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Neuronal activation at the spinal cord and medullary pain control centers after joint stimulation: A c-fos study in acute and chronic articular inflammation
Abstract
Chronic inflammatory pain induces short- and long-term central changes, which have been mainly studied at the spinal cord level. Supraspinal pain control centers intrinsically connected with the dorsal horn are also prone to be affected by chronic inflammatory pain. C-fos expression was used as a neuronal activation marker at spinal and supraspinal levels to i) compare acute and chronic articular inflammation, and ii) analyze the effects of brief innocuous or noxious stimulation of a chronically inflamed joint. Acute articular inflammation was induced by an inflammatory soup with prostaglandin E(2) and bradykinin, both at 10(-5) M. Chronic articular inflammation consisted of 14 days of monoarthritis. Early c-fos expression was studied 4 min after inflammatory soup injection or stimulation of the arthritic joint whereas late c-fos expression was evaluated 2 h after those stimuli. At the spinal cord, the analysis was focused on the dorsal horn (laminae I-V) and supraspinally, five major regions of the endogenous pain control system were considered: the caudal ventrolateral medulla (VLM), the dorsal reticular nucleus (DRt), the ventral reticular nucleus (VRt), the nucleus of the solitary tract (Sol) and the rostroventromedial medulla (RVM). Acute articular inflammation induced early and late increases in c-fos expression at the spinal level and late increases supraspinally whereas the effects of monoarthritis were more moderate and restricted to the spinal cord. When monoarthritic animals were subjected to gentle touch or bending of the joint, early increases in c-fos expression were detected supraspinally, but not at the spinal level. In this region, noxious mechanical stimulation induced late increases in non-inflamed animals and both early and late increases in monoarthritic rats. Supraspinally, noxious stimulation induced only late increases in c-fos expression. The present results show complex differences in the patterns of c-fos expression between the spinal cord and medullary areas of the pain control system during articular inflammation, which indicate that the somatosensory system is differentially affected by the installation of chronic pain.
... 23 Stimulation of the DRt increases nociceptive responses, whereas the reverse occurs upon local blockade. 2,3,23,51 Increased ascending nociceptive input from the spinal cord to the DRt during chronic inflammation 42,48 is likely to activate DRt neurons and trigger pronociceptive actions from the DRt. The role of GABA in the control of DRt pronociceptive effects has not been evaluated. ...
... The role of GABA in the control of DRt pronociceptive effects has not been evaluated. We have previously shown that DRt neurons express GABA B receptors, [40][41][42][43] which in a large proportion are activated in response to noxious peripheral stimulation. 41 Dorsal reticular nucleus neurons expressing GABA B receptors are spinally projecting and nonspinally projecting neurons. ...
... First, we used in vivo microdialysis during the formalin test to relate GABA levels in the DRt with behavioural analysis, a neuroapproach that provided interesting results regarding the release of other neurotransmitters in the DRt, namely during the formalin test. 32 Because GABA B receptors were proposed to play a role in modulation of neuronal activity at the DRt, [40][41][42][43] we then evaluated the effect of lentiviral and pharmacologically mediated interference upon GABA B receptors in the DRt in the formalin test. Finally, we determined the telencephalic and diencephalic GABAergic afferents of the DRt by combining retrograde tracttracing from the DRt with immunodetection of glutamate decarboxylase (GAD), the GABA-synthesizing enzyme. ...
The dorsal reticular nucleus (DRt) plays a key role in facilitation of nociceptive transmission at the spinal cord. In this study, we evaluated the mechanisms involved in GABA-mediated control of the DRt focusing on the role of local GABAB receptors. First, we used in vivo microdialysis to study the release of GABA in the DRt during the course of the formalin test. An increase of GABA levels in comparison with baseline values was detected in the second phase of the test. Because we previously showed that GABAB receptors are expressed by opioidergic DRt neurons, which respond to nociceptive stimuli and inhibit spinally projecting DRt neurons involved in descending pronociception, we then interfered with local GABAB receptors using gene transfer and pharmacological approaches. Lentiviral-mediated knockdown of GABAB1a expression decreased nociceptive responses during the second phase of the test. Local administration of the GABAB receptor antagonist CGP 35348 also decreased nociceptive responses in the second phase of the test, whereas the opposite was detected after injection of the GABAB agonist baclofen. Finally, we determined the GABAergic afferents of the DRt, namely those arising from its main brain afferents, which are located at the telencephalon and diencephalon. For that purpose, we combined retrograde tract-tracing from the DRt with immunodetection of glutamate decarboxylase, the GABA-synthesizing enzyme. The higher numbers of retrogradely labelled glutamate decarboxylase-immunoreactive neurons were located at insular, somatosensory, and motor cortices. Collectively, the results suggest that GABA acting on GABAB receptors may enhance pain facilitation from the DRt during inflammatory pain.
... Tissue injury increases c-fos expression in a number of animal models with pain including after noxious electrical stimulation (Berrocal et al., 2007;Herdegen et al., 1991;Molander et al., 1992), mechanical stimuli (Bullitt, 1990), formalin (Berrocal et al., 2007;Sun et al., 1996) and paw inflammation (Ma and Woolf, 1996;Pinto et al., 2007) and thus c-fos is used as a marker for neuronal activation. In animals following sciatic nerve transection, c-fos expression in the lumbar spinal cord increased in the superficial dorsal horn and deep dorsal horn within hours and lasted for at least 4 weeks (Chi et al., 1993). ...
... On the other hand, supraspinal c-fos expression is scarcely reported compared to the spinal level. In acute and chronic inflammatory pain, c-fos expression in the RVM was increased within 5 min and lasted through 2 h in the RVM as well as the spinal cord (Pinto et al., 2007). With respect to nerve injury, 3-4 days after partial sciatic nerve injury there was increased c-fos expression in the rat frontal cortex, thalamus, PAG and parabrachial nucleus (Jergova et al., 2008;Narita et al., 2003). ...
... In the current study, it was surprising that c-fos expression in the NRM occurred within 35 min after starting SCS and returned to normal 2 h later while that in the spinal cord increased 2 h after SCS but not 5 min. However, transcriptional activation of the c-fos gene occurs within minutes of noxious stimulation (see Harris 1998) and protein expression can begin as early as 7-8 min after stimulation (Pinto et al., 2007). Further, electrical stimulation of peripheral nerves in uninjured animals increases c-fos immunoreactivity in the superficial dorsal horn 30 min after the end of the stimulus (Bullitt et al., 1992), a similar time frame to the current study. ...
Spinal cord stimulation (SCS) is an alternative approach for treatment of neuropathic pain when conservative management is ineffective. Previously we showed both 4 Hz and 60 Hz SCS reduces hyperalgesia in an animal model of neuropathic pain. However, the mechanisms underlying the pain reduction by SCS and how different frequencies of SCS produce the analgesic effect are unclear. To elucidate potential sites modulated by SCS we examined distribution of c-fos in Sprague-Dawley rats with spared nerve injury (SNI) and those without injury in response to SCS. SCS was delivered at one of 3 different frequencies (4 Hz, 60 Hz, and 100 Hz) for 30 min 2 weeks after SNI or in animals without SNI. Animals were perfused either 5 min or 2 h after SCS and c-fos protein examined immunohistochemically. The number of c-fos positive cells significantly increased 5 min (35 min after SCS began) after 4 Hz SCS in the NRM, but not PAG in animals with nerve injury. The number of c-fos positive cells was significantly increased bilaterally 2 h after either 4 Hz or 60 Hz SCS in the spinal cord dorsal horn in the cervical enlargement and under the electrode, but not in the lumbar enlargement in animals with nerve injury. In uninjured animals 4 Hz SCS increased c-fos expression at the electrode site and lumbar enlargement when compared to animals implanted with the electrode who did not receive SCS. 100 Hz SCS had no effect on c-fos expression. Thus, at the time points examined in this model, low frequency SCS likely activates supraspinal and spinal mechanisms to produce analgesia, while higher frequencies activate spinal mechanisms.
... The anatomical data reviewed above are easy to conciliate with functional findings showing that the magnitude and duration of behavioral nociceptive responses is more intense when the VLMlat is stimulated in comparison with stimulations directed to more medial VLM areas. The VLMlat contains neurons that respond to noxious activation of the joints (Pinto et al., 2007). Incidentally, it must be noted that the magnitude of activation of VLMlat neurons, as measured by the expression of the c-fos protooncogene, is directly correlated with the magnitude of activation of lamina I neurons (Pinto et al., 2006), which reinforces the functional relevance of the above mentioned lamina I-VLMlat-lamina I loop. ...
... Indeed, GABA B receptors knock-down at the DRt, mediated by lentiviral vectors, or the pharmacological blockade, via the local administration of a GABA B antagonist, significantly attenuated formalin-induced pain behavior while the local administration of a GABA B agonist induced the opposite (Martins et al., 2015a). The effect of GABA is likely due to disinhibition of the DRt spinally projecting neurons since a large proportion of GABA B receptors are expressed by local opioidergic neurons inhibiting DRt spinally projecting neurons (Pinto et al., 2007(Pinto et al., , 2008aMartins et al., 2008). GABA might be released from local interneurons but also from insular, somatosensory and motor cortices (Figure 2) which represent the most important afferent pathways to the DRt and they are GABAergic (Martins et al., 2015a). ...
