Peripheral and central alterations affecting spinal nociceptive processing and pain at adulthood in rats exposed to neonatal maternal deprivation.

The nociceptive system of rodents is not fully developed and functional at birth. Specifically, C fibers transmitting peripheral nociceptive information establish synaptic connections in the spinal cord already during the embryonic period that only become fully functional after birth. Here, we studied the consequences of neonatal maternal deprivation (NMD, 3 h/day, P2-P12) on the functional establishment of C fiber-mediated neurotransmission in spinal cord and of pain-related behavior. In vivo recording revealed that C fiber-mediated excitation of spinal cord neurons could be observed at P14 only in control but not in NMD rats. NMD was associated with a strong alteration in the expression of growth factors controlling C nociceptor maturation as well as two-pore domain K+ channels known to set nociceptive thresholds. In good agreement, C-type sensory neurons from NMD animals appeared to be hypoexcitable but functionally connected to spinal neurons, especially those expressing TRPV1 receptors. In vivo and in vitro recordings of lamina II spinal neurons at P14 revealed that the NMD-related lack of C fiber-evoked responses resulted from an inhibitory barrage in the spinal cord dorsal horn. Eventually, C-type sensory-spinal processing could be recovered after a delay of about 10 days in NMD animals. However, animals remained hypersensitive to noxious stimulus up to P100 and this might be due to an excessive expression of Nav1.8 transcripts in DRG neurons. Together, our data provide evidence for a deleterious impact of perinatal stress exposure on the maturation of the sensory-spinal nociceptive system that may contribute to the nociceptive hypersensitivity in early adulthood.

Corticosterone analgesia is mediated by the spinal production of neuroactive metabolites that enhance GABAergic inhibitory transmission on dorsal horn rat neurons.

Corticosterone (CORT) is a glucocorticoid produced by adrenal glands under the control of the hypothalamic-pituitary-adrenal axis. Circulating CORT can enter the central nervous system and be reduced to neuroactive 3α5α-reduced steroids, which modulate GABAA receptors. In the dorsal spinal cord, GABAergic transmission modulates integration of nociceptive information. It has been shown that enhancing spinal inhibitory transmission alleviates hyperalgesia and allodynia. Therefore, the spinal neuronal network is a pivotal target to counteract pain symptoms. Thus, any increase in spinal 3α5α-reduced steroid production enhancing GABAergic inhibition should reduce nociceptive message integration and the pain response. Previously, it has been shown that high levels of plasma glucocorticoids give rise to analgesia. However, to our knowledge, nothing has been reported regarding direct non-genomic modulation of neuronal spinal activity by peripheral CORT. In the present study, we used combined in vivo and in vitro electrophysiology approaches, associated with measurement of nociceptive mechanical sensitivity and plasma CORT level measurement, to assess the impact of circulating CORT on rat nociception. We showed that CORT plasma level elevation produced analgesia via a reduction in C-fiber-mediated spinal responses. In the spine, CORT is reduced to the neuroactive metabolite allotetrahydrodeoxycorticosterone, which specifically enhances lamina II GABAergic synaptic transmission. The main consequence is a reduction in lamina II network excitability, reflecting a selective decrease in the processing of nociceptive inputs. The depressed neuronal activity at the spinal level then, in turn, leads to weaker nociceptive message transmission to supraspinal structures and hence to alleviation of pain.

Differential neuropathic pain sensitivity and expression of spinal mediators in Lewis and Fischer 344 rats.

BACKGROUND: Altered hypothalamo-pituitary-adrenal (HPA) axis activity may be accompanied by a modulation of pain sensitivity. In a model of neuropathic pain (chronic constriction injury, CCI) we investigated the onset and maintenance of mechanical allodynia/hyperalgesia and the expression of biochemical mediators potentially involved in spinal cell modulation in two rat strains displaying either hypo- (Lewis-LEW) or hyper- (Fischer 344-FIS) reactivity of the HPA axis. RESULTS: Mechanical pain thresholds and plasmatic corticosterone levels were assessed before and during periods of 4 or 21 days following CCI surgery. At the end of the respective protocols, the mRNA expression of glial cell markers (GFAP and Iba1) and glutamate transporters (EAAT3 and EAAT2) were examined. We observed a correlation between the HPA axis reactivity and the pain behavior but not as commonly described in the literature; LEW rats seemed to be less sensitive than FIS from 4 to 14 days after the CCI surgery when looking at the mechanical allodynia/hyperalgesia. However, the biochemical spinal markers expression we observed is conflicting. CONCLUSION: We did not find a specific causal relation between the pain behavior and the glial cell activation or the expression of the glutamate transporters, suggesting that the interaction between the HPA axis and the spinal activation pattern is more complex in a context of neuropathic pain.

