Differential effects of intrathecally administered delta and mu opioid receptor agonists on formalin-evoked nociception and on the expression of Fos-like immunoreactivity in the spinal cord of the rat

In this study we demonstrate that Drosophila calcium/calmodulin-dependent protein kinase II (CaMKII) is capable of complex regulation by autophosphorylation of the three threonines within its regulatory domain. Specifically, we show that autophosphorylation of threonine-287 in Drosophila CaMKII is equivalent to phosphorylation of threonine-286 in rat alpha CaMKII both in its ability to confer calcium independence on the enzyme and in the mechanistic details of how it becomes phosphorylated. Autophosphorylation of this residue occurs only within the holoenzyme structure and requires calmodulin (CaM) to be bound to the substrate subunit. Phosphorylation of threonine-306 and threonine-307 in the CaM binding domain of the Drosophila kinase occurs only in the absence of CaM, and this phosphorylation is capable of inhibiting further CaM binding. Additionally, our findings suggest that phosphorylation of threonine-306 and threonine-307 does not mimic bound CaM to alleviate the requirement for CaM binding to the substrate subunit for intermolecular threonine-287 phosphorylation. These results demonstrate that the mechanism of regulatory autophosphorylation of this kinase predates the split between invertebrates and vertebrates.     

Antinociceptive action of vaginocervical stimulation in rat spinal cord: 2-DG analysis

Using [14C] 2-deoxyglucose (2-DG) autoradiography with computerized densitometric analysis, unilateral foot pinch was found to significantly increase the relative optical density in laminae I and II of the ipsilateral, compared to the contralateral, spinal cord at lumbar 5 (L5). However, during vaginocervical mechanostimulation applied concurrently with the unilateral foot pinch, no comparable difference was observed. No response to foot pinch was observed in other laminae of the spinal cord at L5, and no effects comparable to the above were observed at L3. These findings indicate that vaginocervical mechanostimulation suppresses neural responses to noxious foot pinch stimulation selectively at the laminae I and II level of the spinal cord at L5, but not at L3.     

Effect of intrathecally administered local anesthetics on protein phosphorylation in the spinal cord

To elucidate the biochemical mechanisms of spinal anesthesia, we studied the effects of procaine and tetracaine on protein phosphorylation in the mouse spinal cord. Mice were injected intrathecally with either procaine, tetracaine (67 mM/approximately 2%, 10 microL, N = 5/drug), or saline (N = 4/group). Five minutes after injection, animals were killed with a guillotine, and the spinal cord was removed. The caudal 3-cm cord segment was homogenized and centrifuged, and an aliquot of the supernatant was used for phosphorylation assays. Calcium-dependent phosphorylation was initiated by incubating the samples in buffer containing [gamma-32P]ATP at 37 degrees for 30 min. The proteins were electrophoresed using slab gel and two-dimensional electrophoresis, and phosphorylated proteins were visualized by autoradiography. The data demonstrated that spinal anesthesia changes the phosphorylation state of five endogenous substrate proteins with apparent molecular masses of 130 (protein-a), 105 (protein-b), 55 (protein-c), 47 (protein-d), and 33 (protein-e) kDa. In two-dimensional electrophoresis, protein-a resolved into two proteins (a1 and a2). Analysis of variance of the densitometric data suggested a significant effect for the treatment (F(2,16) 735, P < 0.00005). Post hoc comparisons with the saline-treated controls, using the Newman-Keuls test, indicated that local anesthetics significantly affected phosphoproteins (P < 0.05) except for protein-al in the tetracaine-treated group. Further characterization of these phosphoproteins should aid in determining their role in the signal transduction cascade affected by spinal anesthesia.     

Antinociceptive effect of intrathecally administered pituitary adenylate cyclase activating polypeptide (PACAP) on the rat formalin test

Diclofenac (0.5-2 mM) dose- and time-dependently reduces the viability of isolated hepatocytes. This effect cannot be counteracted by the calcium channel blockers diltiazem (0.05-0.1 mM) and verapamil (0.05-0.5 mM), the calmodulin antagonist calmidazolium (0.01 mM) or Quin 2-AM (0.1 mM), an intracellular calcium chelating agent. On the contrary, verapamil even accentuates the toxic effects of diclofenac. It is concluded from these results, that diclofenac causes cell damage by other mechanisms than calcium overload.     

