Gene Expression In Spinal Cord Research Articles

Midkine regulates pleiotrophin organ-specific gene expression: Evidence for transcriptional regulation and functional redundancy within the pleiotrophin/midkine developmental gene family

Midkine (MK) and the highly related cytokine pleiotrophin (PTN) constitute the PTN/MK developmental gene family. The Mk and Ptn genes are essential for normal development of the catecholamine and renin–angiotensin pathways and the synthesis of different collagens. It is not known whether the Ptn and Mk genes regulate each other or whether PTN and MK are functionally redundant in development. We have now compared the levels of expression of Ptn and Mk in genetically deficient Mk −/− and Ptn −/− mice and found highly significant increases in Ptn gene expression in spinal cord, dorsal root ganglia, eye, heart, aorta, bladder, and urethra, but not in brain, bone marrow, testis, and lung of Mk −/− mice compared with wild type mice; a remarkable ∼230-fold increase in Ptn expression levels was found in heart of Mk −/− mice and highly significant but lesser increases were found in six other organs. Differences in levels of Mk gene expression in Ptn −/− mice could not be detected in any of the organs tested. The data demonstrate that MK regulates Ptn gene expression with a high degree of organ specificity, suggesting that Ptn gene expression follows Mk gene expression in development, that the increase in Ptn gene expression is compensatory for the absence of MK in Mk −/− mice, that PTN and MK share a high degree of functional redundancy, and that MK may be very important in the development of heart in mouse.

Targeted retrograde gene delivery into the injured cervical spinal cord using recombinant adenovirus vector

Direct routes of gene administration (intrathecal, intracerebroventricular or intraparenchymal infusion) have been used for effective and sustained gene delivery, but serious concerns exist about possible traumatic injury as well as neural damage that may lead to further tissue necrosis, apoptosis and cell death. We evaluated targeted retrograde gene delivery through the sternomastoid muscle (innervated by the spinal accessory nerves) into the injured cervical spinal cord using a recombinant adenovirus vector. LacZ gene expression in the cervical spinal cord was noted from 3 days to 4 weeks after the injection of vector into the sternomastoid muscles of the rats. Recombinant adenovirus vector was transferred via a retrograde mechanism into the injured cervical spinal cord with high transduction efficacy (80.6–98.9%) over certain adenoviral titer and dosage. Transduction was less efficient when the vector was injected 1 and 2 weeks after spinal cord injury (44.2–56.8%). Our results indicate retrograde delivery of recombinant adenovirus vector is possible immediately after spinal cord injury, and that this method is promising for gene delivery because it is effective, selective, less invasive to the injured spinal cord, has long-lasting gene expression, and is potentially feasible treatment choice for spinal cord injury.

644. Gene Delivery for Chronic Pain Control: Micrcroencapsulated Plasmid DNA Encoding the Anti-Inflammatory Cytokine Gene, Interleukin-10 (IL-10)

Chronic pain control is a major unresolved clinical problem. Spinal cord astrocytes & microglia are critically involved in the creation & maintenance of diverse enhanced pain states via the release of proinflammatory cytokines. Interleukin-10 (IL-10), a potent anti-inflammatory cytokine, suppresses proinflammatory cytokine production & activity. We have previously shown that administration of IL-10 protein directly or via a viral vector encoding it into the spinal cord (intrathecal, i.t.) reverses neuropathic pain in the rat; although direct administration of IL-10 protein showed a very short-term reversal. We sought to determine whether neuropathic pain (hypersensitivity to light touch [allodynia] produced by chronic constriction injury [CCI] of the sciatic nerve) could be reversed by: 1) single or repeated i.t. delivery of naked plasmid DNA encoding rat IL-10 (pIL10), 2) single or repeated i.t. delivery of pIL10 treated with the cationic polymer, polyethyleneimine (PEI- pIL10), and 3) single or repeated i.t. pIL10 encapsulated in micro-particles prepared from FDA-approved biodegradable copolymers of polylactide and polyglycolide (PLGA) (PLGA- pIL10). Lastly, we examined whether i.t. rhodamine-labeled PLGA-micro-particles could be visualized in spinal cord using confocal microscopy in naive rats. Behavioral measures were assessed prior to & at 3 & 10 days post CCI. I.t. injections were given on day 10 post-CCI, consisting of pIL10 (100 ug/injection), control plasmid encoding jellyfish green fluorescent protein (pGFP; 100 ug/injection), vehicle (3% sucrose in phosphate buffered saline), PEI- pIL10 (10 ug/injection) or PLGA- pIL10 (350 ug PGLA/injection). For repeated injections, a 2nd injection was given on day 13. Allodynia was reassessed every 1 to 4 days. Allodynia was stable in control treated rats but was reversed (prolonged reversal lasting 40+ days), in rats given 2 i.t. injections of: 1) pIL10 2) PEI- pIL10 & 3) PLGA-IL10. Single i.t. injections of pIL10, PEI- pIL10 or PLGA- pIL10 produced a brief 3-6 day reversal of allodynia. Importantly, rhodamine-labeled PLGA-micro-particles were visible immediately & at 3 days after i.t. injection. Ongoing studies using i.t. rhodamine-labeled PLGA-micro-particles (either without DNA or with pGFP DNA) are aimed at examining the spread, the distribution in superficial spinal cord or deeper parenchymal layers & gene expression. This approach to pain control represents a dramatic departure from all other available therapies. Support: Avigen & NIH HL56510, DA015656, DA018156 & DA015642.

Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination

Clostridial neurotoxins have assumed increasing importance in clinical application. The toxin's light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health. To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed. LC expressed in differentiated neuronal PC12 cells was shown to induce time- and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products. LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso-Beattie-Bresnahan (BBB) scale (P<0.001) and rotarod assay (P<0.003). Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats. At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected. Spontaneous functional recovery was observed to parallel the cessation of LC gene expression. These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.

Adeno-associated viral vector gene expression in the adult rat spinal cord following remote vector delivery

The current investigation tests whether adeno-associated viral vectors (rAAV) undergo remote delivery to the spinal cord via peripheral nerve injection as previously demonstrated with adenoviral vectors. The sciatic nerves of adult rats ( n = 10) were injected with either an rAAV (rAAVCMV-lacZ) or adenoviral (AdCMV-lacZ) vector (1.4 × 10 7 particles/ml). After 21 days, the rAAV group demonstrated significantly higher spinal cord viral expression than the adenoviral group ( P < 0.024). A second group of rats was injected with rAAV expressing the green fluorescence protein (GFP) reporter gene. GFP was detected 21 days after unilateral sciatic nerve injection in the neurons of the dorsal root ganglion and spinal cord. The codistribution of the viral genome and transgene in CNS neurons was confirmed with in situ hybridization. In summary, rAAV genes are expressed in CNS neurons following peripheral nerve injection at levels exceeding those seen following remote adenovirus injection.

Effect of 17β-estradiol on gene expression in lumbar spinal cord following sciatic nerve crush injury in ovariectomized mice

Previously, we observed that estrogen treatment enhances regeneration of the sciatic nerve after crush injury [Brain Res. 943 (2002) 283]. In this research, we studied expression of estrogen receptors and effects of estrogen on gene expression in the lumbar spinal cord, following sciatic nerve crush injury. Using the Atlas Mouse 1.2 Array, changes in the expression of 267 of 1176 genes were registered 4 days after nerve injury. Those genes that exhibited a change in signal intensity ratios of 2-fold or greater were selected as up-regulated (42) or down-regulated (21). In estrogen treated mice, we have observed up-regulation of the genes known to control apoptosis, cell proliferation, and growth, which might account for the positive effects of estrogen on the regeneration of motor neurons. Immunohistochemical staining revealed estrogen receptor-α and estrogen receptor-β localized in the nucleus and cytoplasm of lumbar motor neurons, and in the regenerating neurites of the sciatic nerve. Expression of estrogen receptor-α and estrogen receptor-β mRNA in lumbar spinal cord was shown by traditional RT-PCR. Using real-time quantitative RT-PCR, we demonstrated increased expression of estrogen receptors-α and -β mRNA on the injured side of the lumbar spinal cord. Western blot analysis showed the accumulation of ERs in regenerating sciatic nerve, and revealed a 40% increase of activated ERK1/2 in estrogen treated mice, compared to placebo. Our findings indicate that: (i) axotomized motor neurons increase expression of estrogen receptors-α and -β mRNA, (ii) estrogen mediates the expression of genes which accelerate the growth and maturation of axons, and (iii) estrogen receptors are transported from the perikaryon into regenerating neurites, and estrogen promotes regeneration locally through the non-genomic ERK-activated signaling pathway.

Altered expression of transcripts for alpha-tubulin and an unidentified gene in the spinal cord of phenyl saligenin phosphate treated hens (Gallus gallus).