Polycaprolactone/Polylactic Acid Membrane Fails to Prevent Laminectomy-induced Sprouting of CGRP- and SP-immunopositive Nerve Fibres in the Dura mater lumbalis of Rats. / Polycaprolacton/Polylactid-Membran kann Laminektomie-induziertes Wachstum von CGRP- und SP-immunpositiven Nervenfasern in der Dura mater lumbalis von Ratten nicht verhindern.

BACKGROUND CONTEXT: Mechanisms and prevention of failed back surgery syndromes are rarely known in the clinical context. It has been shown that laminectomy induces outgrowth of putative nociceptive peptidergic afferents in the dura mater lumbalis of rats. PURPOSE: We aimed to investigate whether the application of a polycaprolactone/polylactic acid membrane (Mesofol) after surgery inhibits sensory hyperinnervation. MATERIALS/METHODS: Adult Lewis rats were assigned to three groups: Control (no manipulation), Laminectomy and Laminectomy + Mesofol. Six weeks post-surgery, the durae were removed, immunohistochemically stained for CGRP- and SP-positive afferents and their density quantified. RESULTS: In controls, CGRP- and SP-positive neurons were predominantly found in ventral but rarely observed in dorsal parts of the dura. Following laminectomy, the density of afferents significantly increased ventrally, resulting in a dense network of nerve fibers. In dorsal regions, neuronal sprouting of was observed. Covering the dura with Mesofol after laminectomy had no impact on nerve fibre outgrowth. CONCLUSION: Application of Mesofol neither prevents nor significantly diminishes the laminectomy-induced increase in the density of peptidergic afferents.

The density of nociceptive SP- and CGRP-immunopositive nerve fibers in the dura mater lumbalis of rats is enhanced after laminectomy, even after application of autologous fat grafts.

A considerable number of patients complain about pain after lumbar surgery. The spinal dura mater has been debated as a possible source of this pain. However, there is no information if laminectomy influences the nociceptive sensory innervation of the dura. Therefore, we quantitatively evaluated the density of SP- and CGRP-immunopositive nerve fibers in the dura mater lumbalis in an animal model of laminectomy. Twelve adult Lewis rats underwent laminectomy, in six of them the exposed dura was covered by an autologous fat graft. Further six animals without surgical treatment served as controls. Six weeks after surgery, the animals were perfused and the lumbar dura was processed immunohistochemically for the detection of CGRP- and SP-containing nerve fibers. In controls, the peptidergic nerve fibers were found predominantly in the ventral but rarely in the dorsal dura mater lumbalis. After laminectomy, the density of SP- and CGRP-immunopositive neurons significantly increased in ventral as well as in dorsal parts of the dura. Axonal spines could be observed in some cases at the site of laminectomy. The application of autologous fat grafts failed to inhibit the significant increase in the density of peptidergic afferents. Thus, we have provided the first evidence that laminectomies induce an increase in the density of putative nociceptive SP- and CGRP-immunopositive neurons in the lumbar dura mater ascribable to an axonal sprouting of fine nerve fibers. This effect was not prevented by using autologous fat grafts. It is conceivable that the neuronal outgrowth of nociceptive afferents is a cause of low back pain observed after lumbar surgery.

Maternal separation stress leads to resilience against neuropathic pain in adulthood.

Early life stress (ELS) leads to a permanent reprogramming of biochemical stress response cascades that may also be relevant for the processing of chronic pain states such as neuropathy. Despite clinical evidence, little is known about ELS-related vulnerability for neuropathic pain and the possibly underlying etiology. In the framework of experimental studies aimed at investigating the respective relationships we used the established ELS model of maternal separation (MS). Rat dams and neonates were separated for 3 h/day from post-natal day 2-12. At adulthood, noxious mechanical and thermal thresholds were assessed before and during induction of neuropathic pain by chronic constriction injury (CCI). The potential involvement of spinal glutamatergic transmission, glial cells, pro-inflammatory cytokines and growth factors was studied by using qPCR. MS per se did not modify pain thresholds. But, when exposed to neuropathic pain, MS rats exhibited a marked reduction of thermal sensitivity and a delayed development of mechanical allodynia/hyperalgesia when compared to control animals. Also, MS did not alter glucocorticoid receptor mRNA levels, but prevented the CCI-induced down-regulation of NR1 and NR2 sub-units of the NMDA receptor and of the glutamate transporter EAAT3 as observed at 21 days post-surgery. Additionally, CCI-provoked up-regulation of glial cell markers was either prevented (GFAP for astrocytes) or dampened (Iba1 for microglia) by MS. Pro-inflammatory cytokine mRNA expression was either not affected (IL-6) or reduced (IL-1ß) by MS shortly after CCI. The growth factors GDNF and NGF were only slightly downregulated 4 days after CCI in the MS-treated animals. The changes in glutamatergic signaling, astroglial and cytokine activation as well as neurotrophin expression could, to some extent, explain these changes in pain behavior. Taken together, the results obtained in the described experimental conditions support the mismatch theory of chronic stress where an early life stress, rather than predisposing individuals to certain pathologies, renders them resilient.