Axotomy increases CNTF receptor mRNA in rat spinal cord

Chronic eosinophilic pneumonia (CEP) is a rare disorder of unknown etiology characterized by striking systemic and pulmonary manifestations such as fever, weight loss, blood eosinophilia, characteristic fluffy peripheral opacities on chest radiograph, and a prompt response to corticosteroid therapy. While the initial phase has been well documented, there is very limited information concerning the long-term natural history and treated course of this condition. We report the clinical and laboratory findings together with the long-term follow-up data on 12 patients with classic CEP who were followed up for a mean of 10.2 years (range, 4 to 13 years). The most striking feature of the long-term follow-up was the occurrence of relapses of CEP (often on multiple occasions) when corticosteroid therapy was discontinued or the dose was tapered. In those nine patients in whom steroid withdrawal was commenced, there was a clinical, hematologic, and radiologic relapse in seven (58 percent). However, prompt reinstitution of therapy led to a rapid resolution of symptoms. By contrast, two patients (17 percent) showed no evidence of relapse when steroid therapy was discontinued. A further three patients (25 percent) are maintained on a regimen of low-dose steroid therapy with no episodes of relapse. Reassuringly, all 12 patients are well at the end of a long period of follow-up. These data suggest that the long-term prognosis for patients with CEP is excellent but the majority will require long-term low-dose oral corticosteroid therapy in order to prevent relapse.     

Astroglial distribution of neurokinin-2 receptor immunoreactivity in the rat spinal cord

Two mouse monoclonal antibodies, 11H9.1 and 1G7.10, raised against the COOH-terminus peptide (359-390) of the rat neurokinin-2 receptor, were used to visualize by light and electron microscope immunocytochemistry the distribution of this receptor in adult rat spinal cord. At all spinal levels, immunoreactivity was mainly observed in two narrow crescentic zones bordering the gray matter of the dorsal and ventral horns, and around the central canal. In the light microscope, this labelling was the densest within the outer part of lamina I facing the dorsal column, where it took the form of minute dots and streaks scattered in the neuropil. In the electron microscope, such a localization was exclusively astrocytic and essentially involved astrocytic leaflets, as indicated by the size and irregular shape of the immunostained processes, their location between and around neuronal profiles, and their occasional display of glial filaments. The diaminobenzidine reaction product showed some predilection for the plasma membrane and was occasionally seen at gap junctions of these labelled processes. Many labelled astrocytic leaflets were observed in the immediate vicinity of axon terminals containing large dense-cored vesicles, and around fibres morphologically identifiable as primary afferent, unmyelinated C-fibres. These observations suggest that astrocytic neurokinin-2 receptors could define the effective sphere of neurokinin A neuromodulation in rat spinal cord, via alterations in the regulation of the extracellular environment and glutamate uptake by astrocytes and/or the release of putative astroglial mediators. The astrocyte neurokinin-2 receptors, activated by extrasynaptic neurokinin A, might thus co-operate with neurokinin-1 and neurokinin-3 neuronal receptors in the modulation of nociceptive information.     

Angiotensin II in the spinal cord of the rat and its sympatho-excitatory effects

Immunohistochemical studies have shown there is a dense angiotensin-like immunoreactivity of terminals in the sympathetic region of the thoracic and lumbar spinal cord. In the present study measurements were made of the concentration of angiotensin in the spinal cord of rats using radioimmunoassay following two different extraction procedures. These gave concentrations of angiotensin as mean of 108 and 161 pg.g-1 tissue wet weight. Angiotensin II given intrathecally or microinjected into the spinal cord caused an increase in postganglionic sympathetic nerve activity which was blocked by prior application of saralasin. Angiotensin III was without effect. Intracellular recordings from sympathetic preganglionic neurones in-vitro in slices of neonate rat spinal cord showed that angiotensin II produced an increase of excitability of the neurones by a slow depolarisation without the generation of action potentials. This effect still occurred in the presence of TTX. Angiotensin II also could increase synaptic activity, both EPSPs and IPSPs as well as a synaptically induced slow depolarisation being observed suggesting that presympathetic interneurones are also sensitive to the peptide. The evidence indicates that if angiotensin is released from nerve terminals surrounding sympathetic neurones it will enhance the gain of the neurone so that it could more easily be discharged by other excitatory inputs.     

