M.A. Ruda

The effects of a non-competitive NMDA receptor antagonist, MK-801, on behavioral hyperalgesia and dorsal horn neuronal activity in rats with unilateral inflammation

&NA; The involvement of NMDA receptors in rats with peripheral inflammation and hyperalgesia was evaluated by administration of the non‐competitive NMDA receptor antagonist, MK‐801. Inflammation and hyperalgesia was induced by intradermal injection of complete Freunds adjuvant (CFA) or carrageenan into the left hind paw. The latency of paw withdrawal from a thermal stimulus was used as a measure of hyperalgesia in awake rats. MK‐801 (1.6 mg/kg, i.p., or 31.5 &mgr;g, intrathecal) significantly attenuated thermal hyperalgesia and reduced its duration in comparison to saline‐injected rats (P < 0.05). The receptive field size of nociceptive‐specific and wide‐dynamic‐range neurons in the superficial and deep spinal dorsal horn recorded 24 h after injection of CFA was significantly reduced to 73 ± 6% (P < 0.05, n = 8) and 74 ± 4% (P < 0.05, n = 8) of control values, respectively, by a cumulative dose of 3 mg/kg of MK‐801 (i.v.). MK‐801 (2 mg/kg) prevented the expansion of the receptive fields of dorsal horn neurons recorded 5 ± 0.4 h (n = 5) after intradermal injection of CFA as compared to saline‐injected rats (P < 0.05). MK‐801 had no significant effect on receptive field size of dorsal horn neurons in rats without CFA‐induced inflammation but blocked a transient expansion of the receptive fields induced by 1 Hz, C‐fiber intensity electrical stimulation of the sciatic nerve. The background activity and noxious heat‐evoked response of dorsal horn neurons in rats with CFA‐induced inflammation were primarily inhibited and noxious pinch‐evoked activity was both facilitated and inhibited by the administration of MK‐801. These results support the hypothesis that NMDA receptors are involved in the dorsal horn neuronal plasticity and behavioral hyperalgesia that follows peripheral tissue inflammation.

The intrathecal administration of excitatory amino acid receptor antagonists selectively attenuated carrageenan-induced behavioral hyperalgesia in rats

A single unilateral injection of carrageenan (4.5-6.0 mg in 0.15-0.20 ml saline) into the rat hindpaw induced behavioral hyperalgesia as evidenced by a significant reduction in hindpaw withdrawal latency to a noxious thermal stimulus. The involvement of N-methyl-D-aspartate (NMDA) receptors in this model of hyperalgesia was examined by intrathecal administration of the selective excitatory amino acid (EAA) receptor antagonists: (+/-)-2-amino-5-phosphonopentanoic acid (AP-5), (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), ketamine hydrochloride (ketamine), 7-chlorokynurenic acid (7-Cl kynurenic acid), and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The effects of dizocilpine maleate (MK-801) were studied under the same conditions and published previously (Ren et al., 1992) and the data are presented for comparison. While the withdrawal latencies of the non-injected paws and of the paws of naive rats were not significantly affected by application of the EAA receptor antagonists at doses tested, the paw withdrawal latencies of the carrageenan-injected paws were elevated dose dependently. The rank order of potency of these agents to reduce hyperalgesia was: MK-801 greater than or equal to AP-5 greater than or equal to CPP = 7-Cl kynurenic acid = ketamine much greater than CNQX greater than 0. In contrast, intrathecal injection of the opioid receptor agonists, [D-Ala2,MePhe4,Gly-ol5]enkephalin (DAMGO, mu-selective) and [D-Pen2,D-Pen5] enkephalin (DPDPE, delta-selective), produced antinociception in both injected and non-injected paws. DAMGO was much more potent, while DPDPE was less potent, than MK-801.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuropathic pain from an experimental neuritis of the rat sciatic nerve.

Painful peripheral neuropathies involve both axonal damage and an inflammation of the nerve. The role of the latter by itself was investigated by producing an experimental neuritis in the rat. The sciatic nerves were exposed at mid-thigh level and wrapped loosely in hemostatic oxidized cellulose (Oxycel) that on one side was saturated with an inflammatory stimulus, carrageenan (CARRA) or complete Freunds adjuvant (CFA), and on the other side saturated with saline. In other rats, a myositis was created by implanting Oxycel saturated with CFA into a pocket made in the biceps femoris at a position adjacent to where the nerve was treated. Pain-evoked responses from the plantar hind paws were tested before treatment and daily thereafter. Statistically significant heat- and mechano-hyperalgesia, and mechano- and cold-allodynia were present on the side of the inflamed nerve (CARRA or CFA) for 1-5 days after which responses returned to normal. There were no abnormal pain responses on the side of the saline-treated nerve, and none in the rats with the experimental myositis. The abnormal pain responses were inhibited by N-methyl-D-aspartate receptor blockade with MK-801, but were relatively resistant to the dose of morphine tested (10 mg/kg). Light microscopic examination of CARRA-treated nerves, harvested at the time of peak symptom severity, revealed that the treated region was mildly edematous and that there was an obvious endoneurial infiltration of immune cells (granulocytes and lymphocytes). There was either a complete absence of degeneration, or the degeneration of no more than a few tens of axons. Immunocytochemical staining for CD4 and CD8 T-lymphocyte markers revealed that both cell types were present in the epineurial and endoneurial compartments. The endoneurial T-cells appeared to derive from the endoneurial vasculature, rather than from migration across the nerve sheath. We conclude that a focal inflammation of the sciatic nerve produces neuropathic pain sensations in a distant region (the ipsilateral hind paw) and that this is not due to axonal damage. The neuropathic pain is specific to inflammation of the nerve because it was absent in animals with the experimental myositis and in those receiving sham-treatment. These results suggest that an acute episode of neuritis-evoked neuropathic pain may contribute to the genesis of chronically painful peripheral neuropathies, and that a chronic (or chronically recurrent) focal neuritis might produce neuropathic pain in the absence of significant (or clinically detectable) structural damage to the nerve. The model that we describe is likely to be useful in the study of the neuroimmune factors that contribute to painful peripheral neuropathies.

