functional and neurochemical identity is so far unknown. Many adenosine A1 receptor positive structures are in close contact with isolectin B4 positive C-fiber primary afferents and/or postsynaptic structures containing components of importance for the modulation of nociceptive information.

Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor.

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.

Sp1 is a key regulator of the PDGF beta-receptor transcription.

The mouse PDGF beta-receptor promoter is tightly controlled by NF-Y that binds to a CCAAT box located upstream of the initiation site [1, 2]. In this report, we show that Sp1 plays an essential role in the PDGF beta-receptor transcription. Within the upstream GC rich area there are two Sp1 binding sites located in close proximity to the CCAAT box. Deletion of the GC rich region resulted in a 50% decrease of the transcriptional activity of the promoter, and a complete loss of its responsiveness to over-expression of Sp1. There was an additive effect between NF-Y and Sp I in reporter activity when they were co-transfected together with the promoter-reporter construct. Furthermore, transfection of NF-Y failed to enhance transcriptional activity when the Sp1 binding sites were deleted from the promoter, suggesting an important role for Sp1 in this NF-Y controlled transcription. We have recently reported that c-Myc represses PDGF beta-receptor transcription through its interference with the transactivation activity of NF-Y [3]. In the case of p21(wafl/cip1) transcription, c-Myc was shown to repress its transcription by sequestering Sp1 [4]. However, we could not find any effect of Sp1 in the c-Myc-mediated repression on the PFDGF beta-receptor promoter, since the deletion of SpI binding sites could not attenuate the repression by c-Myc on the promoter activity.

TNF-alpha suppresses the PDGF beta-receptor kinase.

PDGF and TNF-alpha are both known to play important roles in inflammation, albeit frequently by opposing actions. Typically, TNF-alpha can attenuate PDGF beta-receptor signaling. Pretreatment of mouse 3T3 L1 fibroblasts with TNF-alpha greatly diminished their proliferative response to PDGF. However, TNF-alpha affected neither the binding of PDGF-BB to cell surface receptors nor the total amount of PDGF beta-receptor in the cells, but decreased the PDGF-induced in vitro kinase activity of the receptor. The phosphatase inhibitor ortho-vanadate did not prevent this effect. Ortho-phosphate labeling of cells prior to TNF-alpha treatment and PDGF-BB stimulation confirmed a decrease of in vivo phosphorylation of the PDGF beta-receptor. Two-dimensional mapping after tryptic cleavage as well as phosphoamino acid analysis demonstrated a general decrease in phosphorylation of all known tyrosine residues in the PDGF beta-receptor. The exact mechanism for this suppression remains to be clarified.

Alterations in the distribution of stimulus-evoked c-fos in the spinal cord after neonatal peripheral nerve injury in the rat.

Neonatal peripheral nerve injury results in a significant rearrangement of the central terminals of surviving axotomized and adjacent intact primary afferents in the dorsal horn of the spinal cord. This study investigates the ability of these afferents to make functional contacts with dorsal horn cells, using c-fos expression as a marker of synaptic activation. Graded electrical stimulation at A- or C-fiber strength of either the neonatally axotomized sciatic nerve or the adjacent uninjured saphenous nerve was performed in adult rats. Stimulation of the contralateral uninjured nerve served as a control. Quantitative examination of the number and distribution of c-fos-labeled cells in the spinal cord laminae was performed. Electrical stimulation of the previously axotomized sciatic nerve at A-fiber intensity resulted in many labeled profiles in laminae I-V of the lumbar spinal cord on the experimental as compared to the contralateral side. Electrical stimulation of uninjured saphenous nerve or saphenous-nerve-innervated skin (using pin electrodes) at A-fiber intensity did not evoke c-fos. Stimulation of the saphenous nerve at C-fiber intensity, however, resulted in a significant increase in the number and distribution of c-fos-labeled profiles in laminae I-V on the experimental side as compared to the contralateral control side. The results show that the distribution of c-fos-expressing cells after neonatal nerve injury is compatible with the previously demonstrated distribution of sprouting of primary afferents belonging to an uninjured nerve adjacent to an injured nerve, and that the surviving axotomized afferents are capable of transmitting signals to postsynaptic cells. These findings indicate that Abeta afferent stimulation of injured but not uninjured afferents elicits c-fos expression in postsynaptic cells. This may reflect an injury-induced maintenance of a normal developmental process whereby Abeta stimulation elicits c-fos in dorsal horn neurons.

