A biophysical model of cell evolution after cytotoxic treatments: Damage, repair and cell response.

We present a theoretical agent-based model of cell evolution under the action of cytotoxic treatments, such as radiotherapy or chemotherapy. The major features of cell cycle and proliferation, cell damage and repair, and chemical diffusion are included. Cell evolution is based on a discrete Markov chain, with cells stepping along a sequence of discrete internal states from 'normal' to 'inactive'. Probabilistic laws are introduced for each type of event a cell can undergo during its life: duplication, arrest, senescence, damage, reparation, or death. We adjust the model parameters on a series of cell irradiation experiments, carried out in a clinical LINAC, in which the damage and repair kinetics of single- and double-strand breaks are followed. Two showcase applications of the model are then presented. In the first one, we reconstruct the cell survival curves from a number of published low- and high-dose irradiation experiments. We reobtain a very good description of the data without assuming the well-known linear-quadratic model, but instead including a variable DSB repair probability. The repair capability of the model spontaneously saturates to an exponential decay at increasingly high doses. As a second test, we attempt to simulate the two extreme possibilities of the so-called 'bystander' effect in radiotherapy: the 'local' effect versus a 'global' effect, respectively activated by the short-range or long-range diffusion of some factor, presumably secreted by the irradiated cells. Even with an oversimplified simulation, we could demonstrate a sizeable difference in the proliferation rate of non-irradiated cells, the proliferation acceleration being much larger for the global than the local effect, for relatively small fractions of irradiated cells in the colony.

Level of macroautophagy drives senescent keratinocytes into cell death or neoplastic evasion.

Senescence is a non-proliferative state reached by normal cells in response to various stresses, including telomere uncapping, oxidative stress or oncogene activation. In previous reports, we have highlighted that senescent human epidermal keratinocytes have two opposite outcomes: either they die by autophagic programmed cell death or they evade in the form of neoplastic postsenescence emergent (PSNE) cells. Herein, we show that partially reducing macroautophagy in senescent keratinocytes using 3-methyl adenine or anti-Atg5 siRNAs increases the PSNE frequency, suggesting that senescent keratinocytes have to escape autophagic cell death to generate PSNE cells. However, totally inhibiting macroautophagy impairs PSNE and leads to a huge accumulation of oxidative damages, indicating that senescent keratinocytes need to achieve quality-control macroautophagy for PSNE to occur. In accordance, we demonstrate that the progenitors of PSNE cells display a level of macroautophagy slightly lower than that of the average senescent population, which is directly dictated by their level of reactive oxygen species, their level of upregulation of MnSOD, their level of activation of NF-κB transcription factors and their level of dysfunctional mitochondria. Macroautophagy thus has antagonistic roles during senescence, inducing cell death or promoting neoplastic transformation, depending on its level of activation. Taken together, these data suggest that levels of oxidative damages and ensuing macroautophagic activity could be two main determinants of the very initial phases of neoplastic transformation by senescence evasion.

cRel induces mitochondrial alterations in correlation with proliferation arrest.

We have previously shown that overexpressing cRel, a transcription factor of the Rel/NF-kappa B family, concomitantly inhibits proliferation of HeLa cells and makes them resistant against TNF alpha-induced apoptosis. Both effects rely on the upregulation of the manganese superoxide dismutase (MnSOD), a mitochondrial enzyme that converts O(2)(*-) in H(2)O(2). Here we describe additional alterations induced by cRel, namely mitochondrial clustering and accumulation of dense dark granules near the nucleus. These changes preferentially occur in cells that display a sustained cRel expression in the nucleus and that are cell-cycle arrested. As the cell-cycle arrest, these changes are reproduced by directly overexpressing MnSOD or by treating cells with H(2)O(2), suggesting they are due to MnSOD induction and ensuing H(2)O(2) accumulation. We propose that mitochondria cluster because they are damaged by the H(2)O(2) they overproduce. They would then be autophagocytosed and degraded in secondary lysosomes. In support of this scenario, we documented the occurrence of oxidative damage and the presence of lysosomes in the area of mitochondrial clustering. In addition, we identified the dense dark granules as lipofuscin, based on their autofluorescence. Lipofuscin could directly originate from the mitochondrial degradation products that would aggregate and become indigestible because of the presence of H(2)O(2) in the secondary lysosomes. Altogether, our findings show that cRel overexpression in HeLa cells creates, via the induction of MnSOD, an oxidative injury that culminates in mitochondrial degeneration, proliferation blockage, and resistance against TNF alpha-induced apoptosis.