Neonatal maternal separation leads to a dampening of inflammation-related mechanical and thermal hypersensitivity in juvenile rats.

Early life stress (ELS) constitutes a risk factor for the later development of psychopathological disorders partly displaying pain comorbidity. Since ELS may hence be expected to have an impact on pain processing the present study investigates whether ELS could be a factor of vulnerability or resilience against an enhancement of noxious sensitivity in the framework of inflammatory pain in later life. Rats were exposed to the maternal separation (MS) paradigm, an established ELS model. At adulthood, corticosterone levels and anxiety-related behaviors were evaluated. Subsequently, paw edema, noxious mechanical and thermal sensitivity were measured prior and during an inflammation induced by intraplantar injection of Complete Freunds Adjuvant (CFA). The open field test and the corticosterone measures showed no effect of MS. MS did not change mechanical thresholds prior to inflammation but reduced mechanical hyperalgesia after CFA-injection. MS animals did however present shorter latencies to display nocifensive behaviors compared to controls (CON). Furthermore, in CON but not MS, the repetitive noxious heat testing induced a decrease in reaction latencies. Moreover, MS dampened CFA-induced heat hyperalgesia. Altogether, our results suggest that ELS may have a protective impact on inflammatory pain.

The combination of postnatal maternal separation and social stress in young adulthood does not lead to enhanced inflammatory pain sensitivity and depression-related behavior in rats.

The cumulative and match/mismatch hypotheses of stress are still under discussion regarding the effects of early life stress (ELS) on the vulnerability or resilience to psychopathology. In this context, an additional stress in later life (second hit) often leads to stress-related disorders that frequently include comorbid pain states. We previously observed that maternal separation (MS), a model of ELS, reduces vulnerability to neuropathic and inflammatory pain in rats. In the present study, we investigated the effects of an additional later stressor on the vulnerability to inflammatory pain. Sprague Dawley pups were divided into 4 groups: controls (CON, no stress), MS, social stress (SS) and MS+SS. At young adult age (from 7 to 15 weeks), stress- as well as pain-related parameters were evaluated prior and during 21 days following the induction of paw inflammation with complete Freund's adjuvant (CFA). Finally spinal glutamatergic transmission, immunocompetent cells, pro-inflammatory cytokines and growth factors were examined using qPCR. None of the stress conditions had a significant impact on corticosterone levels and anhedonia. In the forced swim test, MS and SS displayed increased passive coping whereas the combination of both stressors revoked this effect. The different stress conditions had no influence on basal mechanical thresholds and heat sensitivity. At 4 days post-inflammation all stress groups displayed lower mechanical thresholds than CON but the respective values were comparable at 7, 10, and 14 days. Only on day 21, MS rats were more sensitive to mechanical stimulation compared to the other groups. Regarding noxious heat sensitivity, MS+SS animals were significantly less sensitive than CON at 10 and 21 days after CFA-injection. qPCR results were mitigated. Despite the finding that stress conditions differentially affected different players of glutamatergic transmission, astrocyte activity and NGF expression, our biochemical results could not readily be related to the behavioral observations, precluding a congruent conclusion. The present results do neither confirm the cumulative nor corroborate or disprove the match/mismatch hypothesis.

Localization of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with painful osteoarthritis and of patients with painless failed total hip arthroplasties.