The involvement of the reticular formation (RF) in the transmission and modulation of nociceptive information has been extensively studied. The brainstem RF contains several areas which are targeted by spinal cord afferents conveying nociceptive input. The arrival of nociceptive input to the RF may trigger alert reactions which generate a protective/defense reaction to pain. RF neurons located at the medulla oblongata and targeted by ascending nociceptive information are also involved in the control of vital functions that can be affected by pain, namely cardiovascular control. The RF contains centers that belong to the pain modulatory system, namely areas involved in bidirectional balance (decrease or enhancement) of pain responses. It is currently accepted that the imbalance of pain modulation towards pain facilitation accounts for chronic pain. The medullary RF has the peculiarity of harboring areas involved in bidirectional pain control namely by the existence of specific neuronal populations involved in antinociceptive or pronociceptive behavioral responses, namely at the rostroventromedial medulla (RVM) and the caudal ventrolateral medulla (VLM). Furthermore the dorsal reticular nucleus (also known as subnucleus reticularis dorsalis; DRt) may enhance nociceptive responses, through a reverberative circuit established with spinal lamina I neurons and inhibit wide-dynamic range (WDR) neurons of the deep dorsal horn. The components of the triad RVM-VLM-DRt are reciprocally connected and represent a key gateway for top-down pain modulation. The RVM-VLM-DRt triad also represents the neurobiological substrate for the emotional and cognitive modulation of pain, through pathways that involve the periaqueductal gray (PAG)-RVM connection. Collectively, we propose that the RVM-VLM-DRt triad represents a key component of the “dynamic pain connectome” with special features to provide integrated and rapid responses in situations which are life-threatening and involve pain. The new available techniques in neurobiological studies both in animal and human studies are producing new and fascinating data which allow to understand the complex role of the RF in pain modulation and its integration with several body functions and also how the RF accounts for chronic pain.
... We previously showed that the PAG is strongly affected by the dilation of the cerebral ventricles in the hydrocephalus model used in the present study [21]. Here, the PAG was analyzed to evaluate the expression of the c-Fos protooncogene, as this is an indicator of neuronal activity [39]. Kaolin-injected animals presented a decrease in c-Fos levels in the PAG, mainly at the vlPAG column, which indicates decreased activation of this nucleus. ...
Hydrocephalus is characterized by enlargement of the cerebral ventricles, accompanied by distortion of the periventricular tissue. Patients with hydrocephalus usually experience urinary impairments. Although the underlying etiology is not fully described, the effects of hydrocephalus in the neuronal network responsible for the control of urination, which involves periventricular areas, including the periaqueductal gray (PAG) and the noradrenergic locus coeruleus (LC). In this study, we aimed to investigate the mechanisms behind urinary dysfunction in rats with kaolin-induced hydrocephalus. For that purpose, we used a validated model of hydrocephalus—the rat injected with kaolin in the cisterna magna—also presents urinary impairments in order to investigate the putative involvement of noradrenergic control from the brain to the spinal cord Onuf’s nucleus, a key area in the motor control of micturition. We first evaluated bladder contraction capacity using cystometry. Since our previous characterization of the LC in hydrocephalic animals showed increased levels of noradrenaline, we then evaluated the noradrenergic innervation of the spinal cord’s Onuf’s nucleus by measuring levels of dopamine β-hydroxylase (DBH). We also evaluated the expression of the c-Fos protooncogene, the most widely used marker of neuronal activation, in the ventrolateral PAG (vlPAG), an area that plays a major role in the control of urination by its indirect control of the LC via pontine micturition center. Hydrocephalic rats showed an increased frequency of bladder contractions and lower minimum pressure. These animals also presented increased DBH levels at the Onuf´s nucleus, along with decreased c-Fos expression in the vlPAG. The present findings suggest that impairments in urinary function during hydrocephalus may be due to alterations in descending noradrenergic modulation. We propose that the effects of hydrocephalus in the decrease of vlPAG neuronal activation lead to a decrease in the control over the LC. The increased availability of noradrenaline production at the LC probably causes an exaggerated micturition reflex due to the increased innervation of the Onuf´s nucleus, accounting for the urinary impairments detected in hydrocephalic animals. The results of the study provide new insights into the neuronal underlying mechanisms of urinary dysfunction in hydrocephalus. Further research is needed to fully evaluate the translational perspectives of the current findings.
... At the early stages of OA, pain is thought to be initiated by the activation and sensitization of nociceptive fibers because of pathological changes in the injured joint (Fingleton et al., 2015;Steen Pettersen et al., 2019). However, at later stages central mechanisms also contribute to OA pain (Gwilym et al., 2009;Lluch et al., 2018;Pinto, Lima, & Tavares, 2007). Recently, it has become clear that macrophages have important functions in the regulation of pain. ...
... It remains to be ascertained if the increase in the expression of the cathecolaminergic enzymes TH and DBH account for the reduced activation of dorsal horn neurons along with decreased behavioral nociresponses in the formalin test in hydrocephalic animals. We elected the formalin-induced activation of the c-Fos protooncogene at the spinal cord, since it is considered a more functional method that allowed us to use the same animal to study behavior and Fos expression, and there is usually a correlation between nociceptive activation and behavioral responses [45,46]. It is important to perform intrathecal deliveries of α2-adrenoreceptors agonists to evaluate if the function of this receptor is preserved at the spinal cord. ...
Pain transmission at the spinal cord is modulated by noradrenaline (NA)-mediated actions that arise from supraspinal areas. We studied the locus coeruleus (LC) to evaluate the expression of the cathecolamine-synthetizing enzyme tyrosine hydroxylase (TH) and search for local oxidative stress and possible consequences in descending pain modulation in a model of hydrocephalus, a disease characterized by enlargement of the cerebral ventricular system usually due to the obstruction of cerebrospinal fluid flow. Four weeks after kaolin injection into the cisterna magna, immunodetection of the catecholamine-synthetizing enzymes TH and dopamine-β-hydroxylase (DBH) was performed in the LC and spinal cord. Colocalization of the oxidative stress marker 8-OHdG (8-hydroxyguanosine; 8-OHdG), with TH in the LC was performed. Formalin was injected in the hindpaw both for behavioral nociceptive evaluation and the immunodetection of Fos expression in the spinal cord. Hydrocephalic rats presented with a higher expression of TH at the LC, of TH and DBH at the spinal dorsal horn along with decreased nociceptive behavioral responses in the second (inflammatory) phase of the formalin test, and formalin-evoked Fos expression at the spinal dorsal horn. The expression of 8-OHdG was increased in the LC neurons, with higher co-localization in TH-immunoreactive neurons. Collectively, the results indicate increased noradrenergic expression at the LC during hydrocephalus. The strong oxidative stress damage at the LC neurons may lead to local neuroprotective-mediated increases in NA levels. The increased expression of catecholamine-synthetizing enzymes along with the decreased nociception-induced neuronal activation of dorsal horn neurons and behavioral pain signs may indicate that hydrocephalus is associated with alterations in descending pain modulation.
... Our results showed some differences from those of Chen, which showed that transcranial focused ultrasound reduced the number of c-fos-positive cells [37]; the difference may reflect the different animal models adopted. Tissue injury, such as paw inflammation [48], sciatic nerve transection [49], and chronic constriction injury of the sciatic nerve [50], can also increase c-fos expression. A previous study found that the expression of c-fos increased in rats with seizures [51]. ...
Although neurocircuits can be activated by focused ultrasound stimulation, it is unclear whether this is also true for spinal cord neurocircuits. In this study, we used low-intensity focused ultrasound (LIFU) to stimulate lumbar 4–lumbar 5 (L4–L5) segments of the spinal cord of normal Sprague Dawley rats with a clapper. The activation of the spinal cord neurocircuits enhanced soleus muscle contraction as measured by electromyography (EMG). Neuronal activation and injury were assessed by EMG, western blotting (WB), immunofluorescence, hematoxylin and eosin (H&E) staining, Nissl staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), somatosensory evoked potentials (SEPs), motor evoked potentials (MEPs), and the Basso–Beattie–Bresnahan locomotor rating scale. When the LIFU intensity was more than 0.5 MPa, LIFU stimulation induced soleus muscle contraction and increased the EMG amplitudes (P<0.05) and the number of c-fos- and GAD65-positive cells (P<0.05). When the LIFU intensity was 3.0 MPa, the LIFU stimulation led to spinal cord damage and decreased SEP amplitudes for electrophysiological assessment (P<0.05); this resulted in coagulation necrosis, structural destruction, neuronal loss in the dorsal horn by H&E and Nissl staining, and increased expression of GFAP, IL-1β, TNF-α, and caspase-3 by IHC, ELISA, and WB (P<0.05). These results show that LIFU can activate spinal cord neurocircuits and that LIFU stimulation with an irradiation intensity ≤1.5 MPa is a safe neurostimulation method for the spinal cord.
... To identify the virus-EGFP positive cells in the SCG, TH expression was detected by immunohistochemical staining. The immunofluorescence of c-Fos (Servicebio, Wuhan, China) was used to evaluate the neuronal activity (21). The fluorescent dye DAPI (4 ′ ,6diamidino-2-phenylindole) was used to locate the position of the nucleus. ...
Background: Circadian rhythms have a considerable impact on the daily physiology of the heart, and their disruption causes pathology. Several studies have revealed that circadian disruption impaired cardiac remodeling after myocardial infarction (MI); however, the underlying brain-heart mechanisms remain unknown. We aim to discuss whether circadian disruption facilitates cardiac remodeling after MI by activating sympathetic nervous system.
Methods: Rats were randomly divided into three groups: Sham group (Sham), MI group (MI), and MI+ circadian disruption group (MI+Dis); rats were treated with pseudorabies virus (PRV) injections for trans-synaptic retrograde tracing; rats were randomly divided into two groups: MI+ circadian disruption + Empty Vector+ clozapine N-oxide (CNO) (Empty Vector), and MI+ circadian disruption + hM4D(Gi)+ CNO [hM4D(Gi)].