Rel/NF-kappaB transcription factors protect against tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by up-regulating the TRAIL decoy receptor DcR1.

Rel/nuclear factor (NF)-kappaB transcription factors play a major role in the regulation of programmed cell death. A few anti-apoptotic Rel/NF-kappaB target genes have been characterized; they act either downstream in the apoptotic pathway or upstream, for example at the tumor necrosis factor (TNF) receptor level. We found using DNA arrays, reverse transcription-polymerase chain reaction, and immunofluorescence that Rel/NF-kappaB factors up-regulate DcR1, a receptor for TNF-related apoptosis-inducing ligand (TRAIL), a cytokine of the TNF family that induces apoptosis in tumor cells. Four related receptors bind TRAIL, two death receptors (DR4 and DR5) that signal apoptosis and two decoy receptors (DcR1 and DcR2) that act as dominant negative inhibitors of TRAIL-mediated apoptosis. DcR1 is devoid of an intracellular domain and is anchored at the cell surface membrane by a glycophospholipid. Our results indicate that overexpression of cRel or activation of endogenous Rel/NF-kappaB factors by TNFalpha in HeLa cells up-regulates DcR1 without changing the expression of DcR2, DR4, and DR5 and makes cells resistant against TRAIL-induced apoptosis. This resistance is a consequence of DcR1 up-regulation, because it was abolished when DcR1 was removed from the cell surface by a phosphatidylinositol phospholipase C. Therefore, Rel/NF-kappaB transcription factors could regulate the sensitivity of cells to TRAIL, by controlling the ratio of TRAIL-decoy to -death receptors.

Antiproliferative and antiapoptotic effects of crel may occur within the same cells via the up-regulation of manganese superoxide dismutase.

Rel/nuclear factor kappaB transcription factors were shown to have either pro- or antiapoptotic as well as pro- or antiproliferative functions, and it is often assumed that the outcome of their activation depends on the cell type or cellular context. Inconsistent with this assumption, we show here that cRel is able in one cell type to inhibit proliferation, protect against apoptosis induced by tumor necrosis factor alpha (TNF-alpha) + cycloheximide (CHX), and increase the basal rate of apoptosis. Both the effects of proliferation inhibition and protection against TNF-alpha + CHX-induced apoptosis are massive and occur in the same cells. Using reverse transcription-PCR, Western blot and immunofluorescence, and transactivation assays, we found that the manganese superoxide dismutase (MnSOD), an enzyme that converts O2*- in H2O2, is up-regulated by cRel through a kappaB site in intron 2. Inhibition of MnSOD induction by antisense oligonucleotides and overexpression of MnSOD respectively reverts and mimics both the antiproliferative and antiapoptotic effects of cRel, suggesting that they both occur via the induction of this gene. On one hand, MnSOD could improve the efficiency of cRel-overexpressing cells in eliminating toxic O2*- produced on TNF-alpha treatment, explaining why they escape TNF-alpha-induced apoptosis. On the other hand, cRel-overexpressing cells should accumulate H2O2. We present evidence linking this H2O2 accumulation to the proliferation arrest induced by cRel. Therefore, different effects on proliferation and apoptosis could arise from the induction of MnSOD and thus coexist in cRel-overexpressing cells.

Spinal opioid analgesia: how critical is the regulation of substance P signaling?