Phenyl saligenin phosphate (PSP) induces a central-peripheral distal axonopathy in domestic fowl that develops 7-21 days after a single exposure. Neurotoxic esterase (NTE) is the initial molecular target for this neurotoxicity. PSP has to covalently bind to NTE and chemically "age" for induction of axonopathy. It was hypothesized that exposure to PSP results in early changes in spinal cord gene expression that do not occur with phenylmethylsulfonyl fluoride, a non-neuropathic compound that also inhibits NTE, or DMSO controls. Targeted display was used to screen approximately 15,000 gel bands. Three candidate genes were identified, but only the transcript designated P1 showed decreased expression following PSP exposure (2 mg/kg i.m.) in subsequent Northern blot and in situ hybridization experiments in samples taken <48 h after exposure. Additional experiments revealed that a approximately 2.5 kb alpha-tubulin transcript had decreased expression at 12-48 h after PSP exposure, with maximum change at 48 h (33%, p = 0.0479). A approximately 4.5 kb alpha-tubulin transcript had increased expression at 12 h (38%, p = 0.0125) and decreased expression at 48 h (28%, p = 0.0576). In situ hybridization on spinal cord revealed neuronal expression of P1 and alpha-tubulin transcripts. Decreased expression of transcripts for P1 and alpha-tubulin was present at 12 and 48 h, respectively. This decrease occurred in all neurons, not just those whose axons degenerate. Results suggest that (1) in PSP-induced OPIDN (organophosphorus-induced delayed neurotoxicity) some gene transcript expression changes are associated with initiation of axonopathy, and (2) PSP modulates spinal cord gene expression in neuronal types that do not undergo axonal degeneration.

Remote adenoviral gene delivery to the spinal cord: contralateral delivery and reinjection.

This study characterizes the distribution of adenoviral genes in the spinal cord after viral vector injection into the sciatic nerve. It also evaluates the ability of repeated adenoviral sciatic nerve injections to prolong gene expression in the spinal cord. Rat sciatic nerves were unilaterally coinjected with the retrograde tracer Fluoro-Gold (Fluorochrome, Inc., Denver, CO) and the adenoviral vector Ad5RSVntLacZ. The distribution of adenoviral gene expression in the spinal cord was compared with that of Fluoro-Gold. Next, levels of gene expression in the sciatic nerve and spinal cord were compared after single and repeated injections of Ad5RSVntLacZ. Finally, remote spinal cord gene expression in naive animals was compared with expression in animals that had been pretreated with subcutaneous Ad5RSVntLacZ inoculation. Viral gene expression was detected in all quadrants of the spinal cord gray matter, whereas Fluoro-Gold was detected only in the ipsilateral ventral horn (n = 5). This remote delivery was blocked by sciatic nerve transection (n = 10). Viral gene expression occurred in the sciatic nerve after both initial and repeated injections, whereas remote gene expression in the spinal cord was observed only after primary sciatic nerve injection (n = 24; P < 0.003). As with repeated sciatic nerve injections, subcutaneous inoculation with Ad5RSVntLacZ blocked subsequent remote spinal cord gene delivery (n = 8; P < 0.05). Remote viral gene delivery occurs in neurons without direct sciatic nerve projections but is dependent on intact peripheral nerves. Repeated injections fail to boost spinal cord gene expression, because of immune recognition of reinjected virus.

Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays.

In order to obtain insight into the aetiology and pathogenesis of amyotrophic lateral sclerosis (ALS), high-density gene discovery arrays (GDA human version 1.2) containing 18 400 non-redundant EST cDNAs pooled from different tissue libraries have been used to monitor gene expression in lumbar spinal cord from ALS cases compared with controls. Quantitative filter analysis revealed differential expression of cDNAs normalized to internal standards. These candidates have been further investigated and their expression in spinal cord characterized in a panel of ALS and control subjects. Significant differential expression was obtained for 14 genes, 13 being elevated (up to six-fold) and one decreased (by 80%) in ALS. Amongst those elevated in ALS were thioredoxin and glial fibrilary acid protein, which have already been shown to be up-regulated in ALS, thus supporting the reliability of this approach. The other differentially regulated transcripts confirmed in the expression studies represent potential candidates in ALS pathogenesis being involved in antioxidant systems, neuroinflammation, the regulation of motor neurone function, lipid metabolism, protease inhibition and protection against apoptosis. The use of the GDA system has greatly facilitated the screening and retrieval of sequence information and has generated useful information on the cascade of molecular events occurring in ALS and potentially may highlight new candidates playing a role in the aetiology and progression of this disease.