Results: Circadian disruption significantly facilitated cardiac remodeling after MI with lower systolic function, larger left ventricular volume, and aggravated cardiac fibrosis. Cardiac sympathetic remodeling makers and serum norepinephrine levels were also significantly increased by circadian disruption. PRV virus-labeled neurons were identified in the superior cervical ganglion (SCG), paraventricular nucleus (PVN), and suprachiasmatic nucleus (SCN) regions. Ganglionic blockade via designer receptors exclusively activated by designer drugs (DREADD) technique suppressed the activity of sympathetic nervous system and significantly alleviated the disruption-related cardiac dysfunction.
Conclusion: Circadian disruption adversely affected cardiac remodeling after MI possibly by activating sympathetic nervous system, and suppressing sympathetic activity can attenuate this disruption-related cardiac dysfunction.
... It was conceptualized based on analyzed samples of inflamed human tissues (Steen et al., 1995), introduced to basic pain research as a tool to activate and sensitize peripheral nociceptors (Steen et al., 1992(Steen et al., , 1996, and used extensively to induce animal models of patholog- ical pain conditions associated with allodynia and hyperalgesia (De Felice et al., 2013;Oshinsky and Gomonchareonsiri, 2007). For example, IS has been shown to be efficacious at inducing sensitization upon administration to sensory fibers innervating a host of anatomical structures including: cutaneous tissue and nerve fibers (Katz and Gold, 2006;Kessler et al., 1992;Meyer et al., 1991;Michaelis et al., 1998;Rivera et al., 2000), joints (Pinto et al., 2007), colonic tissue (Brumovsky et al., 2009;Feng and Gebhart, 2011;Su and Gebhart, 1998), the cornea (Parra et al., 2014), and the dura mater (Burstein et al., 1998b;Strassman et al., 1996). Almost unanimously, IS effectively activates and sensitizes primary afferent nociceptors, rendering them hyper-excitable, spontaneously active, and responsive to previously sub-threshold mechanical and thermal stimuli (Chen et al., 2007;Grossmann et al., 2009;Ma et al., 2006;Meyer et al., 1991;Michaelis et al., 1998). ...
Advances in our understanding of the human pain experience have shifted much of the focus of pain research from the periphery to the brain. Current hypotheses suggest that the progression of migraine depends on abnormal functioning of neurons in multiple brain regions. Accordingly, we sought to capture functional brain changes induced by the application of an inflammatory cocktail known as inflammatory soup (IS), to the dura mater across multiple brain networks. Specifically, we aimed to determine whether IS alters additional neural networks indirectly related to the primary nociceptive pathways via the spinal cord to the thalamus and cortex. IS comprises an acidic combination of bradykinin, serotonin, histamine and prostaglandin PGE2 and was introduced to basic pain research as a tool to activate and sensitize peripheral nociceptors when studying pathological pain conditions associated with allodynia and hyperalgesia. Using this model of intracranial pain, we found that dural application of IS in awake, fully conscious, rats enhanced thalamic, hypothalamic, hippocampal and somatosensory cortex responses to mechanical stimulation of the face (compared to sham synthetic interstitial fluid administration). Furthermore, resting state MRI data revealed altered functional connectivity in a number of networks previously identified in clinical chronic pain populations. These included the default mode, sensorimotor, interoceptive (Salience) and autonomic networks. The findings suggest that activation and sensitization of meningeal nociceptors by IS can enhance the extent to which the brain processes nociceptive signaling, define new level of modulation of affective and cognitive responses to pain; set new tone for hypothalamic regulation of autonomic outflow to the cranium; and change cerebellar functions.
... Chronic pain can occur during RA, and the inflammation involved is usually followed by increased behavioral responses to innocuous (allodynia) and noxious (hyperalgesia) stimulation (Pinto et al., 2007;Edwards et al., 2011). Hyperalgesia is induced by the action of mediators released in response to inflammation in the inflamed tissue. ...
Rheumatoid arthritis is an inflammatory autoimmune condition and tumor necrosis factor-α (TNF-α) plays an important role in its pathophysiology. In vitro, (E)-N'-(3,4-dimethoxybenzylidene)-N-methylbenzohydrazide (LASSBio-1359) has exhibited anti-TNF-α properties and in vivo these effects are mediated via activation of adenosine receptor. This work investigates the antinociceptive action of LASSBio-1359 in murine models of acute and chronic inflammatory pain. Male mice received an intraperitoneal injection of LASSBio-1359 and then were evaluated in formalin- and carrageenan-induced paw edema assays. Complete Freund's Adjuvant (CFA) was used to induce a mouse model of monoarthritis. These mice were treated with LASSBio-1359 by oral gavage to evaluate thermal and mechanical hyperalgesia. TNF-α and i-NOS expression, as well as histological features were also analyzed. The time of reactivity to formalin in the neurogenic phase was reduced from 56.3 ± 6.0 s to 32.7 ± 2.2 s and 23.8 ± 2.6 s following treatment with LASSBio-1359 at doses of 10 mg/kg and 20 mg/kg, respectively. A reversal of the antinociceptive action of LASSBio-1359 was observed in inflammatory phase following treatment with ZM 241385, an adenosine A2A antagonist. Carrageenan-induced thermal and mechanical hyperalgesia were reduced after treatment with LASSBio-1359. Similarly, CFA-induced thermal and mechanical hyperalgesia were reduced following treatment with LASSBio-1359 (25 and 50 mg/kg). Levels of TNF-α and iNOS expression increased in the monoarthritis model and were normalized in animals treated with LASSBio-1359 which was also associated with beneficial effects in the histological analysis. These results suggest that LASSBio-1359 represents an alternative treatment for monoarthritis.
... 32 The IEG c-fos is also activated in the spinal cord in models of chronic pain and inflammation. 33,34 Their expression is postulated to be the first signal of long-term changes in these neurons which will lead to chronic pain. Thus, it is likely that c-fos is activating similar cascades of gene expression in tinnitus and chronic pain, leading to changes that would underlie the symptomatology of these conditions. ...
Introduction:
Salicylate at high doses induces tinnitus in humans and experimental animals. However, the mechanisms and loci of action of salicylate in inducing tinnitus are still not well known. The expression of Immediate Early Genes (IEG) is traditionally associated with long-term neuronal modifications but it is still not clear how and where IEGs are activated in animal models of tinnitus.
Objectives:
Here we investigated the expression of c-fos and Egr-1, two IEGs, in the Dorsal Cochlear Nucleus (DCN), the Inferior Colliculus (IC), and the Posterior Ventral Cochlear Nucleus (pVCN) of rats.
Methods:
Rats were treated with doses known to induce tinnitus in rats (300mg/kg i.p. daily, for 3 days), and c-fos and Egr-1 protein expressions were analyzed using western blot and immunocytochemistry.
Results:
After administration of salicylate, c-fos protein expression increased significantly in the DCN, pVCN and IC when assayed by western blot. Immunohistochemistry staining showed a more intense labeling of c-fos in the DCN, pVCN and IC and a significant increase in c-fos positive nuclei in the pVCN and IC. We did not detect increased Egr-1 expression in any of these areas.
Conclusion:
Our data show that a high dose of salicylate activates neurons in the DCN, pVCN and IC. The expression of these genes by high doses of salicylate strongly suggests that plastic changes in these areas are involved in the genesis of tinnitus.
... The intermediate NTS is the major site of termination of carotid and aortic baroreceptor fibers (Ciriello et al., 1994) and is targeted by ascending fibers from the spinal cord (Gamboa-Esteves et al., 2001; Potts et al., 2002). Marked neuronal activation is elicited at the NTS during hypertension (Chan and Sawchenko, 1998) and after noxious stimulation (Pinto et al., 2007). The neurotransmitters that modulate BP and pain control at the NTS remain to be evaluated. ...
N-methyl-D-aspartate receptors (NMDAR) have a role in cardiovascular control at the nucleus tractus solitarii (NTS), eliciting increases or decreases in blood pressure (BP), depending on the area injected with the agonists. In spite of the association between cardiovascular control and pain modulation, the effects of manipulating NMDAR in pain responses have never been evaluated. In this study, we decreased the expression of NMDAR in the NTS using gene transfer to target receptor subunits and evaluate long-term effects. Seven days after the injection of lentiviral vectors containing the NR1a subunit cDNA of NMDAR, in antisense orientation, into the intermediate NTS of Wistar rats, BP was measured, and the formalin test of nociception was performed. The antisense vector induced a decrease of NR1 expression in the NTS and elicited BP rises and hypoalgesia. Antisense vectors inhibited formalin-evoked c-Fos expression in the spinal cord, indicating decreased nociceptive activity of spinal neurons. Using a time-course approach, we verified that the onset of both the increases in BP and the hypoalgesia was at 4 days after vector injection into the NTS. The injection of NMDA into the NTS reversed the effects of antisense vectors in pain behavioral responses and spinal neuronal activation and decreased BP and heart rate. The present study shows that the NR1 subunit of the NMDAR at the NTS is critical in the regulation of tonic cardiovascular and nociceptive control and shows an involvement of the nucleus in the modulation of sustained pain.