Although opioids can reduce stimulus-evoked efflux of Substance P (SP) from nociceptive primary afferents, the consequences of this reduction on spinal cord nociceptive processing has not been studied. Rather than assaying SP release, in the present study we examined the effect of opioids on two postsynaptic measures of SP release, Fos expression and neurokinin-1 (NK-1) receptor internalization, in the rat. The functional significance of the latter was first established in in vitro studies that showed that SP-induced Ca(2+) mobilization is highly correlated with the magnitude of SP-induced NK-1 receptor internalization in dorsal horn neurons. Using an in vivo analysis, we found that morphine had little effect on noxious stimulus-evoked internalization of the NK-1 receptor in lamina I neurons. However, internalization was reduced when we coadministered morphine with a dose of an NK-1 receptor antagonist that by itself was without effect. Thus, although opioids may modulate SP release, the residual release is sufficient to exert maximal effects on the target NK-1 receptors. Morphine significantly reduced noxious stimulus-induced Fos expression in lamina I, but the Fos inhibition was less pronounced in neurons that expressed the NK-1 receptor. Taken together, these results suggest that opioid analgesia predominantly involves postsynaptic inhibitory mechanisms and/or presynaptic control of non-SP-containing primary afferent nociceptors.

Neurons in the dorsal column white matter of the spinal cord: complex neuropil in an unexpected location.

It is common to think of gray matter as the site of integration in neural circuits and white matter as the wires that connect different groups of neurons. The dorsal column (DC) white matter, for example, is the spinal cord axonal pathway through which a topographic map of the body is conveyed to the somatosensory cortex. We now describe a network of neurons located along the midline of the DCs. The neurons are present in several mammals, including primates and birds, and have a profuse dendritic arbor that expresses both the neuron-specific marker, microtubule-associated protein-2, and the neurokinin-1 receptor, a target of the neuropeptide, substance P. Electron microscopy and double immunostaining for synaptophysin and a marker of gamma-aminobutyric acid-ergic terminals documented a rich synaptic input to these neurons. Finally, injection of a gamma-aminobutyric acid type A receptor antagonist or of substance P into the cerebrospinal fluid of the rat spinal cord induced Fos expression and internalization of the neurokinin-1 receptor in these neurons, respectively, indicating that the DC neurons are under tonic inhibitory control and can respond to neurotransmitters that circulate in the cerebrospinal fluid.

Expression of transcription factor c-Rel and apoptosis occurrence in polydactylous and syndactylous limb buds of the talpid3 mutant chick embryo.

The chicken proto-oncogene c-rel encodes a transcription factor of the Rel/NF-kappa B family. We have previously shown that c-rel mRNAs accumulate in different types of apoptotic cells of the chick embryo, especially in mesenchymal cells within the four cell death areas of the limb bud: the anterior and posterior necrotic zones, the opaque patch and the interdigital necrotic zones. This study aimed to further establish the involvement of c-Rel in apoptosis of the developing limb by investigating its expression in the talpid3 mutant which was originally shown to be defective in apoptosis. However, our preliminary examinations highlighted the apparent presence of apoptotic cells in talpid3 embryos. Hence, we performed a systematic study of the occurrence of apoptosis in mutant and control embryos by the TUNEL method. The results revealed that apoptosis does occur in talpid3 embryos but with altered spatial and temporal patterns. This suggests that the talpid3 mutation does not affect a gene involved in apoptosis per se but rather in the determination of the pattern of apoptosis. Neither the expression of c-Rel nor that of its I kappa B alpha inhibitor are grossly modified in talpid3 limb buds, suggesting that the talpid3 mutation does not affect any of these genes. They are mostly expressed in epidermal, endodermal and striated muscle cells in control and in talpid3 limb buds as well. C-Rel was also detected in some scarce mesenchymal cells that could be apoptotic, in both control and mutant embryos. The only slight difference between control and talpid3 limbs lies in the perichondrium which is not fully differentiated in talpid3 embryos: c-Rel and I kappa B alpha are only faintly expressed in talpid3 perichondrial cells, whereas they are both detected in control perichondrial cells. Taken together, these results suggest that c-Rel could participate in several developmental processes, especially in the differentiation of perichondrial cells, besides its already documented involvement in apoptosis and haematopoeisis.

The avian transcription factor c-Rel is expressed in lymphocyte precursor cells and antigen-presenting cells during thymus development.