NMDA and non-NMDA receptor antagonists protect against excitotoxic injury in the rat spinal cord

The neuroprotective properties of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine (MK-801) and the non-NMDA antagonists 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) and alpha-methyl-4-carboxyphenylglycine (MCPG) were evaluated against neuronal injury produced by the intraspinal injection of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Forty-nine animals were divided into eight groups in order to evaluate the effects of different drug combinations: (a) NMDA; (b) NMDA + MCPG; (c) NMDA + NBQX; (d) NMDA + MK-801; (e) AMPA; (f) AMPA + MCPG; (g) AMPA + MK-801; and (h) AMPA + NBQX. Drugs were microinjected into spinal segments T12-L3 through a micropipette attached to a Hamilton microliter syringe. Spinal cords were evaluated after a survival period of 48 h at which time NMDA and AMPA were found to produce morphological changes over the concentration ranges of 125-500 mM and 75-500 microM, respectively. Neuronal loss following injections of NMDA + MK-801 or AMPA + NBQX was significantly less than that following injections of NMDA or AMPA alone. By contrast, neuronal loss following co-injections of NMDA or AMPA with inappropriate antagonists, i.e., NMDA + NBQX/MCPG or AMPA + MCPG/MK-801, was not significantly different from that produced by NMDA or AMPA. The results suggest that elevations in spinal levels of glutamate followed by prolonged activation of NMDA and AMPA receptor subtypes initiate an excitotoxic cascade resulting in neuronal injury. Blockade of NMDA and AMPA effects by MK-801 and NBQX respectively confirms the well documented neuroprotective effects of these drugs and lends support to the potential importance of NMDA and especially AMPA receptor antagonists as therapeutic agents in the treatment of acute spinal cord injury.     

Spinal root avulsion-induced upregulation of osteopontin expression in the adult rat spinal cord

Osteopontin (OPN) is a secretory adhesive glycoprotein that is expressed in various tissues and plays a role in inflammation and tissue repair. It has been suggested that OPN plays a role in inflammation and wound healing after spinal cord injury; however, the expression of OPN and its function in the spinal cord under normal conditions and following spinal motoneuron injury have not been well characterized. Here we examined the expression of OPN mRNA before and after spinal root avulsion. OPN mRNA was detected at a low level in the normal spinal cord in a Northern blot analysis, but dramatically increased following avulsion. In situ hybridization and immunohistochemical studies demonstrated that OPN was present only in a subset of spinal motoneurons before avulsion. After avulsion, the number of OPN-expressing motoneurons increased, although the total number of motoneurons was reduced. OPN expression also became apparent in activated microglia/macrophages and astrocytes. These data suggest that the upregulation of OPN after spinal root avulsion is involved in two events, the protection of neurons and the post-traumatic inflammatory response in microglia/macrophages and astrocytes.     

Interactions between metabotropic and ionotropic glutamate receptor agonists in the rat spinal cord in vivo

Microelectrophoretic application of the non-selective metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and the group I selective mGluR agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG] potentiated the responses of rat spinal neurones to the cyclically-ejected ionotropic excitatory amino acid (EAA) agonists NMDA, AMPA and kainate in vivo. Potentiation was not selective between the three ionotropic responses and was paralleled by an enhancement of background activity in spontaneously active cells. "Correcting" spike count data for this increase in background activity showed that the EAA responses were not potentiated beyond the apparent enhancement of cell excitability. Neither mGluR agonist produced potentiation when NMDA/AMPA cycling was superimposed on background discharge held constant with kainate. It is concluded that potentiation produced by both (1S,3R)-ACPD and (RS)-3,5-DHPG is secondary to an enhancement of cell excitability rather than being due to a specific interaction with ionotropic EAA receptors. The mechanism of excitability enhancement cannot be determined by extracellular recording, but group I mGluRs are most likely to be responsible.     