... Coronal frozen sections, 40 m thick, were obtained and serially collected in four series. Each series was immunohistochemically processed for GABA, GAD 65/67 or KCC2 using the avidin-biotin-peroxidase method, whereas the fourth was stained with formol-thionin for laminae delimitation, as described previously [28]. The sections processed for immunohistochemistry were treated with 1% hydrogen peroxidase for 10 min for inhibition of endogenous peroxidase activity before incubation in a blocking solution of 10% normal serum in 0.3% Triton X 25% in PBS (PBST) with 0.1 M glycine for 2 h. ...
neuronal hyperactivity at the spinal cord during mechanical hyperalgesia induced by diabetes may result from a decrease in the local expression of the potassium chloride co-transporter 2 (KCC2), which shifts the action of the neurotransmitter γ-amminobutiric acid (GABA) from inhibitory to excitatory. In this study, we evaluated the effects of spinal microglia inhibition or brain-derived neurotrophic factor (BDNF) blockade on KCC2 expression, spinal neuronal activity and mechanically induced pain responses of streptozotocin (STZ)-diabetic rats.
four weeks after induction of diabetes, the STZ-diabetic rats received daily intrathecal injections, for 3 days, of minocycline (microglia inhibitor), TrkB/Fc (BDNF sequester) or saline. Behavioural responses to mechanical nociceptive stimulation of STZ-diabetic rats were evaluated by the Randall-Selitto test. The lumbar spinal cord was immunoreacted against the Fos protein (marker of neuronal activation) or KCC2, which was also quantified by western blotting. BDNF levels at the spinal cord were quantified by an enzyme-linked immunosorbent assay (ELISA).
minocycline treatment reversed the mechanical hyperalgesia, increased Fos expression and decreased the KCC2 expression detected in STZ-diabetic rats to control levels. Treatment with TrkB/Fc was less effective, inducing moderate effects in mechanical hyperalgesia and Fos expression and only a partial correction of KCC2 expression. BDNF levels were not increased in STZ-diabetic rats.
this study demonstrates that the microglial activation at the spinal cord contributes to mechanical hyperalgesia and spinal neuronal hyperactivity induced by diabetes, apparently by regulating the KCC2 expression. These effects do not seem to be mediated by BDNF, which is an important difference from other chronic pain conditions. New targets directed to prevent spinal microglia activation should be considered for the treatment of mechanical hyperalgesia induced by diabetes.
... satiety) catecholaminergic neurons of the VLM and the rostral NTS seem to function more specifically as a "danger pathway", in that these neurons respond exclusively to stimuli that signal stress or physiological danger related to deviations from the homeostatic equilibrium. The VLM catecholaminergic (referred to as adrenergic C1 and noradrenergic A1) neurons respond to virtually all dangerous physiological conditions evaluated, including cancer (Mravec et al. 2009), pathogenic bacteria in the gut (Gaykema et al., 2004;Goehler et al., 2005), hypovolemia and hypotension (Dun et al., 1993, Murphy et al., 1994, hypoxia (Smith et al., 1995), glucoprivic challenge (Ritter & Dinh, 1994;Ritter et al., 2006), intragastric gavage of poison-like bitter taste receptor ligands, (Yamamoto & Sawa, 2000), anxiogenic doses of yohimbine (Myers et al., 2005), joint inflammation (Pinto et al. 2007), opiate withdrawal (Laorden et al., 2002), seizures (Kantor et al., 1996, Silveira et al., 2000, pharmacologic levels of CCK (Schreihofer et al., 1997), and inescapable stress (Goebel et al., 2009). However, these VLM and rostral NTS catecholaminergic neurons do not respond to non-dangerous viscerosensory stimuli, such as satiating ingestion of sweetened milk or a meal (Rinaman et al., 1998), even when they produce substantial stomach distension. ...
Immune challenges can lead to marked behavioral changes, including fatigue, reduced social interest, anorexia, and somnolence, but the precise neuronal mechanisms that underlie sickness behavior remain elusive. Part of the neurocircuitry influencing behavior associated with illness likely includes viscerosensory nuclei located in the caudal brainstem, based on findings that inactivation of the dorsal vagal complex (DVC) can prevent social withdrawal. These brainstem nuclei contribute multiple neuronal projections that target different components of autonomic and stress-related neurocircuitry. In particular, catecholaminergic neurons in the ventrolateral medulla (VLM) and DVC target the hypothalamus and drive neuroendocrine responses to immune challenge, but their particular role in sickness behavior is not known. To test whether this catecholamine pathway also mediates sickness behavior, we compared effects of DVC inactivation with targeted lesion of the catecholamine pathway on exploratory behavior, which provides an index of motivation and fatigue, and associated patterns of brain activation assessed by immunohistochemical detection of c-Fos protein. LPS treatment dramatically reduced exploratory behavior, and produced a pattern of increased c-Fos expression in brain regions associated with stress and autonomic adjustments paraventricular hypothalamus (PVN), bed nucleus of the stria terminalis (BST), central amygdala (CEA), whereas activation was reduced in regions involved in exploratory behavior (hippocampus, dorsal striatum, ventral tuberomammillary nucleus, and ventral tegmental area). Both DVC inactivation and catecholamine lesion prevented reductions in exploratory behavior and completely blocked the inhibitory LPS effects on c-Fos expression in the behavior-associated regions. In contrast, LPS-induced activation in the CEA and BST was inhibited by DVC inactivation but not by catecholamine lesion. The findings support the idea that parallel pathways from immune-sensory caudal brainstem sources target distinct populations of forebrain neurons that likely mediate different aspects of sickness. The caudal medullary catecholaminergic projections to the hypothalamus may significantly contribute to brain mechanisms that induce behavioral "fatigue" in the context of physiological stressors.
... Secondary hyperalgesia is a phenomenon of increased pain report and sensitivity to nociceptive stimuli at sites distant to the primary injury, and is thought to involve central sensitization (11). The literature relating to models of musculoskeletal pain suggest that OA is associated with enhanced nociceptive transmission at the dorsal horn (12,13), a hallmark of secondary hyperalgesia. This enhanced excitability of dorsal horn neurons to nociceptive inputs is termed central sensitization, and is manifested by 1) increased response to input from an injured or inflamed region, 2) increased response from regions adjacent to or remote from the injured/inflamed region, and 3) expansion of the receptive field of the spinal cord neuron (14). ...
The groin pain experienced by patients with hip osteoarthritis (OA) is often accompanied by areas of referred pain and changes in skin sensitivity. We aimed to identify the supraspinal influences that underlie these clinical manifestations that we consider indicative of possible central sensitization.
Twenty patients with hip OA awaiting joint replacement and displaying signs of referred pain were recruited into the study, together with age-matched controls. All subjects completed pain psychology questionnaires and underwent quantitative sensory testing (QST) in their area of referred pain. Twelve of 20 patients and their age- and sex-matched controls underwent functional magnetic resonance imaging (MRI) while the areas of referred pain were stimulated using cold stimuli (12 degrees C) and punctate stimuli (256 mN). The remaining 8 of 20 patients underwent punctate stimulation only.
Patients were found to have significantly lower threshold perception to punctate stimuli and were hyperalgesic to the noxious punctate stimulus in their areas of referred pain. Functional brain imaging illustrated significantly greater activation in the brainstem of OA patients in response to punctate stimulation of their referred pain areas compared with healthy controls, and the magnitude of this activation positively correlated with the extent of neuropathic-like elements to the patient's pain, as indicated by the PainDETECT score.
Using psychophysical (QST) and brain imaging methods (functional MRI), we have identified increased activity with the periaqueductal grey matter associated with stimulation of the skin in referred pain areas of patients with hip OA. This offers a central target for analgesia aimed at improving the treatment of this largely peripheral disease.
... Previous studies have demonstrated that VLM catecholaminergic neurons, and especially the rostrally situated C1 adrenergic neurons, respond to potentially dangerous physiological challenges. These neurons respond to hypovolemia and hypotension (Dun et al., 1993;Murphy et al., 1994), hypoxia (Smith et al. 1995) glucoprivic challenge (Ritter and Dinh, 1994;Ritter et al., 2006), intragastric gavage of bitter taste receptor ligands, (Hao et al., 2009), yohimbine (Myers et al., 2005), joint inflammation (Pinto et al., 2007), opiate withdrawal (Laorden et al., 2002), seizures (Kanter et al., 1996;Silviera et al., 2000), and pharmacologic levels of CCK (Myers and Rinaman, 2002). Notably, many of these catecholaminergic neurons project directly to the paraventricular nucleus of the hypothalamus (PVN; Buller and Day, 1996;reviewed in Rinaman, 2007), which coordinates autonomic and neuroendocrine response to stress. ...
Caudal brainstem viscerosensory nuclei convey information about the body's internal state to forebrain regions implicated in feeding behavior and responses to immune challenge, and may modulate ingestive behavior following immune activation. Illness-induced appetite loss might be attributed to accentuated "satiety" pathways, activation of a distinct "danger channel" separate from satiety pathways, or both. To evaluate neural substrates that could mediate the effects of illness on ingestive behavior, we analyzed the pattern and phenotypes of medullary neurons responsive to consumption of a preferred food, sweetened milk, and to intraperitoneal lipopolysaccharide challenge that reduced sweetened milk intake. Brainstem sections were stained for c-Fos, dopamine beta-hydroxylase, phenylethanolamine-N-methyltransferase, and glucagon-like peptide-1 (GLP-1) immunoreactivity. Sweetened milk intake activated many neurons throughout the nucleus of the solitary tract (NTS), including A2 noradrenergic neurons in the caudal half of the NTS. LPS challenge activated a similar population of neurons in the NTS, in addition to rostral C2 adrenergic and mid-level A2 noradrenergic neurons in the NTS, many C1 and A1 neurons in the ventrolateral medulla, and in GLP-1 neurons in the dorsal medullary reticular nucleus. Increased numbers of activated GLP-1 neurons in the NTS were only associated with sweetened milk ingestion. Evidence for parallel processing was reflected in the parabrachial nucleus, where sweetened milk intake resulted in activation of the inner external lateral, ventrolateral and central medial portions, whereas LPS challenge induced c-Fos expression in the outer external lateral portions. Thus, signals generated in response to potentially dangerous physiological conditions seem to be propagated via specific populations of catecholaminergic neurons in the NTS and VLM, and likely include a pathway through the external lateral PBN. The data indicate that immune challenge engages multiple ascending neural pathways including both a distinct catecholaminergic "danger" pathway, and a possibly multimodal pathway derived from the NTS.