Differential expression of 14 genes in amyotrophic lateral sclerosis spinal cord detected using gridded cDNA arrays

In order to obtain insight into the aetiology and pathogenesis of amyotrophic lateral sclerosis (ALS), high‐density gene discovery arrays (GDA human version 1.2) containing 18 400 non‐redundant EST cDNAs pooled from different tissue libraries have been used to monitor gene expression in lumbar spinal cord from ALS cases compared with controls. Quantitative filter analysis revealed differential expression of cDNAs normalized to internal standards. These candidates have been further investigated and their expression in spinal cord characterized in a panel of ALS and control subjects. Significant differential expression was obtained for 14 genes, 13 being elevated (up to six‐fold) and one decreased (by 80%) in ALS. Amongst those elevated in ALS were thioredoxin and glial fibrilary acid protein, which have already been shown to be up‐regulated in ALS, thus supporting the reliability of this approach. The other differentially regulated transcripts confirmed in the expression studies represent potential candidates in ALS pathogenesis being involved in antioxidant systems, neuroinflammation, the regulation of motor neurone function, lipid metabolism, protease inhibition and protection against apoptosis. The use of the GDA system has greatly facilitated the screening and retrieval of sequence information and has generated useful information on the cascade of molecular events occurring in ALS and potentially may highlight new candidates playing a role in the aetiology and progression of this disease.

Spinal cord genes enriched in rat dorsal horn and induced by noxious stimulation identified by subtraction cloning and differential hybridization

Persistent nociceptive input increases neuronal excitability and induces a program of gene expression in the dorsal spinal cord. The alteration in gene expression commences with phosphorylation and induction of immediate early genes and proceeds to target genes. Only a few target genes have been identified as yet. The present report uses a polymerase chain reaction-based subtraction cloning procedure to obtain an “anatomically focused” complementary DNA library enriched in transcripts related to sensory spinal cord (rat dorsal horn minus ventral horn). A subset of clones from this library ( n=158) was screened to verify dorsal horn enrichment and to identify those regulated by carrageenan-induced peripheral inflammation. Molecular classes which displayed enriched expression included a proto-oncogene not previously associated with sensory processes, two regulators of the Rho/Rac pathway which controls cell shape, and three genes involved in cytoskeletal regulation and scaffolding. Additional transcripts coded for proteins involved in intercellular communication or intracellular function. Within the set of 158 transcripts, one known and two unknown genes were induced by persistent noxious input. The known gene codes for the secreted cysteine proteinase inhibitor, cystatin C, suggesting that modulation of extracellular proteolytic activity occurs. Since it is secreted, cystatin C may also provide a cerebrospinal fluid bio-marker for persistent pain states. Using a combined anatomical and functional approach, we have extended the molecular repertoire of genes expressed and induced in second-order neurons or supporting glial cells in several new directions, with particular emphasis on regulation of cell morphology and plasma membrane dynamics. Some of these proteins reveal new pathways for information signaling in the sensory half of the spinal cord and require further research to understand their role in the adult spinal cord. The induced genes may provide new molecular targets for therapeutic development and provide new probes for investigating the dynamic state of cellular activity that occurs during persistent pain states.

Selective upregulation of the flip-flop splice variants of AMPA receptor subunits in the rat spinal cord after hindpaw inflammation

Glutamate receptors are involved in spinal nociceptive transmission and the development of persistent inflammatory hyperalgesia. It is unclear, however, whether there are changes in glutamate receptor gene expression associated with tissue injury. In the present study, we used reverse transcription–polymerase chain reaction (RT–PCR) to examine the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor gene expression in the rat spinal cord by inflammation. Inflammation was introduced into the hindpaw by intraplantar injection of 0.2 ml of complete Freund’s adjuvant (CFA). At 2 h–14 d after inflammation, total RNAs from L4,5 spinal cord were used for RT–PCR with primers targeted at eight flip-flop splice variants of the AMPA receptor subunits. It was found that the GluR1-flop mRNA was up-regulated at 2 h–5 h ( P<0.05), down-regulated at 3 d ( P=0.05), and returned to control levels at 7 d following inflammation. The GluR2-flip and GluR3-flop mRNAs were up-regulated at 5 h–1 d ( P<0.05) and returned to control levels at 3 d after inflammation. The GluR1-flip mRNA was not detected in the samples and the mRNAs for other splice variants did not exhibit significant changes. Immunocytochemical analysis of GluR1 and GluR2 subunits indicate that the protein translation products of these subunits were also increased in the spinal cord. These results demonstrate an increased expression of AMPA receptor subunits that correlates with the acute phase of CFA-induced inflammation and hyperalgesia. Selective changes in the expression of the flip-flop splice variants of the AMPA receptor suggest a reorganization of the composition of the AMPA receptor complex and its involvement in the development of inflammatory hyperalgesia.