Transcription factors of the Rel/NF-kappaB family are widely involved in the immune system. In this study, we investigate the in vivo expression of the avian protein c-Rel in the T-cell lineage during thymus development. The majority of thymocytes do not express the c-Rel protein. However, lymphocyte precursor cells that colonize the thymus express the c-Rel protein shortly after their homing in the organ and before they begin to differentiate. c-Rel is also detected in different subsets of antigen-presenting cells such as epithelial cells, dendritic cells, and macrophages. In vitro studies have shown that Rel/NF-kappaB proteins are sequestered in an inactive form in the cytoplasm by interaction with the IkappaBalpha inhibitory protein. By immunocytochemistry, we show that in vivo c-Rel is localized in the cytoplasm of antigen-presenting cells but in both the cytoplasm and nucleus of lymphocyte precursor cells. The cytoplasmic localization of c-Rel in antigen-presenting cells correlates with a high expression of IkappaBalpha, whereas the nuclear localization of c-Rel in lymphocyte precursor cells correlates with a much lower expression of IkappaBalpha. These results suggest that c-Rel might be constitutively activated in lymphocyte precursor cells.

The contribution of supraspinal, peripheral and intrinsic spinal circuits to the pattern and magnitude of Fos-like immunoreactivity in the lumbar spinal cord of the rat withdrawing from morphine.

Withdrawal from morphine evokes increases in Fos-like immunoreactivity in the spinal cord, particularly in the superficial dorsal horn, laminae I/II. To determine the origin of the increased Fos-like immunoreactivity, we selectively targeted central or peripheral opioid receptors with naloxone-methiodide, an antagonist that does not cross the blood-brain barrier, or induced withdrawal after eliminating possible sources of input to the superficial dorsal horn. To induce tolerance, we implanted rats with morphine or placebo pellets (75 mg, six pellets over three days). On day 4, withdrawal was precipitated and after 1 h, the rats were killed, their spinal cords removed and 50 microm transverse sections of the spinal cord immunoreacted with a rabbit polyclonal antiserum directed against the Fos protein. In placebo-pelleted rats, none of the different procedures, viz. spinal transection, unilateral dorsal rhizotomy (L4-S2), neonatal capsaicin treatment or direct intrathecal opioid antagonist injection, induced expression of the Fos protein. However, both spinally transected and rhizotomized withdrawing animals showed significant increases in Fos-like immunoreactivity in laminae I/II, compared to intact withdrawing rats. Neonatal treatment with capsaicin, which eliminates C-fibres, did not alter Fos-like-immunoreactivity. Selective withdrawal of morphine from peripheral opioid receptors by naloxone-methiodide did not induce Fos-like immunoreactivity in the lumbar spinal cord greater than that recorded in nonwithdrawing rats. However, intrathecal injection of naloxone-methiodide increased Fos-like immunoreactivity in laminae I/II and the ventral horn to a greater extent than did subcutaneous injection of naloxone. We hypothesize that the increased Fos expression after systemic withdrawal in spinally-transected rats results from a loss of descending inhibitory control that is activated during withdrawal. The increase in withdrawal-induced Fos-like immunoreactivity after rhizotomy may be secondary to loss of inhibitory controls exerted by large diameter primary afferents or to deafferentation-induced reorganization in the dorsal horn. Since capsaicin did not alter the magnitude of Fos-like immunoreactivity in withdrawing rats, we conclude that hyperactivity of opioid receptor-laden C-fibres is not a necessary contributor to the withdrawal-induced increase in Fos-like immunoreactivity in laminae I and II. Taken together with the results recorded after intrathecal injection of naloxone-methiodide in tolerant rats, we conclude that the pattern of lumbar spinal cord Fos expression following systemic withdrawal is primarily a consequence of increased activity in opioid receptor-containing circuits intrinsic to the dorsal horn and that the magnitude of Fos expression is normally dampened by supraspinal and primary afferent-derived inhibitory inputs.

The avian transcription factor c-Rel is induced and translocates into the nucleus of thymocytes undergoing apoptosis.