... Acute inflammation induced by intra-articular injection of a solution of PGE 2 and bradykinin induces a strong neuronal activation both at the VLM and the spinal cord. This suggests that at the initial phases of inflammation, descending inhibition from the VLM fails to inhibit the strong nociceptive transmission arising from the spinal cord (Pinto et al., 2007). With chronic inflammation (Complete Freund's adjuvant in the knee joint), innocuous stimulation of the affected paw gives rise to an inverse correlation between c-fos expression in the VLM and dorsal horn, suggesting that descending inhibition is sufficient to suppress spinal activation. ...
The dorsal horn of the spinal cord is the location of the first synapse in pain pathways, and as such, offers a very powerful target for regulation of nociceptive transmission by both local segmental and supraspinal mechanisms. Descending control of spinal nociception originates from many brain regions and plays a critical role in determining the experience of both acute and chronic pain. The earlier concept of descending control as an "analgesia system" is now being replaced with a more nuanced model in which pain input is prioritized relative to other competing behavioral needs and homeostatic demands. Descending control arises from a number of supraspinal sites, including the midline periaqueductal gray-rostral ventromedial medulla (PAG-RVM) system, and the more lateral and caudal dorsal reticular nucleus (DRt) and ventrolateral medulla (VLM). Inhibitory control from the PAG-RVM system preferentially suppresses nociceptive inputs mediated by C-fibers, preserving sensory-discriminative information conveyed by more rapidly conducting A-fibers. Analysis of the circuitry within the RVM reveals that the neural basis for bidirectional control from the midline system is two populations of neurons, ON-cells and OFF-cells, that are differentially recruited by higher structures important in fear, illness and psychological stress to enhance or inhibit pain. Dynamic shifts in the balance between pain inhibiting and facilitating outflows from the brainstem play a role in setting the gain of nociceptive processing as dictated by behavioral priorities, but are also likely to contribute to pathological pain states.
... a1-Adrenoceptors are located mainly on intrinsic neurons in the dorsal horn and, through inhibition of K þ channels and activation of Ca þþ currents, exert strong excitatory actions on nociceptive inputs. Their upregulation in models of primary afferent fiber injury and neuropathic pain has been well documented (Fuchs, Meyer, & Raja, 2001;Hord, Denson, Stowe, & Haygood, 2001;Millan, 1997;Nicholas, Hokfelt, & Pieribone, 1996;Pieribone, Nicholas, Dagerlind, & Hö kfelt, 1994), and there is evidence that persistent activation of supraspinal sites by inflammatory pain actually increases the spinal sensitivity to pain (Guo et al., 2006;Pinto, Lima, & Tavares, 2007;Porreca, Ossipov, & Genhart, 2002;Urban & Gebhart, 1999;Smith, Beyer, & Brandt, 2006). The intense a1 pronociceptive response and the relatively small a2 antinociceptive response may contribute to this increased sensitivity. ...
Previous work from our lab showed that stimulation of the lateral hypothalamus (LH) produces analgesia (antinociception) in a model of thermal nociceptive pain. This antinociceptive effect is mediated by alpha2-adrenoceptors in the spinal cord dorsal horn. However, a concomitant, opposing hyperalgesic (pro-nociceptive) response also occurs, which is mediated by alpha1-adrenoceptors in the dorsal horn. Antinociception predominates but is attenuated by the pronociceptive response. To determine whether such an effect occurs in a model of inflammatory pain, we applied mustard oil (allyl isothiocyanate; 20 microl) to the left ankle of female Sprague-Dawley rats. We then stimulated the LH using carbamylcholine chloride (carbachol; 125 nmol). The foot withdrawal latencies were measured. Some rats received intrathecal alpha-adrenoceptor antagonists to determine whether the opposing alpha-adrenoceptor response was present. Mustard oil application produced hyperalgesia in the affected paw, while the LH stimulation increased the foot withdrawal latencies for the mustard oil paw as compared to the control group. Following carbachol microinjection in the LH, WB4101, an alpha1-adrenoceptor antagonist, produced significantly longer foot withdrawal latencies compared to saline controls, while yohimbine, an alpha2-antagonist, decreased the foot withdrawal latencies from 10 min postinjection (p < .05). These findings support the hypothesis that the LH-induced nociceptive modulation is mediated through an alpha-adrenoceptor opposing response in a model of inflammatory pain.
... Coronal frozen sections, 40 m thick, were obtained and serially collected in four series. Each series was immunohistochemically processed for GABA, GAD 65/67 or KCC2 using the avidin-biotin-peroxidase method, whereas the fourth was stained with formol-thionin for laminae delimitation, as described previously [28]. The sections processed for immunohistochemistry were treated with 1% hydrogen peroxidase for 10 min for inhibition of endogenous peroxidase activity before incubation in a blocking solution of 10% normal serum in 0.3% Triton X 25% in PBS (PBST) with 0.1 M glycine for 2 h. ...
Painful diabetic neuropathy is associated to hyperexcitability and spontaneous hyperactivity of spinal cord neurons. The underlying pathophysiological mechanisms are not clear. Increases in excitatory neurotransmission at the spinal cord, involving glutamate and SP, seem to account for the abnormal neuronal activity, but inhibitory influences were never evaluated. This study aims to analyse the expression of GABA, its synthesizing enzyme glutamic acid decarboxylase (GAD) and the potassium chloride cotransporter (KCC2), in the spinal dorsal horn of streptozotocin (STZ)-induced diabetic rats. Four weeks after saline or STZ (60mg/kg) injection, animals were sacrificed and the spinal segments L2-L3 were removed and immunoreacted for GABA, GAD and KCC2, or processed for western blotting for KCC2. Densitometric quantification was performed in the superficial dorsal horn (laminae I, II and III) of immunoreacted sections and in the immunoblots. STZ rats presented a significant increase of GABA expression in laminae II and III when compared with control animals, while no differences were detected in GAD expression. A significant decrease in KCC2 expression was detected by immunohistochemistry in laminae I and II, which was confirmed by immunoblotting. Increased GABA levels, along with decrease in KCC2 expression, may underlie the abnormal neuronal activity detected in the spinal cord of diabetic rats. Reduction in KCC2 expression was shown to lead to increases in intracellular chloride concentration and, in such condition, GABA binding to GABA(A) receptor induces membrane depolarization, provoking neuronal excitation rather than inhibition. Based on these findings, we propose that a loss of GABA-mediated inhibitory tone at the spinal cord may result in neuronal hyperexcitability and spontaneous hyperactivity during diabetes.
The neurotransmitter glycine is an agonist at the strychnine‐sensitive glycine receptors. In addition, it has recently been discovered to act at two new receptors, the excitatory glycine receptor and metabotropic glycine receptor. Glycine's neurotransmitter roles have been most extensively investigated in the spinal cord, where it is known to play essential roles in pain, itch, and motor function. In contrast, less is known about supraspinal glycinergic functions, and their contributions to pain circuits are largely unrecognized. As glycinergic neurons are absent from cortical regions, a clearer understanding of how supraspinal glycine modulates pain could reveal new pharmacological targets. This review aims to synthesize the published research on glycine's role in the adult brain, highlighting regions where glycine signaling may modulate pain responses. This was achieved through a scoping review methodology identifying several key regions of supraspinal pain circuitry where glycine signaling is involved. Therefore, this review unveils critical research gaps for supraspinal glycine's potential roles in pain and pain‐associated responses, encouraging researchers to consider glycinergic neurotransmission more widely when investigating neural mechanisms of pain.
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Synopsis
The chapter describes the nociceptive system of the joint, and the generation of joint pain upon diseases in the joint. The generation of joint pain upon joint diseases considers three levels, namely pathological changes in the joint which cause hyperalgesia and pain, changes in the nociceptive system such as peripheral and central sensitization and neuropathic changes, and general/systemic factors which influence the nociceptive processing.
Visceral and somatic types of pain have been reported to manifest crucial differences not only in the experience, but also in their peripheral and central processing. However, the precise neuronal mechanisms that responsible for the modality-specific transmission of pain signals, especially at the supraspinal level, remain unclear. Very little is known also about the potential involvement of such mechanisms in the development of viscero-somatic hyperalgesia. Therefore, in the present study performed on urethane-anesthetized adult male Wistar rats we examined responses of neurons in the caudal ventrolateral medulla (CVLM)-the first site for supraspinal processing of both internal and external pain signals-to visceral (colorectal distension, CRD) and somatic (squeezing of the tail) noxious stimulations and evaluated alterations in response properties of these cells after the induction of colitis. It has been found out that the CVLM of healthy control rats, along with harboring of cells excited by both stimulations (23.7%), contained neurons that were activated by either visceral (31.9%) or somatic noxious stimuli (44.4%). In inflamed animals, the percentages of the visceral and somatic nociceptive cells were decreased (to 18.3% and 34.3%, correspondingly) and the number of bimodal neurons was increased (up to 47.4%); these alterations were associated with substantially enhanced responses of both the modality-specific and convergent CVLM neurons not only to CRD, but also to squeezing of the tail. Under these conditions, visceral and somatic pain stimuli induced similar changes in arterial blood pressure and respiratory rate, whereas in the absence of intestinal inflammation noxious CRD and tail stimulation evoked predominantly divergent autonomic reactions. The data obtained can benefit to a deeper understanding of the neuronal mechanisms that underlie differential supraspinal processing of visceral and somatic noxious stimuli and can potentially contribute to the realization of specific cardiovascular and respiratory accompaniments inherent to a particular type of pain. Therewith, results of the study elucidate colitis-induced alterations in these mechanisms, which may be responsible for the combined development of visceral hypersensitivity and somatic hyperalgesia.