The time-course of abeta-evoked c-fos expression in neurons of the dorsal horn and gracile nucleus after peripheral nerve injury.

We have examined the mechanisms underlying Abeta-evoked c-fos expression in the dorsal horn and gracile nucleus following either sciatic nerve section or crush injury. The results indicate that in the spinal cord Abeta-evoked c-fos does not depend on primary afferent sprouting but is associated with the disconnection from the peripheral target since its expression in the dorsal horn reverts to normal after crush injury when regeneration occurs but persists after cut and ligation where regeneration is prevented. In contrast, however, Abeta-evoked c-fos expression in the gracile nucleus may be under some other control since its expression appears independent of peripheral nerve regeneration.

Somatotopic redistribution of c-fos expressing neurons in the superficial dorsal horn after peripheral nerve injury.

The functional somatotopic reorganization of the lumbar spinal cord dorsal horn after nerve injury was studied in the rat by mapping the stimulus-evoked distribution of neurons expressing proto-oncogene c-fos. In three different nerve injury paradigms, the saphenous nerve was electrically stimulated at C-fibre strength at survival times ranging from 40 h to more than six months: 1) Saphenous nerve stimulation from three weeks onwards after ipsilateral sciatic nerve transection resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous territory in laminae I-II, and an expansion of the saphenous territory into the denervated sciatic territory until 14 weeks postinjury. 2) Saphenous nerve stimulation from five days onwards after ipsilateral sciatic nerve section combined with saphenous nerve crush resulted in an increase in the number of Fos-immunoreactive neurons within the dorsal horn saphenous nerve territory, and an expansion of the saphenous nerve territory into the denervated sciatic nerve territory. 3) Stimulation of the crushed nerve (without previous adjacent nerve section) at five days, but not at eight months resulted in a temporary increase in the number of Fos-immunoreactive neurons within the territory of the injured nerve, and no change in area at either survival time. The results indicate that nerve injury results in an increased capacity of afferents in an adjacent uninjured, or regenerating nerve, to excite neurons both in its own and in the territory of the permanently injured nerve in the dorsal horn. The onset and duration of the increased postsynaptic excitability and expansion depends on the types of nerve injuries involved. These findings indicate the complexity of the central changes that follows in nerve injuries that contain a mixture of uninjured, regenerating and permanently destroyed afferents.

Distribution of c-fos expressing dorsal horn neurons after electrical stimulation of low threshold sensory fibers in the chronically injured sciatic nerve.

The distribution of proto-oncogene c-Fos protein-immunoreactive cells in the spinal cord dorsal horn was studied after electrical stimulation at A alpha/A beta-fiber intensity of normal and previously injured sciatic nerves in urethane anesthetized rats. No or only occasional Fos protein-like immunoreactive cells were seen after stimulation of the normal uninjured nerve or after nerve transection without stimulation. Electrical nerve stimulation at 3, 12, and 21 days after sciatic nerve transection resulted in substantial increases in the numbers of Fos protein-like immunoreactive cell nuclei in each of Rexed's laminae I-V. Combined demonstration of Fos protein-like immunoreactivity and of glial fibrillary acidic protein-like immunoreactivity (astroglia) or OX-42 immunoreactivity (microglia), indicated that the observed Fos protein-like response was confined to neurons and not to astroglia or microglia. Combined demonstration in the spinal cord of Fos protein-like immunoreactive neurons and neurons labeled retrogradely with Fluoro-Gold from the gracile nucleus showed that some of the Fos protein-like immunoreactive neurons in Rexed's laminae III and IV contributed to the postsynaptic dorsal column pathway. The results indicate that stimulation at A alpha/A beta-fiber intensity of a previously injured nerve gives rise to an abnormally increased activation pattern of postsynaptic neurons in the dorsal horn, some of which contribute to the postsynaptic dorsal column pathway.

Colocalization of Fos- and glucocorticoid receptor-immunoreactivities is present only in a very restricted population of dorsal horn neurons of the rat spinal cord after nociceptive stimulation.

Immunocytochemical techniques were used to study the colocalization of proto-oncogene c-fos protein (Fos)-like immunoreactivity and of glucocorticoid receptor immunoreactivity in the spinal cord dorsal horn of adult male rats after nociceptive stimulation. About 5-10% of the stimulus evoked Fos-immunoreactive profiles colocalized glucocorticoid receptor immunoreactivity. The results suggest the possibility of a corticosteroid action in a small neuronal population of the dorsal horn implicated in processing nociceptive inputs.

c-fos expression in gracilothalamic tract neurons after electrical stimulation of the injured sciatic nerve in the adult rat.