This study investigates the involvement of the avian transcription factor c-Rel in thymocyte apoptosis occurring either in vivo or in organotypic culture. In vivo, only a few cortical thymocytes express the c-Rel protein. Their number, localization and morphology resemble that of apoptotic cells evidenced by TUNEL staining. In organotypic culture, the expression of c-Rel is induced in medullary thymocytes as apoptosis is triggered. This induction would be post-transcriptional since no increase in the c-rel gene expression is detected. Moreover, c-Rel translocates into the nucleus of medullary thymocytes during the time course of apoptosis. This translocation is preceded by a decrease in ikba expression, the gene which encodes the avian homologue of IkappaBalpha. Altogether these results suggest that the proto-oncogene c-rel could take an active part in apoptosis of cortical thymocytes occurring in vivo during T-cell selection as well as in experimentally-induced apoptosis of medullary thymocytes.

The differential contribution of capsaicin-sensitive afferents to behavioral and cardiovascular measures of brief and persistent nociception and to Fos expression in the formalin test.

Intraplantar injection of dilute formalin evokes brief (Phase 1) and persistent (Phase 2) increases in primary afferent activity, pain behavior, and cardiovascular responses, and induces spinal cord Fos-like immunoreactivity (Fos-LI). Although previous studies demonstrated that the destruction of small diameter primary afferents with neonatal capsaicin treatment decrease formalin-evoked nociception, these studies only evaluated behavioral responses, and did not distinguish between Phase 1 and 2. To address these questions, we simultaneously evaluated formalin-evoked pain behavior (flinching of the afflicted paw), cardiovascular responses (heart rate and mean arterial pressure), and lumbar spinal cord Fos expression in control rats and in rats treated with capsaicin (100 mg/kg) one day postpartum. We found that neonatal capsaicin-treated rats, compared to controls, exhibited similar cardiovascular responses and slightly less flinching behavior during Phase 1. During Phase 2, however, capsaicin-treated rats exhibited 59% less flinching and 45% smaller heart rate responses. Also, in capsaicin-treated rats, we counted 59% fewer Fos-labeled neurons in the spinal cord. These results indicate that capsaicin-sensitive afferents contribute to formalin-evoked behavioral and cardiovascular responses and to spinal cord neuronal responses. The differential effect of neonatal capsaicin on nociception during Phase 1 and Phase 2 suggests that sensitization mechanisms during Phase 1 do not contribute to the magnitude of nociceptive responses during Phase 2.

Differential contribution of the two phases of the formalin test to the pattern of c-fos expression in the rat spinal cord: studies with remifentanil and lidocaine.

Injection of formalin in the rat hindpaw produces two phases of nociceptive behavior. Although it is generally agreed that the first phase results from direct chemical activation of nociceptive primary afferent fibers, the factors that contribute to the second phase are not established. In the present study, we monitored the expression of the c-fos protein to evaluate whether the pattern of activity of dorsal horn neurons differs as a result of ongoing afferent activity during the two phases. To selectively block the first or second phase, we respectively used remifentanil, a potent and short acting opiate agonist, and QX-314, a quaternary derivative of lidocaine, which does not cross the blood brain barrier. We also evaluated the effect of eliminating nociceptive behavior in both phases using both remifentanil and lidocaine or a combination of local anesthetics, bupivicaine and quaternary lidocaine. In all groups, formalin (5%, 50 microliters) was injected subcutaneously into the plantar surface of the hindpaw. To assess the nociceptive behavior produced by formalin, we monitored the number of flinches. Injection of remifentanil during the first phase completely blocked the first phase formalin-evoked nociceptive behavior, and had no effect on the second phase. Injection of lidocaine during the interphase completely blocked second phase nociceptive behavior. As expected, when remifentanil was administered during the first phase and lidocaine during the second phase, all formalin-evoked nociceptive behavior was blocked. The same was true for rats that received injections of bupivicaine and lidocaine during phases 1 and 2, respectively. In laminae I-II of the L4-L5 segment, the magnitude of the decrease in Fos expression was comparable for remifentanil (26.5%) and lidocaine (27.3%); the decrease was greater when both remifentanil and lidocaine were administered (50.5%), and even greater when bupivicaine and lidocaine were used (74.2%). In laminae V-VI, remifentanil, by itself, decreased c-fos expression by 39.4%; for lidocaine alone, the decrease was 58.4%. We did not observe further significant decreases when both remifentanil and lidocaine, or bupivacaine and lidocaine were injected (69.7% and 74.6%, respectively). Our results not only provide strong evidence that activity during the second phase is necessary for maintaining the maximal expression of c-fos in the spinal cord, but also reveal significant regional differences in the central patterns of activity generated during the two phases. These results also confirm our previous reports that c-fos expression is not eliminated when the behavioral manifestation of the noxious stimulus is completely blocked.