Using immunohistochemical methods we determined the presence of SP- and CGRP-immunopositive nerve fibers in the hip joint of patients with femoral neck fracture (controls, group 1), painful osteoarthritis (group 2), and painless failed total hip arthroplasties (group 3). Immunoreactive nerve fibers were found in the soft tissue of the fossa acetabuli as well as in the subintimal part of the synovial layer in the hip joint capsule of groups 1 and 2. In the capsule of controls the innervation density had a median of 5.7fibers/cm(2) for CGRP-ir and 3.2fibers/cm(2) for SP-ir afferents. In the osteoarthritic group, the density significantly increased to a median of 15.6fibers/cm(2) for CGRP-ir and 8.2fibers/cm(2) for SP-ir neurons (p=0.05). Patients with failed hip arthroplasties completely lacked these neuropeptide containing afferents. Innervation density in the fossa acetabuli of osteoarthritc patients showed a median of 14.1fibers/cm(2) for CGRP-ir and 5.9fibers/cm(2) for SP-ir afferents. From these data we assume that the hip joint capsule and the soft tissue of the fossa acetabuli are important triggers of nociception. This is supported by the fact, that patients with loosened total hip arthroplasties, where we failed to detect SP- and CGRP-immunoreactive fibers, did not feel pain. The upregulation of SP- and CGRP-positive neurons in response to arthritic stages suggests a mechanism involving neuropeptides in the maintenance of a painful degenerative joint disease and in mediating noxious stimuli from the periphery. Furthermore, these findings help to explain clinical observations, such as effectiveness of local therapy to control hip pain with intraarticular injection, synovectomy and denervation procedures.

Chronic Social Stress Time-Dependently Affects Neuropathic Pain-Related Cold Allodynia and Leads to Altered Expression of Spinal Biochemical Mediators.

Clinical data have shown that chronic exposure to stress may be accompanied by an enhancement of inflammation-related pain sensitivity. In this context, little is however known on the impact of stress on neuropathic pain. In the present study we addressed this issue by combining the chronic constriction injury (CCI) model with an ongoing social stress (OSS) paradigm. Cold plate and von Frey tests were performed in 48 rats divided into four groups: OSS exposed to OSS, CCI subjected to chronic nerve constriction, OSS+CCI with a combination of neuropathy and stress and CON, a control group lacking any manipulation. While we did not observe any stress-related differences in mechanical sensitivity throughout the observation period, CCI rats were more sensitive to cold stimulation than OSS+CCI in the initial phase of neuropathy. A switch was observed at a later stage, leading to a hypersensitivity of the OSS+CCI compared to the CCI rats. At this time point we investigated the spinal mRNA expression of neuron and glia related molecules potentially involved in neuropathic pain and stress. The combination of psychosocial stress and neuropathic pain seemed to enhance glial cell activation, pro-inflammatory cytokine and neurotrophic factor mRNA levels, rather than glutamatergic transmission. Our data show that long lasting social stress may lead to time-dependent alteration of neuropathy-related cold pain sensitivity while mechanically-induced pain remains unchanged.

Gabapentin reduces the mechanosensitivity of fine afferent nerve fibres in normal and inflamed rat knee joints.

The antiepileptic drug gabapentin has been shown to have an antihyperalgesic effect following central administration. This electrophysiological investigation examined whether peripherally administered gabapentin could modulate the mechanosensitivity of primary afferents innervating normal and kaolin/carrageenan inflamed rat knee joints. Close intraarterial injection of gabapentin (0.01, 1 and 100mg/kg) dose-dependently reduced afferent firing rate in both normal and acutely inflamed rat knees in response to normal and hyper-rotation of the joint. Thus, in addition to its central mode of action, peripheral administration of gabapentin reduces nociception locally and this may prove to be beneficial in the treatment of various pain syndromes including inflammatory arthritis.

The neurokinin-2 receptor is not involved in the sensitization of primary afferents of the rat knee joint.

Using electrophysiological methods, we aimed in the present study to determine whether the NK(2) receptor is involved in the sensitization of articular afferents of the rat. Impulse activity from 27 single fine nerve fibres innervating knee joints was recorded during non-noxious and noxious joint rotations. Close intraarterial application of the NK(2) receptor agonist [beta-Ala(8)]NKA(4-10) at doses of 0.2-200 nmol did not sensitize the afferents from normal knee joints to mechanical stimuli whereas the application of substance P (20 nmol) increased their mechanosensitivity. These data further support the hypothesis that the NK(2) receptor is not involved in the sensitization of primary afferents in normal knee joints to mechanical stimuli.

Glucocorticoid-mediated enhancement of glutamatergic transmission may outweigh anti-inflammatory effects under conditions of neuropathic pain.