Depression of C fiber-evoked activity by intrathecally administered reactive red 2 in rat thalamic neurons

To investigate the possible role of spinal purinoceptors in nociception, the potent P2-purinoceptor antagonist reactive red 2 was studied in rats under urethane anesthesia in which nociceptive activity was elicited by electrical stimulation of afferent C fibers in the sural nerve and recorded from single neurons in the ventrobasal complex of the thalamus. Intrathecal (i.t.) application of reactive red 2 (6-200 micrograms) caused a dose-dependent reduction of the evoked activity in thalamic neurons. The estimated ED50 was 30 micrograms, and the maximum depression of nociceptive activity amounted to about 70% of the control activity at a dose of 100 micrograms. Morphine, administered i.t. at a maximally effective dose (80 micrograms), inhibited the evoked nociceptive activity by only up to 55% of the control activity. An i.t. co-injection of reactive red 2 (100 micrograms) and morphine (80 micrograms) caused a maximum reduction of the evoked thalamic activity by up to 85% of the control activity, thus, exceeding significantly the effect elicited by either drug alone. Similarly, i.t. co-injection of almost equipotent dosages of reactive red 2 (30 micrograms) and morphine (30 micrograms) caused a maximum reduction of the evoked activity by up to 72% of the control activity, which again exceeded significantly the effect of either drug alone. The results suggest that in rats reactive red 2 exerts antinociception by blockade of P2-purinoceptors in the spinal cord and, hence, support the idea that ATP may play an important role in spinal transmission of nociceptive signals. An activation of the spinal opioid system does not seem to contribute to the effect of reactive red 2 but might act additive or even synergistically with its antinociceptive action.     

Histochemistry in rat brain and spinal cord with an antibody directed at the cholecystokininA receptor

The role of D2/D3 receptors in striatum was electrophysiologically examined in vitro in chloralose-anesthetized rats. In addition, in vitro patch clamp method with rat brain slices was followed. Stimulations of the substantia nigra pars compacta (SN) in vivo elicited spike generation which was inhibited by microiontophoretically applied domperidone, a D2 antagonist. These domperidone-sensitive neurons were activated by microiontophoretic application of D2 agonists such as talipexole, quinpirole and bromocriptine as well as the D2 agonist, 7-OH-DPAT. They were also excited by either intravenous injection of bromocriptine or talipexole in a dose-dependent manner. Furthermore, the SN-induced increases in neuronal firing were blocked during microiontophoretic application of domperidone. In patch clamp whole-cell recording large-sized cells, identified visually under Ramanosky microscope, were depolarized with repetitive firing on bath application of talipexole and 7-OH-DPAT at a current clamp mode. Talipexole-induced depolarization in the large-sized cell was similarly observed in the presence of TTX and high Mg2+ in Ca(2+)-free physiological solution. In contrast, the medium-sized cells were hyperpolarized on bath application of talipexole without being affected by 7-OH-DPAT. These findings suggest that the large-sized cells, which were presumably cholinergic interneurons, are activated by dopamine derived from the SN via D2 and/or D3 receptors, while the medium-sized cells are inhibited by dopamine via D2 receptors.     

Retinoid-X receptor signalling in the developing spinal cord

Retinoids regulate gene expression through the action of retinoic acid receptors (RARs) and retinoid-X receptors (RXRs), which both belong to the family of nuclear hormone receptors. Retinoids are of fundamental importance during development, but it has been difficult to assess the distribution of ligand-activated receptors in vivo. This is particularly the case for RXR, which is a critical unliganded auxiliary protein for several nuclear receptors, including RAR, but its ligand-activated role in vivo remains uncertain. Here we describe an assay in transgenic mice, based on the expression of an effector fusion protein linking the ligand-binding domain of either RXR or RAR to the yeast Gal4 DNA-binding domain, and the in situ detection of ligand-activated effector proteins by using an inducible transgenic lacZ reporter gene. We detect receptor activation in the spinal cord in a pattern that indicates that the receptor functions in the maturation of limb-innervating motor neurons. Our results reveal a specific activation pattern of Gal4-RXR which indicates that RXR is a critical bona fide receptor in the developing spinal cord.     