Contribution of sacral spinal cord neurons to the autonomic and somatic consequences of withdrawal from morphine in the rat.

In this study, we monitored Fos-like immunoreactivity in the sacral spinal cord to identify neurons that are likely to contribute to the autonomic manifestations of opioid antagonist-precipitated withdrawal in morphine-tolerant rats. Injection of systemic antagonist increased the Fos-like immunoreactivity throughout the first sacral segment, particularly in laminae I/II, X, and in the sacral parasympathetic nucleus (SPN). Selective peripheral withdrawal, with a hydrophilic antagonist that does not cross the blood-brain barrier (BBB), induced diarrhea, but no other withdrawal signs were evident. Compared to rats that withdrew systemically, peripherally withdrawal evoked significantly less Fos-like immunoreactivity in laminae V/VI, X and the SPN. By contrast, selective spinal withdrawal, by intrathecal injection of an opioid antagonist that does not cross the BBB, provoked hyperactivity of the hindlimbs and tail, but no diarrhea. These animals demonstrated significantly increased Fos-like immunoreactivity in laminae I/II, V/VI, the SPN, and the ventral horn compared to rats that withdrew systemically. Animals treated neonatally with capsaicin, to eliminate C-fiber input, demonstrated withdrawal behavior similar to intact withdrawing rats, except that the capsaicin-pretreated rats had significantly greater weight loss. However, this group had less Fos-like immunoreactivity in laminae V/VI, X and SPN compared to the intact withdrawing rats. These data suggest that withdrawal from morphine evokes hyperactivity of sacral neurons, particularly those involved in regions that process nociceptive and autonomic information. Peripheral withdrawal is sufficient to induce diarrhea, but it does not fully explain the associated weight loss. Unmyelinated primary afferents may contribute a tonic peripheral inhibition of circuits that regulate gut motility and intestinal fluid transport. Taken together, these data suggest that chronic exposure to opioids induces a latent sensitization in sacral cord neurons that can be manifested as neuronal hyperactivity during withdrawal; this mechanism may underlie withdrawal-induced hyperalgesia and gut hypermotility.

c-Fos expression in the spinal cord and pain-related symptoms induced by chronic arthritis in the rat are prevented by pretreatment with Freund adjuvant.

We previously showed that pathological adjuvant-induced arthritis (AIA), a chronic pain model, is associated with an increase in the number of Fos-like immunoreactivity (Fos-LI) in lumbar spinal cord neurons; maximal Fos-LI corresponds to the peak of pathological and behavioral signs of the disease. On the other hand, AIA is prevented in rats that have previously been injected with diluted complete Freund adjuvant (CFA). Here, the effects of pretreatment with CFA were studied in parallel on both pathological parameters and pain-related tests, and on Fos-LI, 3 weeks after the inoculation of the arthritogenic solution during the acute phase of the disease, that is, when hyperalgesia is maximal. One group of rats (vaccinated group) was pretreated with diluted CFA, 3 weeks and 1 week before inoculation (AIA) (with the concentrated arthritogenic solution), while a second group (arthritic group) was pretreated with the vehicle for the CFA solution and then inoculated with the concentrated arthritogenic solution. Control groups consisted of rats treated with various combinations of either saline or vehicle solutions during the pretreatment periods or at the time of inoculation. The arthritic group developed all pathological symptoms of AIA (i.e., loss of weight, difficulties in walking, increases in paw diameter, and decreases in the vocalization thresholds to mechanical pressure of the hindpaw, and finally an increase in the vocalization response to flexion and extension). In contrast, the vaccinated group did not develop AIA symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of opioids and non-opioids on c-Fos-like immunoreactivity induced in rat lumbar spinal cord neurons by noxious heat stimulation.