Objective To observe the expression changes in substance P (SP) and c-fos in rat spinal cord after acute millimeter-wave (MMW) exposure, and explore the mechanism of thermal hyperalgesia at the spinal level. Methods The back skin of SD rats was exposed to 35 GHz MMW (40W/cm2) for 0s (control group), 30s, 1min, or 3min. The corresponding segment of the spinal cord was taken at 0min, 5min, 10min, 1h and 3h after MMW irradiation for total RNA and protein extraction. The expressions of SP and c-fos mRNA were measured by real-time RT-PCR, and the expression of c-fos protein was detected by Western blotting. Results No significant difference was found between the control group and irradiation groups in SP and c-fos mRNA expression in the corresponding segment of spinal cord after MMW irradiation for 30s. After MMW irradiation for 1min, the SP and c-fos mRNA expressions in the corresponding segment of spinal cord increased significantly at 10min time point, and then decreased to the level of control group. After MMW irradiation for 3min, the SP and c-fos mRNA expression in the corresponding segment of spinal cord increased significantly at 5min, 10min and 1h time points, and decreased to the level of control group at 3h. No significant change was found in c-fos protein expression in the corresponding segment of spinal cord after MMW irradiation for 30s and 1min. After MMW irradiation for 3min, the c-fos protein expression in the corresponding segment of spinal cord increased significantly at 5min and 10min time point, and then decreased to the level of control group. Conclusion The increase of SP expression in rat skin after MMW irradiation may be related to the increase of SP and c-fos expressions in the corresponding segment of the spinal cord induced by thermal pain stimulation.
The rostroventral medial medulla (RVM) is part of a rapidly acting spino-bulbo-spinal loop that is activated by ascending nociceptive inputs and drives descending feedback modulation of spinal nociception. In the adult rat, the RVM can facilitate or inhibit dorsal horn neuron inputs but in young animals descending facilitation dominates. It is not known whether this early life facilitation is part of a feedback loop. We hypothesised that the newborn RVM functions independently of sensory input, before the maturation of feedback control. We show here that noxious hindpaw pinch evokes no fos activation in the RVM or the periaqueductal grey (PAG) at postnatal day (P)4 or P8 , indicating a lack of nociceptive input at these ages. Significant fos activation was evident at P12, P21 and in adults. Furthermore, direct excitation of RVM neurons with microinjection of DL-homocysteic acid (DLH) did not alter the net activity of dorsal horn neurons at P10, suggesting an absence of glutamatergic drive, while the same injections caused significant facilitation at P21. In contrast, silencing RVM neurons at P8 with microinjection of lidocaine, inhibited dorsal horn neuron activity, indicating a tonic descending spinal facilitation from the RVM at this age. The results support the hypothesis that early life descending facilitation of spinal nociception is independent of sensory input. Since it is not altered by RVM glutamatergic receptor activation it is likely generated by spontaneous brainstem activity. Only later in postnatal life can this descending activity be modulated by ascending nociceptive inputs in a functional spinal-bulbo-spinal loop. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The dorsal reticular nucleus (DRt) plays a key role in facilitation of nociceptive transmission at the spinal cord. In this study we evaluated the mechanisms involved in GABA-mediated control of the DRt focusing on the role of local GABAB receptors. First, we used in vivo microdialysis to study the release of GABA in the DRt during the course of the formalin test. An increase of GABA levels in comparison to baseline values was detected in the second phase of the test. Since we previously showed that GABAB receptors are expressed by opioidergic DRt neurons which respond to nociceptive stimuli and inhibit spinally-projecting DRt neurons involved in descending pronociception, we then interfered with local GABAB receptors using gene-transfer and pharmacological approaches. Lentiviral-mediated knockdown of GABAB1a expression decreased nociceptive responses during the second phase of the test. Local administration of the GABAB receptor antagonist CGP 35348 also decreased nociceptive responses in the second phase of the test, whereas the opposite was detected after injection of the GABAB agonist baclofen. Finally, we determined the GABAergic afferents of the DRt namely those arising from its main brain afferents, which are located at the telencephalon and diencephalon. For that purpose, we combined retrograde tract-tracing from the DRt with immunodetection of GAD, the GABA-synthetizing enzyme. The higher numbers of retrogradely-labelled GAD-immunoreactive neurons were located at insular, somatosensory and motor cortices. Collectively, the results suggest that GABA acting on GABAB receptors may enhance pain facilitation from the DRt during inflammatory pain.
Pain in knee osteoarthritis (OA) has historically been attributed to peripheral pathophysiology; however, the poor correspondence between objective measures of disease severity and clinical symptoms suggests that non-local factors, such as altered central processing of painful stimuli, also contribute to clinical pain in knee OA. Consistent with this notion, recent evidence demonstrates that patients with knee OA exhibit increased sensitivity to painful stimuli at body sites unaffected by clinical pain.
In order to further investigate the contribution of altered pain processing to knee OA pain, the current study tested the hypothesis that symptomatic knee OA is associated with enhanced sensitivity to experimental pain stimuli at the knee and at remote body sites unaffected by clinical pain. We further anticipated that pain sensitivity would differ as a function of the OA symptom severity. Older adults with and without symptomatic knee OA completed a series of experimental pain assessments. A median split of the Western Ontario and McMaster Universities Index of Osteoarthritis (WOMAC) was used to stratify participants into low vs high OA symptom severity.
Compared to controls and the low symptom group, individuals in the high symptom group were more sensitive to suprathreshold heat stimuli, blunt pressure, punctuate mechanical, and cold stimuli. Individuals in the low symptomatic OA group subgroup exhibited experimental pain responses similar to the pain-free group on most measures. No group differences in endogenous pain inhibition emerged.
These findings suggest that altered central processing of pain is particularly characteristic of individuals with moderate to severe symptomatic knee OA.
Osteoarthritis is one of the most frequent, disabling and costly pathologies of modern society. One of the main aims of osteoarthritis management is pain control and functional ability improvement. The exact cause of osteoarthritis pain remains unclear. In addition to the pathological changes in articular structures, changes in central pain processing or central sensitization appear to be involved in osteoarthritis pain. The latter calls for a broader approach to the management of patients with osteoarthritis. Yet the scientific literature offers few information addressing the treatment of central sensitization specifically in osteoarthritis patients. Interventions like cognitivebehavioral therapy and neuroscience education potentially target cognitive-emotional sensitization (and descending facilitation), while centrally acting drugs and exercise therapy can improve endogenous analgesia (descending inhibition) in patients with osteoarthritis. Future studies should assess these new treatment avenues.
Glyceryl trinitrate (GTN) infusion is a reliable method to provoke migraine-like headaches in humans. Previous studies have simulated this human model in anaesthetized or in awake rodents using GTN doses 10,000 times higher than used in humans. The relevance of such toxicological doses to migraine is not certain. Anaesthesia and low blood pressure caused by high GTN doses both can affect the expression of nociceptive marker c-fos. Therefore, our aim was to simulate the human GTN migraine model in awake rats using a clinically relevant dose.
Awake rats were infused with GTN (4 µg/kg/min, for 20 min, i.v.), a dose just 8 times higher than in humans. mRNA and protein expression for c-fos were analysed in the trigeminal vascular system at various time points using RT-PCR and immunohistochemistry, respectively.
A significant upregulation of c-fos mRNA was observed in the trigeminal nucleus caudalis at 30 min and 2 h that was followed by an upregulation of Fos protein in the trigeminal nucleus caudalis at 2 h and 4 h after GTN infusion. Pre-treatment with sumatriptan attenuated the activation of Fos at 4 h, demonstrating the specificity of this model for migraine.
We present a validated naturalistic rat model suitable for screening of acute anti-migraine drugs.
Targeting supraspinal pain control centers by gene transfer is known to induce sustained analgesia. In this study, we evaluated the effects of injecting a Herpes Simplex Virus type 1 vector which expresses enkephalin (HSV-ENK vector) in the lateralmost part of the caudal ventrolateral medulla (VLMlat), a pain control center that exerts mainly descending inhibitory effects on pain modulation. Overexpression of enkephalin at the VLMlat reduced the number of flinches during the early and delayed phases of the formalin test and decreased c-fos expression in the spinal cord. These antinociceptive effects were detected at 2 and 10days after injection of HSV-ENK in the VLMlat and were completely reversed by local administration of naloxone. Virally driven-enkephalin was expressed from transduced neurons located in the VLMlat and, at lower extent, in the rostral ventromedial medulla. Our results show that HSV-mediated expression of enkephalin in the VLMlat induced antinociceptive effects, likely due to an enhancement of the opioidergic input to the VLMlat which accounted for descending inhibition of the nociceptive transmission at the spinal cord. This study also demonstrates the value of HSV-1 derived vectors to manipulate, in a sustained and directed manner, pain modulatory pathways in the brain, which is important in the study of supraspinal pain control circuits.