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.

Neurochemistry and neural circuitry in the dorsal horn.

Publisher Summary This chapter discusses the wealth of immunocytochemical data about the neurochemicals (the classical neurotransmitters and the neuropeptides) that are found in the mammalian spinal cord. The chapter focuses on those regions of the spinal cord that are likely to be concerned with somatosensation rather than movement or autonomic function. Relay nuclei in the central nervous system characteristically include three major functional components: the terminals of primary afferent neurons that relay signals from distant sites; intrinsic neurons, which include output neurons that transmit signals to distant sites, and local circuit neurons that modify sensory transmission in a restricted region; and the axon terminals of extrinsic neurons arising from distant sites. The spinal dorsal horn is no exception to this common organization. The progress which has been made toward integrating the immunocytochemical data with the emerging picture of the spinal circuitry derived from anatomical and electrophysiological work are also discussed.

Primary sensory neurons exhibit altered gene expression in a rat model of neuropathic pain

&NA; Using a number of complementary anatomical and molecular techniques, we studied the effects of chronic constriction injury (CCI), a model of partial nerve injury that elicits behavioral hyperalgesia, on primary sensory neurons in the rat. Dorsal root ganglia taken from animals with CCI were analyzed for alterations in mRNA levels encoding growth‐associated protein‐43 (GAP‐43), calcitonin gene‐related peptide (CGRP), galanin (GAL), neuropeptide Y (NPY), substance P (SP), and vasoactive intestinal polypeptide (VIP). We found that GAP‐43 expression increased 3‐fold, peaking between 7 and 14 days after development of the CCI. However, within this same 7–14 day time frame, both CGRP and SP mRNAs fell to half their normally abundant constitutive levels of expression. The most dramatic change in expression occurred for GAL, NPY and VIP mRNAs which all rose rapidly (day 3) from non‐detectable levels. Similar alterations in gene expression have been described after complete sciatic nerve transection or crush.

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.

Axonal and transneuronal transport of wheat germ agglutinin demonstrated by immunocytochemistry

Immunocytochemical methods at the light and electron microscopic level were used to examine the anterograde and retrograde transport of wheat germ agglutinin. Following anterograde transport to sensory nerve terminals in the central nervous system, wheat germ agglutinin can be localized to neuronal cell bodies in the area of terminal labeling. The transneuronal transport of wheat germ agglutinin demonstrates the existence of a mechanism whereby axonally transported glycomacromolecules might exert neurotrophic effects on postsynaptic cells.

Ultrastructural characterization of axonal endings in the substantia gelatinosa which take up [3H]serotonin

Serotonergic axonal endings in layers I and II of the dorsal horn of the medulla were identified by autoradiography. In adult cats, pretreated with a monoamine oxidase inhibitor, tritiated serotonin ([3H]5-HT) was topically applied onto the surface of the caudal medulla. Light autoradiographs from 1 micrometer sections demonstrated silver grains in both layers I and II. In EM autoradiographs, two categories of axonal endings were labeled by [3H]5-HT uptake: dome-shaped endings which form a single synapse and scalloped endings which form multiple synapses. Each category was further divided into several types based on morphological criteria. The [3H]5-HT-labeled endings synapse primarily on small caliber dendritic shafts and spines, with the dome-shaped endings forming both symmetrical and asymmetrical synapses and the scalloped endings forming only asymmetrical synapses. Dome-shaped endings were most common and two types were found in layers I and II while a third type was found only in layer II. Layer I contained a single type of scalloped ending while layer II contained three types of scalloped endings. In a series of experiments designed to provide another approach to identifying serotonergic endings, 5,6-dihydroxytryptamine, a serotonin neurotoxin, was either topically applied onto the caudal medulla or injected into the fourth ventricle. Following treatment with the neurotoxin, blackened degenerating dome-shaped and scalloped endings similar to those labeled in the [3H]5-HT uptake experiments were found in layers I and II. The presence of serotonergic endings in layer I suggests that some of these endings synapse on the dendrites of layer I projection neurons where they may inhibit the output of the projection neuron directly. Serotonergic endings in layer II may modulate the activity of layer II interneurons by synapsing directly on these interneurons. The interneurons in layer II may function by mediating the transfer of inputs from primary endings in these layers to layer I projection neurons.

Enkephalin immunoreactive stalked cells and lamina IIb islet cells in cat substantia gelatinosa

Neurons in lamina II of the lumbar spinal cords of colchicine-pretreated cats were stained immunocytochemically for enkephalin. Two morphological types were found. The most common type had the light microscopic characteristics of stalked cell. The other type was found in the deep part of the lamina and had the light and electron microscopic characteristics of the lamina IIb islet cell.