This study evaluated Fos-like immunoreactivity in rat lumbar spinal cord neurons following peripheral noxious heat stimulation and the modifications induced by pharmacological agents. Under urethane anaesthesia, the hindpaw was stimulated by dipping it in a regulated temperature bath at various temperatures (44-65 degrees C) and for various durations (5 s to 2 min). There was no Fos-like immunoreactivity in lumbar spinal cord neurons when the paw was stimulated at 44 degrees C for 15 s. From 46 to 52 degrees C, the number of Fos-like immunoreactivity neurons increased with increasing stimulation temperature, but was decreased at 65 degrees C as compared to 52 degrees C. At 52 degrees C, the number of Fos-like immunoreactivity neurons increased with the duration of stimulation. Fos-like immunoreactive neurons in the L4 segment were almost exclusively located in laminae I-II. On the basis of the results of the latter experiments, we chose a stimulation of 52 degrees C for 15 s to perform pharmacological investigations. The number of Fos-like immunoreactive neurons induced by the heat stimulation was significantly decreased by pretreatment with morphine (42, 64 and 75% decrease as compared to control values after 2.5, 5 and 7.5 mg/kg i.v. respectively), and these effects were blocked by naloxone. When various stimulation intensities (46-52 degrees C) were used, the effects of morphine (5 mg/kg i.v.) were most marked when the temperature was highest. In morphine-tolerant rats, morphine (5 mg/kg i.v.) was half as potent in decreasing Fos-like immunoreactivity induced by the heat stimulation than in non-tolerant rats. RB 101, a systemically active mixed inhibitor of enkephalin-metabolising enzymes, significantly decreased Fos-like immunoreactivity induced by heat stimulation (19, 29 and 48% decreases as compared to control values at 10, 20 and 40 mg/kg i.v. respectively) and these effects were blocked by naloxone. Aspirin (150 mg/kg i.v.), proacetaminophen (300 mg/kg i.v.) and tizanidine, a centrally acting myorelaxant (0.25-1 mg/kg i.v.), had no effect on the number of Fos-like immunoreactivity neurons induced by heat stimulation. The use of immunochemistry of the c-Fos protein as a pharmacological test in order to gauge antinociceptive effects at the dorsal horn level is discussed.

Intense cold noxious stimulation of the rat hindpaw induces c-fos expression in lumbar spinal cord neurons.

This study evaluated Fos-like immunoreactivity in lumbar spinal cord neurons following intense cold stimulation and then the modifications induced by opioid administration. Under urethane anaesthesia, the rat's right foot was stimulated by holding it in a regulated temperature bath at 15, 10, 0, -10, -15, -17.5 or -20 degrees C. There was no or little Fos-like immunoreactivity in lumbar spinal cord neurons when the paw was at temperatures between 15 and -10 degrees C (0-5 Fos-like immunoreactive neurons/section). The threshold to induce consistent c-fos expression was -15 degrees C. From -15 to -20 degrees C, the number of Fos-like immunoreactive neurons increased with decreases in temperature. At -20 degrees C, Fos-like immunoreactive neurons were numerous in L3 and L4 segments, in laminae I-II (approximately 60 Fos-like immunoreactive neurons/section) and to a lesser extent in laminae V-VI (approximately 20). Almost no Fos-like immunoreactivity was present in laminae III-IV (< 5). At -20 degrees C, the number of Fos-like immunoreactive neurons increased with the duration of the stimulation. The number of Fos-like immunoreactive neurons induced by the cold stimulation temperatures was significantly decreased by pretreatment with 10 mg/kg s.c. morphine and moderately decreased by 5 mg/kg s.c. This effect was antagonized by the combined administration of morphine (10 mg/kg s.c.) and naloxone (2 mg/kg s.c.). Naloxone (2 mg/kg s.c.) significantly increased the number of Fos-like immunoreactive neurons induced by -20 degrees C as compared to saline-injected rats. This study showed that Fos-like immunoreactivity distribution is in good agreement with the location of neurons receiving noxious inputs and that the threshold to induce c-fos expression with cold was unexpectedly low at -15 degrees C. Taking into account, on the one hand, previous investigations using the same technique using noxious heat stimulation and, on the other hand, electrophysiological and psychophysiological studies using cold stimulation in animals and humans, our results suggest that Fos-like immunoreactivity induced by extremely cold stimulation, which seems to reproduce frostbite, may reflect activation of nociceptors due to vasoconstriction.

