Analysis of gene expression following spinal cord injury in rat using complementary DNA microarray.

To identify genes that were altered by spinal cord injury (SCI), we used complementary DNA microarray consisting 1176 rat genes. Rats were subjected to contusive injury of the thoracic spinal cord. Sham animals received only a laminectomy. Twenty-four hours later, spinal cord was dissected out, a 32P labeled probe was prepared and hybridized to the microarray. We identified three genes that showed a greater than 2-fold increase in SCI tissue, heat shock 27-kDa protein, tissue inhibitor of metalloproteinase-1 and epidermal fatty acid-binding protein. Seven genes, lecithin:cholesterol acyltransferase, dipeptidyl aminopeptidase related protein, phospholipase C delta 4, plasma membrane Ca2+-ATPase isoform 2, G-protein GO alpha subunit, GABA transporter 3, and neuroendrocrine protein 7B2 were down-regulated greater than 50% in SCI tissue. Changes in expression of these genes were confirmed by reverse transcription-polymerase chain reaction. These genes may play a role in the response to tissue damage or repair following SCI.

Increased Fos induction in adult rats that experienced neonatal peripheral inflammation.

The response to noxious stimulation was compared in adult rats that had peripheral inflammation as neonates and untreated rats. On postnatal day 1, rat pups experienced complete Freund's adjuvant (CFA)-induced inflammation of the left hind paw. At 8 weeks of age, these rats and neonatal untreated rats received a bilateral injection of CFA into their hind paws. Fos-like immunoreactivity (Fos-LI) was used as a measure of neuronal activity in dorsal horn nociceptive pathways. A significant increase in Fos-LI was found on the left side of the lumbar spinal cord of neonatal treated rats as compared to neonatal untreated rats. These results suggest that the experience of neonatal peripheral inflammation may result in an increase in the response of spinal cord neurons to peripheral inflammation as adults.

Descending modulation of opioid-containing nociceptive neurons in rats with peripheral inflammation and hyperalgesia.

Inflammation and hyperalgesia induce a dramatic up-regulation of opioid messenger RNA and peptide levels in nociceptive neurons of the spinal dorsal horn. Descending axons modulate nociceptive transmission at the spinal level during inflammatory pain, and may play a role in the development of persistent pain. The role of descending bulbospinal pathways in opioid-containing nociceptive neurons was examined. Removal of descending inputs to the spinal cord was performed by complete spinal transection at the midthoracic level. Seven days after spinal transection, rats received a unilateral hindpaw injection of complete Freund's adjuvant, a noxious stimulus that produces inflammation and hyperalgesia. Tissues from the L4 and L5 segments of the spinal cord were removed and analysed by northern blotting and immunocytochemistry. Spinal transection resulted in a further increase in both dynorphin and enkephalin messenger RNA content following complete Freund's adjuvant injection. There was a similar distribution and number of dynorphin-immunoreactive cells in transected rats compared to rats which received sham surgery. These data suggest that increased dynorphin messenger RNA ipsilateral to inflammation, in rats without descending axons, was due to increased expression within the same cells and not to recruitment of additional dynorphin-expressing cells. This reflects a greater dynamic response of nociceptive neurons to noxious stimuli in the absence of descending modulation. Therefore, the net effect of descending afferents on spinal nociceptive circuits may be to reduce the response of opioid-containing neurons to noxious stimulation from the periphery.

Descending modulation of Fos expression after persistent peripheral inflammation.

The effects of spinal transection on Fos protein expression following complete Freund's adjuvant-induced hindpaw inflammation and hyperalgesia were studied. Fos-like immunoreactivity (LI) was used as a measure of neuronal activity in dorsal horn nociceptive pathways. Induction of Fos-LI in spinally transected rats with 3 days of hindpaw inflammation was significantly increased in ipsilateral lumbar (L 4,5) spinal cord, when compared with control animals with similar inflammation but an intact spinal cord. The contralateral side of spinally transected rats also showed significant induction of Fos-LI compared to the non-inflamed side of control rats. Since Fos induction provides an indication of the level of neuronal activity in response to a given stimulus, these results suggest that the net effect of brain stem descending pathways is to dampen central hyper-excitability in the spinal cord induced by tissue inflammation and hyperalgesia.

Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury.