Chronic Pain and Gene Expression in Spinal Cord

Chronic Pain and Gene Expression in Spinal Cord

Nociceptive regulation of GABA B receptor gene expression in rat spinal cord

Activation of γ-aminobutyric acid (GABA) neurotransmission evokes antinociceptive responses in laboratory animals. The recent cloning of GABA B receptor gene products makes it possible to examine the regulation of this receptor system as it relates to the mediation of pain-related sensory information. Inasmuch as acute and chronic pain alter the expression of a number of nociception-related receptors, and because such changes are important components in the regulation of pain, the present study was undertaken to determine whether GABA B receptor gene expression is altered in sensory systems following a peripheral nociceptive stimulus. Solution hybridization-nuclease protection assays conducted 24 h after formalin injection into the right hindpaw of the rat revealed a significant bilateral increase in GABA B R1 and R2 receptor expression in the dorsal lumbar spinal cord, and a significant increase in GABA B R1 receptor mRNA in the ipsilateral lumbar dorsal root ganglion. These findings indicate an activity-dependent, differential regulation of GABA B R1 and R2 receptor gene expression in spinal sensory systems in response to chemogenic nociceptive activation, suggesting that GABA B receptor plasticity may play an important role in regulating the mediation, and perception, of chronic pain.

In vivo adenovirus-mediated gene transfer and expression in ischemic rabbit spinal cord

Purpose : In an attempt to study whether ischemic spinal cord expresses a foreign gene in vivo, a replication-defective adenoviral vector containing the Escherichia coli lacZ gene was directly injected into the ischemic spinal cord of rabbits, and temporal and spatial profiles of the exogenous gene expression were compared with that of the control spinal cord. Methods : Thirty-nine Japanese domesticated white rabbits weighing 2 to 3 kg were used in this study and were divided into two subgroups, a 15-minute ischemia group and a sham control group. The adenoviral vector was directly injected into lumbar spinal cord by a needle from dorsal spine just after the infrarenal aortic occlusion in the case of ischemia. Animals were allowed to recover at ambient temperature and were killed at 1, 2, 4, and 7 days after reperfusion (n = 3 at each time point). Results : In the control rabbit, adenoviral vector was transferred into the spinal cord, and the lacZ gene was expressed at dorsal astroglia and anterior motor neurons at 1 to 7 days of reperfusion. After 15 minutes of ischemia, the lacZ gene was expressed at 2 and 4 days of reperfusion in dorsal astroglia and anterior motor neurons, which were positive for Fas antigen. Conclusion : This result suggests that it is possible to transfer and express the lacZ gene in ischemic motor neurons, which eventually show apoptotic change with induction of Fas antigen, and also suggests a great potential of gene therapy for paraplegic patients in the future. (J Vasc Surg 1999;30:542-50.)

Characterization of Adenoviral Gene Expression in Spinal Cord after Remote Vector Delivery

Characterization of Adenoviral Gene Expression in Spinal Cord after Remote Vector Delivery

Characterization of adenoviral gene expression in spinal cord after remote vector delivery.

Recent work has established that the remote injection of attenuated adenoviral vectors may result in central nervous system (CNS) gene expression. These studies suggest that virus passes through peripheral nerves into the CNS. The present experiment attempts to characterize this phenomenon systematically. Spinal cord cells staining for the reporter gene beta-galactosidase were histologically quantified after microinjection of the viral vector Ad5RSVntLacZ into rat footpad, muscle, or sciatic nerve. The effects of injection location, titer, and time, as well as nerve crush and dexamethasone, were examined. Sciatic nerve viral vector injection results in significantly higher CNS uptake than intramuscular and subcutaneous injections (P < 0.05). Nerve crush injury caused a time-dependent reduction in spinal cord gene uptake after sciatic nerve adenoviral injection (P < 0.05). Neuronal staining reaches its peak at 6 days after injection (P < 0.002). Peripheral nerve delivery to the CNS increases with augmented titers (P < 0.03). Finally, gene expression is augmented by administration of dexamethasone (P < 0.0001). Remote adenoviral vector injection represents a potential method for spinal cord gene therapy that avoids any manipulation of CNS tissue.