Painful diabetic neuropathy (PDN) induces neuronal hyperactivity at the spinal cord and periaqueductal gray (PAG), a key area in descending nociceptive modulation. Since the PAG uses relay stations at serotoninergic and noradrenergic brainstem areas, we determined the serotonin and noradrenaline levels at the spinal cord of streptozotocin-diabetic rats and at those brainstem areas (serotoninergic rostroventromedial medulla and noradrenergic A(5) and A(7) cell groups). Since, during diabetes, the levels of insulin growth factor 1 (IGF1) decrease, reducing its neurotrophic effect in the brain, we also studied the effects of IGF1 treatment. One week after diabetes induction, subcutaneous injections of IGF1 (2.5mg/kg) were performed during 3 weeks. Body weights, glycemia, and mechanical nociception were weekly evaluated until the end of the study, the time when the animals were subjected to a modified formalin test to study chemical allodynia. Serotonin and noradrenaline levels were quantified by ELISA at the spinal cord, whereas at the brainstem, the quantification was performed by immunohistochemistry against, respectively, tryptophan hydroxylase (TpH) or tyrosine hydroxylase (TH). STZ-diabetic rats exhibited mechanical hyperalgesia and chemical allodynia, along with higher spinal levels of serotonin and noradrenaline and higher numbers of neurons expressing TpH at the RVM and TH at the A(5) noradrenergic cell group. Treatment with IGF1 prevented the behavioral signs of PDN and reversed the neuronal hyperactivity at the spinal cord and ventrolateral PAG and the neurochemical changes at the spinal cord and at the brainstem. Based on the facilitatory role of serotoninergic and noradrenergic descending modulation during chronic pain, the increased serotonin and noradrenaline innervation of the dorsal horn in STZ-diabetic rats may probably account for enhanced pain during PDN. The benefits of IGF1 in PDN are probably due to blockade of the increased peripheral input to the somatosensory system, but direct central actions cannot be discarded. The value of IGF1 in PDN treatment deserves further evaluation.
Unlabelled:
Hypertension-associated hypoalgesia is widely recognized in acute pain conditions. In chronic pain states, however, the relationship between blood pressure and pain sensitivity is still ill-defined, with different authors reporting negative, positive, or even no relationship at all. This work addresses this issue, using complete Freund's adjuvant (CFA)-induced monoarthritis in different models of hypertension: Spontaneous (spontaneously hypertensive rats, SHR), induced by infusion of angiotensin II (ANG) or 1,3-dipropyl-8-sulfophenylxanthine (DPSPX, an adenosine receptors' antagonist), and renal artery ligation (RAL). Nociceptive responses associated with monoarthritis were evaluated by different behavioral tests (von Frey, ankle-bend and CatWalk) and by quantification of Fos expression at the dorsal horn upon noxious stimulation. In all hypertension models, higher von Frey thresholds and lower Fos expression were detected in hypertensive rats with chronic inflammatory pain, as compared to normotensive monoarthritic rats. In SHR and DPSPX, but not ANG or RAL models, hypertensive animals displayed lower inflammation than normotensives. Ankle-bend and CatWalk results indicated lower pain sensitivity in hypertensive rats only in SHR and DPSPX models. The present study shows the importance of using multiple models of hypertension, and evaluating pain responses by various methods, to better understand the complexity of the interactions between pain and cardiovascular regulatory systems.
Perspective:
This study used different models of hypertension to investigate whether chronic pain alters the normal integration of cardiovascular and pain regulatory systems. A complete understanding of the mechanisms underlying the complex interactions between these systems may disclose future therapeutic approaches to treat hypertension/chronic pain comorbidity states.
Painful diabetic neuropathy may be due to impairments in descending modulation of nociceptive transmission at the spinal cord. In the present study, streptozotocin diabetic rats (STZ rats) with neuropathic symptoms (mechanical hypersensitivity) were used to perform a time-course evaluation of neuronal activity at the spinal dorsal horn and at the periaqueductal grey matter (PAG), a major brainstem area of pain modulation. The expression of Fos protein, a marker of nociceptive activation, progressively increased at the spinal dorsal horn at 4 and 10 weeks. At the PAG, increases in Fos expression were detected until the 4th week, with a reversal to baseline values at 10 weeks in all areas except the ventrolateral PAG. Co-localisation of Fos with NeuN ascertained the neuronal nature of Fos-expressing cells at the spinal cord and PAG. Four weeks after diabetes induction, the effects of gabapentin (i.p. injection of 50mg/kg, daily during 3 days) were assessed. Gabapentin decreased Fos expression at the spinal cord and PAG and reversed mechanical hypersensitivity. The present study shows that diabetic neuropathy is accompanied by a progressive increase of the spontaneous neuronal activity at the spinal cord. Changes in descending modulation of nociceptive transmission from the PAG are likely to occur during diabetic neuropathy, probably with exacerbation of facilitatory actions. The effects of gabapentin in reversing the behavioural signs of diabetic neuropathy and neuronal hyperactivity in the spinal cord and PAG reinforce the central causes of diabetic neuropathy and point to the central targets of the drug.
Osteoarthritis (OA) is a painful and degenerating progressive disease of the joints which affects millions of patients worldwide. The cause of OA is largely unknown. Among the potential therapies for the symptomatic treatment of OA, the intra-articular administration of a specific bradykinin (BK) B2 receptor antagonist has been reported to produce a long lasting analgesic effect in patients affected by knee OA. BK is a vasodilator and inflammatory nonapeptide which is generated in OA synovium. It contributes to the initiation and maintenance of inflammation, to exciting and sensitizing sensory nerve fibres, thus producing pain, and to activating synoviocytes and chondrocytes which are the main cells involved in the homeostasis of synovial fluid and cartilage, respectively. Moreover, BK synergistically potentiates the effects produced by pro-inflammatory cytokines. Biochemical and preclinical evidence supporting the therapeutic relevance of B2 receptor blockade in OA pathophysiology are reviewed in this publication.
During chronic pain, the supraspinal pain modulatory system undergoes plastic changes with enhancement of facilitation transmission at the spinal cord. The changes induced by chronic pain at descending modulation often affect opioidergic modulation, and were never described for a key facilitatory component of the system, the dorsal reticular nucleus (DRt). Neurochemical characterization of the DRt-spinal pathway showed that delta-opioid receptors are positioned as to indirectly modulate the activity of non-projecting DRt neurons, whereas neurons expressing mu-opioid receptors project to the spinal dorsal horn or act as interneurons, the latter of which co-expressing GABA(B) receptors. In monoarthritic rats, the expression of mu-opioid receptors decreased in the DRt whereas the levels of endogenous enkephalin remained unaltered. To increase the opioidergic inhibition of the DRt, we locally injected selective agonists of delta- and mu-opioid receptors or a viral vector containing the human preproenkephalin transgene. Injection of the Herpes Simplex viral vector encoding preproenkephalin induced thermal hypoalgesia in non-inflamed animals and hyperalgesia in monoarthritic rats. The opioid agonists [D-Ala(2), Glu(4)]-deltorphin (DELT) and [D-Ala(2), NMePhe(4)Gly-ol(5)]-enkephalin (DAMGO) induced thermal hyperalgesia in both non-inflamed and monoarthritic rats, but with lower doses in the latter group. The present study shows that opioidergic neurons at the DRt are modulated by GABAergic cells herein controlling the descending facilitation of pain transmission. The DRt exhibits plastic changes during chronic inflammatory pain, with decrease opioid receptor expression which may account for increased descending facilitation during chronic pain.
Investigators have demonstrated pressor areas in the medullas of various species. The present study precisely localized the pressor area in the caudal medulla of the rat and determined its projections to the caudal and rostral ventrolateral medulla. The caudal medulla first was mapped grossly in rats with injections (30 nl) of glutamate (30-, 15-, and 7.5-nmol doses) placed 0.5, 1.0, and 1.5 mm caudal to the calamus scriptorius, 1.0, 1.5, and 2.0 mm lateral to the midline, and 1.8, 1.7, and 1.6 mm ventral to the dorsal medullary surface, respectively, and their arterial pressures were recorded. One of these nine injections showed significant increases in arterial pressure. We micromapped this area with a total of 27 injections of glutamate (10 nl; 5 nmol) placed 300 μm apart at 3 different dorsoventral levels. This micromapping study pinpointed the precise location of caudal pressor area (CPA) neurons in a restricted region lateral to the caudal end of the lateral reticular nucleus and ventromedial to the medullary dorsal horn near the level of the pyramidal decussation. Injections of glutamate into this spot, 1.0 mm caudal to the calamus scriptorius, 2.0 mm lateral to the midline, and 1.7 mm ventral from the dorsal surface of the medulla, induced significant increases in arterial pressure. The neuroanatomic connections of neurons in the CPA to the ventrolateral medulla were then investigated with iontophoretic injections of either the anterograde tracer biotinylated dextran amine (BDA) made into the CPA or the retrograde tracer Fluoro-Gold (FG) injected into either the caudal or rostral ventrolateral medulla. BDA injections resulted in bouton-laden fibers throughout both caudal and rostral portions of the ventrolateral medulla. Either of the F G injections resulted in numerous spindle-shaped neurons interspersed between the longitudinal fiber bundles running through the CPA area. The proximity of the CPA neurons to the A1 catecholaminergic cell group is discussed.
Four experiments examined the role of the nucleus tractus solitarius (NTS) and subjacent nucleus reticularis ventralis (NRV) in the production of antinociception. Experiment 1 showed that microinjection of glutamate (50 nmol) into the caudal NTS resulted in inhibition of the tail-flick reflex, hypotension, and mild bradycardia, whereas microinjection of glutamate into the rostral NTS resulted only in hypotension and mild bradycardia. Microinjections of glutamate into the NRV resulted in inhibition of the tail-flick reflex, hypertension, and mild bradycardia. Experiment 2 demonstrated that the magnitude of the antinociceptive and cardiovascular responses resulting from glutamate microinjections into both the depressor and pressor regions were dose dependent. Experiment 3 showed that the antinociceptive effects resulting from microinjections of glutamate into either the depressor or pressor regions could not be antagonized by phentolamine (30 micrograms), methysergide (30 micrograms), or naloxone (30 micrograms) alone, but the combined intrathecal administration of phentolamine and methysergide (7.5, 15, or 30 micrograms of each) attenuated the antinociception resulting from microinjection of glutamate into either depressor or pressor regions in a dose-dependent fashion. Experiment 4 showed that systemic administration of hexamethonium blocked the pressor response produced by microinjection of glutamate into the NRV but did not reduce the antinociceptive effect of the microinjection. These findings are consistent with a role for the NTS and NRV in the production of antinociception.