The number of c-fos protein-like immunoreactive (Fos-LI) cells in the gracile nucleus was determined after electrical stimulation at A alpha/A beta-fiber strength of the normal and of the previously injured sciatic nerve in adult rats. No Fos-LI cells were seen after electrical stimulation of the noninjured sciatic nerve, or after sciatic nerve injury without electrical stimulation. However, stimulation 21 days after sciatic nerve transection resulted in numerous Fos-LI cells in the ipsilateral gracile nucleus. Combined Fos immunocytochemistry and retrograde labeling from the thalamus showed that the majority (76%; range = 70-80%) of the cells in the gracile nucleus that expressed Fos-LI after nerve injury projected to the thalamus. The results indicate that morphological, biochemical, and physiological alterations in primary sensory central endings and second-order neurons, which have earlier been demonstrated in the dorsal column nuclei after peripheral nerve injury, are accompanied by changes in the c-fos gene activation pattern after stimulation of the injured sciatic nerve. A substantial number of the c-fos-expressing neurons project to the thalamus.

Changing pattern of c-FOS expression in spinal cord neurons after electrical stimulation of the chronically injured sciatic nerve in the rat.

Immunocytochemical technique was used to study the distribution of c-FOS protein immunoreactive cells in the spinal cord and gracile nuclei 2 h after electrical stimulation of the sciatic nerve in ketamine/xylazine/acepromazine-anesthetized adult rats. Quantitative examination of the c-fos-labeled cells in the spinal cord laminae was made in unoperated and sham operated controls, after sciatic nerve transection without electrical stimulation, and after electrical stimulation at C-fiber or A alpha/beta-fiber intensity, both in normal animals and at various survival times after chronic sciatic nerve injury (transection and ligation) or crush. Unoperated animals showed very few c-fos-labeled cells, and sham operated controls showed labeled cells located mainly outside the sciatic nerve projection territory. A small increase in number of c-fos protein positive cells was seen after sciatic nerve transection without electrical stimulation. Stimulation of the normal sciatic nerve at C-fiber intensity resulted in c-fos protein-positive cells within the sciatic projection territory in the ipsilateral dorsal horn. Labeled cells were seen in all spinal cord laminae except lamina IX, with the vast majority in lamina I and outer lamina II. No labeled cells were seen in the gracile nucleus. Stimulation at A alpha/beta fiber intensity resulted in no or only a very small number of c-fos-positive neurons. Electrical stimulation of the injured sciatic nerve at C-fiber intensity, using the uninjured contralateral side as control, resulted in significant decreases in c-fos-immunoreactive cells in lamina I plus the outer portion of lamina II at 12 and 39 days survival after injury. A non-significant decrease was seen in these laminae also after 21 days. Significant increases were seen in laminae III and IV at 21 days. Decreases in laminae V, VI and more ventral laminae were significant at 21 and 39 days after injury. At longer survival times, the difference between the normal and injured side seen weeks after injury tended to disappear. Stimulation at A alpha/beta fiber intensity 21 days after injury resulted in increases in the numbers of labeled cells in ipsilateral laminae II, III and IV and in the gracile nucleus. Sciatic nerve stimulation after crush injury resulted in more variable side differences, with tendencies for the same alterations as those noted after chronic transection-ligation.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in microglial cell numbers in the spinal cord dorsal horn following brachial plexus transection in the adult rat.

The effect of peripheral nerve transection on the size of the microglial cell population in cytoarchitecturally distinct regions of the spinal cord dorsal horn of rats was evaluated at selected intervals 2 through 35 days after unilateral brachial plexotomy. The identification of cells was verified by electron microscopic examination of a representative random sample of cells included in the counts. Microglial cell numbers were increased in laminae I, II as well as the arbitrarily defined deeper laminae 3.5 days after surgery. Although microglial cell numbers in laminae I were within normal range 35 days after axotomy, those of the more ventrally located laminae remained significantly greater than control values for the duration of the experimental period. These findings demonstrate that: 1) microglial cell proliferation in the dorsal horn is an early event in the central changes that are attendant to peripheral nerve injury 2) the time course of the response varies in cytoarchitecturally different regions.