Peripheral nerve trauma induces the expression of genes presumed to be involved in the process of nerve degeneration and repair. In the present study, an in vivo paradigm was employed to identify molecules which may have important roles in these processes. A cDNA library was constructed with RNA extracted from rat dorsal root ganglia (DRG) 3 days after a sciatic nerve crush. After differential hybridization to this library, several cDNAs were identified that encoded mRNAs that were upregulated in the DRG ipsilateral to the crush injury, as opposed to the contralateral or naive DRG. Approximately 0.15% of all the clones screened were found to be induced. This report presents the types of induced sequences identified and characterizes one of them, DA11. The 0.7 kb DA11 full length cDNA clone contains a 405 nucleotide open reading frame that encodes a putative protein of 15.2 kDa (135 amino acid residues) and is a member of the family of fatty acid binding proteins (FABP). The DA11 protein differs by one amino acid residue from the sequence of the C-FAPB protein and by eight residues from the sequence of mal1, proteins found in rat and mouse skin, respectively. Northern and Western blot analyses showed that the DA11 mRNA and protein were induced in the injured DRG. Furthermore, studies using antibodies generated against DA11 found that the DA11-like immunoreactivity was more pronounced in the nuclei of neurons located in the DRG ipsilateral to the sciatic cut than those located in the contralateral DRG. The induction of DA11 mRNA and protein in DRG neurons suggests, for the first time, the involvement of a neuronal FABP in the process of degeneration and repair in the nervous system.

Comparison of c-jun, junB, and junD mRNA expression and protein in the rat dorsal root ganglia following sciatic nerve transection.

The present study was designed to compare the expression of the Jun family of protooncogenes following nerve injury. Adult rats were anesthetized and the sciatic nerve transected. Dorsal root ganglia (DRG) at 1, 2, 3, and 7 days after nerve transection were collected, their total RNA extracted, and Northern blots performed using 32P-labeled oligonucleotide probes. The constitutive expression of c-jun mRNA was very low in DRG. Induction of c-jun mRNA was observed by day 1 after nerve transection, with a sixfold peak at 3 days and a twofold induction still present by day 7. The constitutive expression of junB mRNA was also low in the DRG, and sciatic nerve transection produced only a modest induction (1.7-fold by day 3) in the DRG ipsilateral to the nerve cut. junD mRNA was constitutively expressed at high levels in the DRG, and its level of expression did not in the DRG, and its level of expression did not change after sciatic nerve transection. Immunocytochemistry studies demonstrated a pattern of c-Jun, JunB, and JunD immunoreactivity (IR) associated with the cell nuclei of DRG neurons. c-Jun IR was found at very low levels in the undamaged contralateral DRG neurons, but sciatic nerve transection dramatically increased the number of c-Jun-immunoreactive neurons. Dot blot immunoblotting assay confirmed that the DRG ipsilateral to the sciatic nerve cut contained a higher level of c-Jun protein than the contralateral control DRG. Similar to c-Jun IR, JunB IR was minimal in the undamaged contralateral DRG. However, the DRG ipsilateral to the nerve transection did not show an increase in the number of immunoreactive neurons. JunD protein was expressed at high levels in the contralateral DRG, and this level of expression persisted after sciatic nerve transection in the ipsilateral DRG. DNA gel retardation assay experiments with an AP-1 consensus sequence showed a single DNA-protein complex. This complex was increased in ipsilateral as compared with contralateral DRG extracts. The amount of DNA-protein complex was reduced by c-Jun protein antiserum but was not altered when treated with a Fos antibody. We conclude that c-jun, junB and junD mRNAs and proteins are differentially regulated in the DRG after sciatic nerve transection.

SR13/PMP-22 expression in rat nervous system, in PC12 cells, and C6 glial cell lines.

SR13/PMP-22 is a protein that was identified after screening a sciatic nerve cDNA library. Our study focused on comparing the level and pattern of expression of SR13/PMP-22 protein and RNA. Northern blot analysis revealed that although SR13/PMP-22 mRNA was present in all nervous tissues and cells studied, levels were at least seven fold higher in the sciatic nerve and the spinal cord. During sciatic nerve postnatal development and maturation, the SR13/PMP-22 mRNA was detected at 2 days after birth, reached a maximal level at day 24, and decreased to 1/3 of the maximum in adult animals. Nerve transection reduced the level of SR13/PMP-22 mRNA to less than 5% in the segment distal to the nerve injury. Experiments using in situ hybridization localized the SR13/PMP-22 mRNA in Schwann cells. Schwann cells present in the vicinity or distal to the nerve cut repressed the signal for the message. In situ hybridization experiments also demonstrated that dorsal root ganglia satellite cells contained the message for SR13/PMP-22. The SR13/PMP-22 antisera used in our study showed a complex pattern of staining. As expected, the SR13/PMP-22 antibody peptide 1 immunoreacted with the sciatic nerve sheath. However, immunocytochemistry of the dorsal root ganglia revealed that the staining was contained in the neuron's cell body and processes and also in satellite cells. We also identified immunoreactive cell bodies and fibers in the spinal cord dorsal horn. Tissue culture studies demonstrated that SR13/PMP-22 mRNA is induced in NGF treated PC12 but not in C6 glioma cell lines grown under experimental conditions that stimulated cell growth arrest. Our experiments suggest that SR13/PMP-22 may have some other function(s) in addition to its hypothesized role in peripheral myelination.