Sequential expression of transforming growth factor-beta1 by T-cells, macrophages, and microglia in rat spinal cord during autoimmune inflammation.

Transforming growth factor-beta1 (TGF-beta1) is crucially involved in regulating inflammatory events during experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Despite accumulating evidence for local expression of TGF-beta1 in the inflamed nervous system, uncertainty remains regarding its cellular source. We have investigated the temporospatial distribution of TGF-beta1 gene expression in rat spinal cord during EAE. In actively induced EAE, in situ hybridization revealed strong expression of TGF-beta1 in meningeal and perivascular mononuclear infiltrates at onset of the disease, continued expression in perivascular infiltrates and scattered mononuclear cells at maximal disease severity, and expression in scattered parenchymal cells during recovery. Double labeling studies revealed subpopulations of infiltrating T-cells to be the major source of TGF-beta1 early in the disease, followed by macrophages at peak severity and microglial cells during the recovery phase of EAE. Astrocytes and neurons did not express TGF-beta1. Quantification of mRNA by Northern blot analysis revealed that cellular expression of TGF-beta1 by T-cells, macrophages, and microglia sums up to a long-lasting elevation of TGF-beta1 mRNA extending well into the recovery phase. Our data indicate cellular diversity and suggest functional diversity of TGF-beta1 gene expression during EAE. While TGF-beta1 expressed early in the disease by T-cells may contribute to inflammatory lesion development, microglial cells may potentially contribute to recovery by expressing immunosuppressive TGF-beta1 during remission.

Attenuation of IGF-1 antinociceptive action and a reduction in spinal cord gene expression of its receptor in experimental diabetes

Insulin-like growth factor I (IGF-1) is trophic to sensory, motor and sympathetic neurons. Intrathecal (i.t.) administration of IGF-1 produced analgesic effects when tail flick/withdrawal latency was used as an indicator. This action was blocked by genistein (an inhibitor of tyrosine kinase) but not by atipamezol (an α 2 adrenoreceptor antagonist), naloxone (an opioid antagonist) or glibenclamide (a blocker of ATP sensitive K + channels). Induction of diabetes with streptozotocin (STZ, 55 mg/kg, i.v.) impaired the ability of IGF-1 to elevate nociceptive threshold. This phenomenon was not seen in normal animals rendered hyperglycemic with d-glucose (20 mmol in 2.5 ml of saline, i.p.). PCR-based assay revealed that the lumbar region of the spinal cord expresses mRNA transcripts for IGF-1 and its receptor. The rates of expression of both of these transcripts were reduced during diabetes. The above behavioral and biochemical abnormalities induced by the diabetic state were partially restored following replacement therapy with insulin. Overall, our data suggest that a receptor-linked tyrosine kinase mediates the antinociceptive effect of IGF-1. Additionally, the attenuation in the ability of IGF-1 to elevate nociceptive threshold may be a consequence of reduced gene expression of IGF-1 receptor within the spinal cord.

Oligodendrocyte gene expression in the human fetal spinal cord during the second trimester of gestation.

A comprehensive evaluation of myelination during normal human development is essential to understand the pathology of congenital diseases of white matter. The present study establishes quantitative values for normal oligodendrocyte-specific gene expression during the early stages of myelination in the human fetal spinal cord. Complementary techniques of Northern and immunoblotting were used to determine relative amounts of oligodendrocyte-specific mRNAs and proteins between 12 and 24 gestational weeks. Values were determined for myelin basic protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and proteolipid protein. The relative amount of myelin-associated glycoprotein mRNA was also estimated. To compare gene expression between glial cell types, the relative amounts of mRNA and protein were determined for glial fibrillary acidic protein (GFAP), a cell-type specific marker for astrocytes. All oligodendrocyte-specific genes expressed similar developmental kinetics. Between 12 and 15 gestational weeks, less than a five-fold increase was detected in the expression of these genes and their protein products. Between 15 and 22 gestational weeks, the relative amounts of mRNA and protein for the myelin genes increased more than 80-fold. The kinetics of GFAP expression were similar to those of the myelin-associated genes. Absolute values for the increase in mass of the human fetal spinal cord were also obtained. These results provide data that may aid in the neuropathologic assessment and characterization of myelin disorders in the preterm, neonatal, and pediatric spinal cord.