At the clinical level comorbidity between chronic pain and dysfunctional hypothalamus-pituitary-adrenal (HPA) axis is well established. We aimed to identify causal relationships in a model of neuropathic pain (chronic constriction injury, CCI) by studying the effects of glucocorticoid receptor agonist (dexamethasone) and antagonist (RU-486) administration on pain behavior and spinal biochemical mediators. Daily injections were performed in Sprague Dawley rats. Weight, plasma corticosterone levels and mechanical pain thresholds were assessed before and during 21 days post-CCI. At days four and 21 we investigated the mRNA expression of spinal mediators. In the dexamethasone-injected group, we observed a diminution of body weight and plasma corticosterone levels during the 21 days post surgery period and a more pronounced pain sensitivity until day 7 post-CCI. This enhanced pain sensitivity in the early period following nerve injury was accompanied by a transient increase of the glutamate receptors mGluR5 and NMDA at day 4. However, at this time point we did not observe any effect of the agonist/antagonist injections on the mRNA expression of pro-inflammatory cytokines. The RU-486-injected rats showed a slight mechanical hypoalgesia until 7 days post-CCI, but without any significant correlation with the expression of the measured markers. Our results indicate that glucocorticoid-related modulations of neuropathic pain processing may rather depend on a modification of glutamatergic transmission than on a change in pro-inflammatory cytokine expression.

Plasma glucocorticoids differentially modulate phasic and tonic GABA inhibition during early postnatal development in rat spinal lamina II.

Nociceptive processing is tuned by GABAA receptor-mediated inhibition in the spinal cord dorsal horn that undergoes postnatal maturation in rodents. These GABAergic inhibitory postsynaptic currents (IPSCs) are modulated by 3α5α-reduced steroids during early postnatal development in spinal cord lamina II. Thus an enhanced phasic inhibition is present in neonates and decreases over time. GABA can also activate extrasynaptic receptors, giving rise to tonic inhibition. In this study, we characterized the contribution of plasma corticosterone (CORT) to postnatal maturation of spinal phasic and, for the first time, tonic GABAergic inhibitions. We used Fisher and Lewis rat strains displaying respectively high and low hypothalamic-pituitary-adrenal axis reactivity, compared to control Sprague-Dawley rats. Measured plasma CORT levels were significantly higher in Fisher rats, which also displayed significantly higher mechanical nociceptive thresholds, supporting the hypothesis of an antinociceptive action of CORT. Recorded GABAA IPSCs shortened during maturation in all strains while remaining larger in Fisher rats. Blocking the 5α-reduction of steroids in Fisher rats produced a further decrease of IPSC deactivation time constant. In contrast, GABAA tonic inhibition progressively increased during maturation, without any difference among strains. In conclusion, we show that both phasic and tonic GABAergic inhibitions undergo postnatal maturation in lamina II. Moreover spinal production of 3α5α-reduced steroids that presumably derive from plasma CORT is correlated to spinal GABAA phasic (but not tonic) inhibition and to mechanical nociceptive thresholds.

Pain behavior and spinal cell activation due to carrageenan-induced inflammation in two inbred rat strains with differential hypothalamic-pituitary-adrenal axis reactivity.

Lewis (LEW) and Fischer (FIS) inbred rats were used to study the relationship of hypothalamic-pituitary-adrenal (HPA) axis reactivity with inflammation-related pain behavior. LEW rats are susceptible to the development of autoimmune and chronic inflammatory disorders, whereas FIS rats are resistant. Since contradictory data have previously been collected under conditions of acute inflammation, we investigated the onset and maintenance of thermal and mechanical hyperalgesia and spinal activation of neurons and glia cells in a model of ongoing inflammation in both strains. Hind paw volumes and mechanical and thermal pain thresholds were measured prior to and during one week after intraplantar injection of carrageenan. The activation of nociceptive neurons (FosB), astroglia (GFAP) and microglia (OX-42) in the spinal cord of segments L5/L6 was assessed using immunohistochemistry. Inflammation increased paw volume, pain sensitivity and cell activation in both strains. FIS rats were more sensitive to sensory stimulation and developed a more severe edema on day 1, but recovered faster up to day 7 than LEW rats. At that time a higher amount of activated nociceptive neurons and corticosterone was seen in FIS rats, but microglial activation was more pronounced in LEW rats. Our results suggest a biphasic role of the HPA axis in pain behavior and spinal cell activation associated with ongoing inflammation. In the acute stage, the stronger reaction in FIS rats might be explained by an activating effect of corticosteroids on neutrophil function. Under ongoing inflammatory conditions the immunosuppressive actions of corticosteroids may dominate and lead to a quicker recovery of paw volume and pain sensitivity in FIS rats.