Quantification of axotomy-induced alteration of neuropeptide mRNAs in dorsal root ganglion neurons with special reference to neuropeptide Y mRNA and the effects of neonatal capsaicin treatment.

Alteration in mRNA expression in dorsal root ganglia (DRG) neurons encoding 5 neuropeptides was quantitatively compared in normal rats and in those neonatally treated with capsaicin, a selective neurotoxin which destroys a subpopulation of DRG neurons with unmyelinated axons. Adult rats received a unilateral transection of the sciatic nerve and were killed 7 days later. Oligonucleotide probes specific for the genes encoding neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), galanin (GAL), somatostatin (SOM), and calcitonin gene-related peptide (CGRP) were used for in situ hybridization and RNA blot analysis. Following the nerve cut, RNA blot analysis demonstrated a dramatic induction of NPY, VIP, and GAL mRNA levels from the undetectable constitutive level of expression. Conversely, CGRP and SOM mRNAs, which are constitutively expressed, were reduced 55% and 70%, respectively, following the nerve cut. A unimodal size distribution for neurons expressing NPY mRNA was determined, with a mean cross-sectional area of 1700 microns2 representing 24.4% of DRG neurons ipsilateral to the nerve cut. Neurons expressing VIP mRNA were mainly small sized, with a cross-sectional area of approximately 700 microns2, while those expressing GAL mRNA were both small (approximately 700 microns2) and medium (approximately 1,300 microns2) sized. The percentages of neurons expressing VIP or GAL mRNA were 19.9% and 33.7%, respectively. In neonatal capsaicin-treated rats, there was a 10% reduction in neurons expressing NPY mRNA, a 37% reduction for VIP, and a 27% for GAL mRNA compared to vehicle-treated rats after nerve cut. Capsaicin-sensitive neurons comprised 37% of CGRP neurons and 83% of SOM neurons. These observations suggest that NPY is primarily induced in myelinated primary afferent neurons, while VIP and GAL mRNA induction occurs in a mixed population, a sizeable percentage of which has unmyelinated axons. Additionally, SOM mRNA expression is associated mainly with unmyelinated primary afferents.

Immunohistochemical localization of calretinin in the dorsal root ganglion and spinal cord of the rat.

Calretinin (CR), a recently identified calcium-binding protein, is present in nervous tissue, including sensory pathways, where it may play an important role in regulation of cellular activity. Using immunocytochemistry, we examined the cellular localization of CR in dorsal root ganglia (DRG) and spinal cord of normal rats and after multiple unilateral dorsal root ganglionectomies. In DRG, CR-immunoreactive cell bodies and axons were a small subpopulation (10%) of medium- to large-sized neurons. In the spinal cord, CR-like immunoreactivity (LI) in neurons and fibers was found in all laminae except motoneurons. Dense fiber networks were also found in Clarke's column. The densest staining of both cell bodies and fibers was in the superficial laminae, especially lamina II, and in the lateral spinal and lateral cervical nuclei. CR-immunoreactive fibers were also observed in the fasciculi cuneatus and gracilis. Fasciculus gracilis exhibited the greatest number of labeled axons at the lumbosacral levels, but few labeled axons were found at the rostral thoracic and cervical levels. In contrast, the corticospinal tract at the base of the dorsal column was devoid of CR-immunoreactive fibers. Unilateral multiple lumbar ganglionectomies resulted in a loss of CR-LI in the dorsal columns ipsilateral to the surgery. In the spinal gray matter ipsilateral to the ganglionectomies, CR-LI was reduced in Clarke's column and slightly enhanced in the medial third of lamina II. Our observations demonstrate a unique distribution pattern of CR-LI compared to other calcium-binding proteins in the spinal cord, and suggest a role for CR in nociceptive and proprioceptive pathways.