High levels of c-rel expression are associated with programmed cell death in the developing avian embryo and in bone marrow cells in vitro.

To determine the physiological processes in which the transcription factor c-Rel may act, we have examined its pattern of expression in the avian embryo by in situ hybridization. These studies showed that c-rel is expressed ubiquitously at low levels and at high levels in isolated cells undergoing programmed cell death by apoptosis or autophagocytosis. To further establish a functional link between expression of c-rel and cell death, we examined the biological consequences of c-rel overexpression in vitro. In primary avian fibroblasts, overexpression of c-rel leads to transformation and dramatic life span extension. In contrast, bone marrow cells expressing high levels of c-rel undergo a process of programmed cell death displaying features of both apoptosis and autophagocytic cell death. Thus, these experiments suggest a critical role for c-rel not only in the control of cell proliferation, but also in the induction of cell death.

C-fos expression in rat lumbar spinal cord following peripheral stimulation in adjuvant-induced arthritic and normal rats.

Our previous data reported a maximal expression of the c-fos immediate-early gene in the lumbar spinal cord of the non-stimulated polyarthritic rat neurons, three weeks after Freund's adjuvant injection. The present study utilises c-fos expression to judge the reactivity of spinal neurons to calibrated mechanical pressure applied to the ankle joint, in both normal and arthritic rats under ketamine anesthesia. The results indicate that the number of Fos-like immunoreactive neurons (1) is slightly decreased in ketamine-anesthetized non-stimulated arthritic rats as compared to the non-anesthetized non-stimulated ones, (2) is significantly higher in both stimulated normal and arthritic animals as compared to non-stimulated animals, particularly in laminae I, II, V and VI of L3 and L4, and (3) is significantly increased in stimulated arthritic as compared to stimulated normal rats, in all laminae of lumbar spinal segments. The appearance of 'basal' Fos labeling during the adjuvant-induced arthritic disease and the increased number of Fos-like immunoreactive neurons in stimulated arthritic rats compared to stimulated normal animals indirectly suggests that these neurons are abnormally active and thus involved in the hyperalgesia of arthritic disease. Therefore the use of Fos-like immunoreactivity in the chronic pain model seems to be an appropriate tool to study possible effects of various pharmacological compounds, such as analgesics and anti-inflammatory drugs.

c-fos expression in rat lumbar spinal cord during the development of adjuvant-induced arthritis.

A parallel clinical and behavioral study of adjuvant-induced arthritis in the rat showed four stages in the time-course of the disease: preclinical (first week), acute (weeks 2-4), post-acute (weeks 5-8) and recovery weeks 9-11) [Calvino et al. (1987) Behav. Brain Res. 24, 11-29]. As several studies have reported the expression of the proto-oncogene c-fos in spinal cord neurons following acute noxious peripheral stimuli, the aim of this study was to quantitatively assess Fos-like immunoreactivity in lumbar spinal cord neurons at various times of adjuvant-induced arthritis development, i.e. one, two, three, 11 and 22 weeks post-inoculation. The total number of Fos-like immunoreactive neurons in the lumbar enlargement correlated with the observed development of adjuvant-induced arthritis, i.e. Fos-like immunoreactivity was absent at one week, moderate at two weeks, greatly increased at three weeks, decreased at 11 weeks and returned to control values at 22 weeks. At three weeks, at the peak of Fos-like immunoreactivity distribution and acute stage of hyperalgesia, maximal labeling was observed in L3 and L4 spinal segments. In these segments, the most densely labeled region was the neck (laminae V and VI) of the dorsal horn (55%) and the ventral horn (35%) as compared to the superficial laminae (laminae I and II; 5%) and the nucleus proprius (laminae III and IV; 5%). These data indicate that c-fos expression induced by chronic inflammation is better expressed in deeper laminae than in the superficial ones, and that the number of Fos-positive cells correlates with behavioral studies.(ABSTRACT TRUNCATED AT 250 WORDS)