Identification of macrophage migration inhibitory factor (MIF) in rat spinal cord and its kinetics on experimental spinal cord injury

BACKGROUND: Among spinal cord injuries, secondary injury is considered to be a "reversible" process and seems to be a key target for the treatment of spinal cord injury. Recently, macrophage migration inhibitory factor (MIF) has been reevaluated as being one of the most important cytokines which act during wound healing, proliferation and differentiation of cells. However, the expression of MIF in the spinal cord has not been investigated yet. PURPOSE: The purpose of this paper is to demonstrate the MIF expression in normal rat spinal cord and to evaluate the kinetics of MIF after spinal cord injury. MATERIALS & METHODS: Female Wistar (280-320 g) rats were studied. Spinal cord injury was made by the clip compression method at the level of C7/Th1 (56 g, For 1 min.). The expression of MIF was examined by immunohistochemistry and northern blot analysis. MIF content in the cerebrospinal fluid (CSF) was measured by enzyme-linked immunosorbent assays (ELISA). Furthermore, to examine the MIF function on neuronal cell, cell proliferation assay (MTS assay) was carried out using PC12, pheochromocytoma cell line, and LN444, glioblastoma cell line, in the presence of anti-MIF monoclonal antibody. RESULTS: MIF stain was positive in normal rat spinal cord white matter. The expression of MIF decreased between 1 hour and 6 hours after injury. It was found to have re-appeared 24 hours after injury. The kinetics of MIF mRNA expression showed reverse-correlation with those of the MIF positive stain. MIF content in CSF was found to be elevated soon after injury. MTS assay suggested that MIF had some proliferative function on neuronal cells. CONCLUSION: MIF exists in the rat white matter. And it's immediately released into the CSF and then re-synthesized 24-hr after injury. MIF shows a cell proliferative function on neuronal cells. These results suggest that MIF plays an important role for secondary spinal cord injury.     

Localization of cyclooxygenase-2 and prostaglandin E2 receptor EP3 in the rat lumbar spinal cord

Cyclooxygenase-2 (COX-2) is now considered to be the major constitutively expressed COX isozyme in the central nervous system. The present immunocytochemical study details localization of COX-2 immunoreactivity in rat spinal cord along with the expression of prostaglandin E2 receptor subtype EP3. Prominent COX-2 staining was observed in the nuclear envelope of neurons throughout the spinal cord, especially in the superficial dorsal horn laminae and motoneurons of lamina IX, as well as in glial cells of the white matter. Expression of EP3 receptor was strictly confined to afferent terminal areas in the superficial dorsal horns.     

Spinal opioid mu receptor expression in lumbar spinal cord of rats following nerve injury

The oxidation of inulin with Pt/C as catalyst was studied. Methyl alpha-D-fructofuranoside was used as a model compound for the monomeric unit of inulin. Oxidation occurred selectively at the C-6 position in a high yield (79%). The rate of oxidation and the degree of oxidation obtained for inulin oligosaccharides decreased upon increase of the chain length of the substrate. Inulin could only be oxidized partially: the oxidation degree obtained was 20% of the primary hydroxy groups for inulin with an average dp 30. Possible explanations for these relatively low conversions are discussed. Adsorption and desorption phenomena appear to play and important role during the oxidation process.     

NMDA receptor activation modulates evoked release of substance P from rat spinal cord

The possible modulation exerted by glutamate on substance P (SP) release from the rat spinal cord has been investigated. The N-methyl-D-aspartate (NMDA) receptor agonist, NMDA (1 microM), increased SP basal outflow by 46.5+/-10.9% (n = 3, P<0.01) without changing the evoked release of the peptide. Conversely, NMDA antagonists but not 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) inhibited both electrically-evoked and capsaicin-induced release of SP. In particular, D-2-amino-5-phosphonopentanoate (D-AP5; 50 microM) inhibited electrically-evoked and capsaicin-induced release of SP by 93+/-2.4% and 93.2+/-3.8% (n = 12, P<0.01), respectively. Functional pharmacological evidence is provided for glutamate exerting a positive feedback on SP release evoked by C fibre stimulation via NMDA receptor activation.