Neonatal capsaicin treatment attenuates spinal Fos activation and dynorphin gene expression following peripheral tissue inflammation and hyperalgesia.

An animal model of nociception involving unilateral hindpaw inflammation has been used to examine behavioral, molecular, and biochemical aspects of well-characterized spinal cord neural circuits involved in pain transmission. The neurotoxin capsaicin administered neonatally was used to modify this neuronal system by producing a selective destruction of most small, unmyelinated primary afferent axons. Capsaicin had minimal effects on the behavioral hyperalgesia and edema associated with the hindpaw inflammation and on the constitutive expression of preprodynorphin (PPD) mRNA and preproenkephalin mRNA in the spinal cord. However, the inflammation-induced increases in Fos-like immunoreactivity (Fos-LI) and in PPD mRNA were greatly attenuated by neonatal capsaicin treatment. The data indicate that input from small-diameter unmyelinated primary afferents is important for the stimulus-induced increase in Fos-LI and PPD mRNA. Our finding that neonatal capsaicin reduces the levels of Fos-LI and PPD mRNA in a related fashion in the spinal dorsal horn provides further evidence for a relationship between the protein product of the c-fos protooncogene and regulation of dynorphin gene transcription.

Preproenkephalin mRNA in spinal dorsal horn neurons is induced by peripheral inflammation and is co-localized with Fos and Fos-related proteins.

Increased levels of preproenkephalin mRNA in spinal cord neurons induced by peripheral tissue inflammation were examined using in situ hybridization histochemistry. In addition, in situ hybridization histochemistry was combined with immunocytochemistry to determine whether increases in preproenkephalin mRNA were co-localized in spinal cord neurons with increases in immunoreactivity for Fos and Fos-related proteins coded by the immediate-early proto-oncogene, c-fos, and related genes. Dorsal horn laminae I-II, V-VI and VII showed a greater than 200% increase in preproenkephalin mRNA-labeled neurons on the inflamed side as compared to the contralateral control. Inflammation also induced Fos-like immunoreactivity in cell nuclei, mainly in the superficial laminae I-II and the neck of the dorsal horn (laminae V-VI). Few labeled nuclei were detected on the contralateral side. Inflammation resulted in double-labeling of neurons ipsilateral to the inflamed limb whereas they were almost completely absent on the contralateral side. Double-labeled neurons were most frequently found in laminae V-VI. Double-labeled laminae I-II neurons were concentrated in the medial two-thirds of the dorsal horn, the site that receives innervation from the inflamed limb. There were also many double-labeled neurons in laminae VII. Over 90%, 82% and 69% of all neurons expressing preproenkephalin mRNA co-localized Fos immunoreactivity in laminae V-VI, I-II, and VII, respectively. However, the number of neurons expressing increased Fos immunoreactivity was substantially greater than the subpopulation of double-labeled neurons. Our findings indicated that peripheral inflammation induces an increase in preproenkephalin mRNA levels in spinal cord neurons and that most neurons exhibiting preproenkephalin mRNA labeling also co-localized Fos and Fos-related immunoreactivity. These data are consistent with evidence supporting the role of Fos and Fos-related proteins in the regulation of transcription of the preproenkephalin gene in spinal neurons.

Dynorphin expression and Fos-like immunoreactivity following inflammation induced hyperalgesia are colocalized in spinal cord neurons.

Fos and Fos-related proteins are increased in spinal dorsal horn neurons following noxious stimulation. The laminar location of neurons that exhibit this increase is coincident with those that exhibit an increase in dynorphin in a rat model of peripheral inflammation and hyperalgesia. In order to determine whether the increase in Fos or related proteins and dynorphin occurs in the same dorsal horn neurons, two kinds of double-labeling methods were used: in situ hybridization histochemistry to label dynorphin mRNA autoradiographically, and immunocytochemistry to label Fos and Fos-related proteins, or a double immunocytochemical method that labeled Fos and Fos-related proteins and dynorphin peptide with distinct chromagens. With both methods more than 80% of the neurons in laminae I, II, V and VI exhibiting an increase in either dynorphin mRNA or peptide following peripheral inflammation also colocalized increased nuclear Fos-like immunoreactivity. However, the number of neurons displaying increased Fos-like immunoreactivity was substantially greater than the number of neurons colocalizing increased dynorphin. These data suggest that the activation of nuclear Fos and Fos-related proteins may be related to the induction of dynorphin gene expression in a subpopulation of spinal cord neurons following peripheral inflammation and hyperalgesia.