A laminar cytoarchitectonic scheme of the lower thoracic and lumbosacral segments of the rat spinal cord is presented in which Rexed's principles for the cat are applied. The material consists of 80-micron-thick sections stained with toluidine blue or according to van Gieson and 2-micron-thick sections stained with p-phenylenediamine or toluidine blue. The cytoarchitectonic organization of the rat spinal cord was found to be basically similar to that of the cat, although certain differences exist--for example, in the extension of the laminae. In addition to the laminar scheme, the distribution of certain cell groups, Lissauer's tract, and the pyramidal tract were investigated.
The involvement of cAMP-responsive element-binding protein (CREB) signaling in tissue injury-induced inflammation and hyperalgesia has been characterized by measuring phosphorylation of CREB at serine-133 (CREB Ser133) using a specific antibody. In the unstimulated state, unphosphorylated CREB was observed in most nuclei of spinal neurons except for motor neurons, where only a small portion of neurons were stained. A few dorsal root ganglion (DRG) neurons were also CREB-positive. After a unilateral injection of formalin into the hindpaw, a strong and bilateral phosphorylation of CREB Ser133 was induced, as assessed by both immunohistochemistry and Western blot. PhosphoCREB (pCREB)-positive neurons were found in laminae I, II, V, and X of spinal cord on both sides. CREB phosphorylation was very rapid and reached peak levels within 10 min of formalin treatment, whereas few pCREB-positive neurons were seen in unstimulated spinal cord. The induction of pCREB was predominantly postsynaptic, because only 5% of DRG neurons were labeled after inflammation. In contrast to CREB phosphorylation, the induction of c-Fos expression reached peak levels 2 hr after formalin treatment and c-Fos induction was mainly ipsilateral. Both formalin-evoked CREB phosphorylation and c-Fos expression in the spinal cord were suppressed by pretreatment with the NMDA receptor antagonist MK-801 (3.5 mg/kg, i.p.) or halothane anesthesia.
These results suggest that CREB signaling may play a role in the long-term facilitation of spinal cord neurons after hyperalgesia. Furthermore, our results indicate that CREB phosphorylation may be necessary but not sufficient for c-fos induction.
Calcium ions are the principal second messenger in the control of gene expression by electrical activation of neurons. However, the full complexity of calcium-signaling pathways leading to transcriptional activation and the cellular machinery involved are not known. Using the c-fos gene as a model system, we show here that the activity of its complex promoter is controlled by three independently operating signaling mechanisms and that their functional significance is cell type-dependent. The serum response element (SRE), which is composed of a ternary complex factor (TCF) and a serum response factor (SRF) binding site, integrates two calcium-signaling pathways. In PC12 cells, calcium-regulated transcription mediated by the SRE requires the TCF site and is not inhibited by expression of the dominant-negative Ras mutant, RasN17, nor by the MAP kinase kinase 1 inhibitor PD 98059. In contrast, TCF-dependent transcriptional regulation by nerve growth factor or epidermal growth factor is mediated by a Ras/MAP kinases (ERKs) pathway targeting the TCF Elk-1. In AtT20 cells and hippocampal neurons, calcium signals can stimulate transcription via a TCF-independent mechanism that requires the SRF binding site. The cyclic AMP response element (CRE), which cooperates with the TCF site in growth factor-regulated transcription, is a target of a third calcium-regulated pathway that is little affected by the expression of RasN17 or by PD 98059. Thus, calcium can stimulate gene expression via a TCF-, SRF-, and CRE-linked pathway that can operate independently of the Ras/MAP kinases (ERKs) signaling cascade in a cell type-dependent manner.
In experiment 1, quantitative regional comparisons of the antinociceptive and cardiovascular responses produced by electrical stimulation in the caudal medulla, including regions such as the nucleus tractus solitarius (NTS), nucleus reticularis ventralis (NRV), nucleus reticularis gigantocellularis (NRGC), nucleus reticularis paragigantocellularis (NRPGC), nucleus raphe obscurus (NRO), and medial portions of the lateral reticular nucleus (LRN), were made in the rat. Electrical stimulation in all of these regions resulted in inhibition of the nociceptive tail-flick reflex, although the threshold intensity for inhibition was greater for sites in NTS compared to many sites ventral to the NTS. Antinociception was generally accompanied by an increase in mean arterial blood pressure, with the exception of sites in the NRO, where depressor responses were evoked by stimulation. Detailed comparisons between the NTS and NRV revealed that greater intensities of electrical stimulation were required to produce antinociception for sites in the NTS as compared to the NRV. There were no significant differences in threshold intensities for antinociception as a function of rostrocaudal subdivisions of the NTS, but the lateral subdivision of the NTS was significantly more efficacious than the medial subdivision. This mediolateral difference within NTS was primarily due to stimulation in medial sites producing overt movements in some animals, probably due to stimulation of adjacent midline nuclei or pathways. Within the NRV, thresholds for inhibition of the tail-flick reflex were greater for sites in the dorsal subdivision as compared to the ventral subdivision, which contains spinopetal projections from the NRM. The slopes of the lines of recruitment for inhibition of the tail-flick reflex at stimulation sites in either the NTS or NRV were both very steep, similar to other forms of antinociception. In experiment 2, the pulse duration of electrical stimulation was varied for sites of stimulation in the lateral NTS and NRV to generate strength-duration curves. This experiment confirmed that stimulation sites in the lateral NTS required greater current intensities to inhibit the tail-flick reflex than sites in the NRV. However, the chronaxies derived from the strength-duration functions for the NTS or NRV were both approximately 170 μsec, indicating that the antinociceptive effects in these regions may not be exclusively due to the stimulation of fibers of passage. These results are discussed in terms of the role of the NTS, NRV, and caudal medulla in the modulation of nociceptive responses and cardiovascular function.
This chapter presents the mechanisms and hypotheses of acute to chronic pain. This chapter considers three major stages or phases of pain and proposed that different neurophysiological mechanisms are involved depending on the nature and time course of the originating stimulus. These three phases are: (1) the processing of a brief noxious stimulus; (2) the consequences of prolonged noxious stimulation leading to tissue damage and peripheral inflammation; and (3) the consequences of neurological damage, including peripheral neuropathies and central pain states. However, it is important to point out that these phases are not exclusive, and that at any given time several of the neurophysiological mechanisms that underlie these pain states may coexist in the same individual. However, it is clear that the neurochemistry of the nociceptive system changes with noxious input, and there is increasing evidence that the pharmacology of pain changes as one moves as different transmitter systems are recruited as part of the different mechanisms that underlie the different pain state.
The effect of graded inflammatory stimuli (intraplantar-carrageenan, 0.2, 1, and 6 mg/150 μl) on paw edema and c-Fos protein expression at two levels of the spinoparabrachial pathway, the spinal cord and parabrachial area (PB), were studied. The present study, in awake rats, is an extension of previous study (Bester et al. [1997] J. Comp. Neurol. 383:439–458) which evaluated, in anesthetized rats, the effect of graded cutaneous heat stimulation on c-Fos-expression at the same levels.At the spinal level, the c-Fos-protein-like-immunoreactive (c-Fos-LI) neurons were located primarily in superficial laminae ipsilateral to intraplantar carrageenan. The number of c-Fos-LI neurons increased dose dependently (r = 0.973, n = 24) for carrageenan, from a number close to zero for the saline injection. At the PB level, c-Fos was predominantly expressed contralateral to intraplantar carrageenan. c-Fos-LI neurons were located primarily around the pontomesencephalic junction in (i) a restricted pontine area, centered in the lateral crescent, and including an adjacent part of the outer portion of the external lateral subnucleus, and (ii) the mesencephalic superior lateral subnuclei. The number of c-Fos-LI neurons in the PB area was correlated with that in the superficial laminae (r = 0.935, n = 24) and with the paw edema (r = 0.931, n = 24). No significant changes in c-Fos expression were observed in the nucleus of the solitary tract and ventrolateral medulla.The close correlation between c-Fos expression at both the spinal and PB levels and inflammatory edema provides further evidence for the involvement of spinoparabrachial pathway in inflammatory nociceptive processes. The present results are congruent with the existence of electrophysiologically demonstrated spinoparabrachio-amygdaloid and -hypothalamic nociceptive pathways. J. Comp. Neurol. 397:10–28, 1998. © 1998 Wiley-Liss, Inc.
The purpose of this study was to compare effects of various anesthetic drugs on expression of Fos protein in the supraspinal neurons of rats. Optimum anesthesia was maintained under monitoring of EEG. Degree of Fos expression was evaluated by counts of Fos-immunoreactive (ir) neurons. Urethane induced the largest amount of Fos-ir neurons (n = 8439), while fentanyl/midazolam the smallest (n = 112). α-Chloralose, halothane, pentobarbital sodium and urethane/α-chloralose induced amounts in between. The results indicate that fentanyl/midazolam is the most recommendable anesthetic drug in the Fos expression study.
Control of pain perception, essential for organism surviving and recovery from disease, is exerted by higher brain centers integrating nociception with emotional and cognitive information and modulating the brainstem-spinal feedback loops that regulate spinal nociceptive transmission. Development of chronic pain deregulates the forebrain-brainstem-spinal pain control system, which leads to neuroplasticity and disruption of a balanced brain-spinal communication. Targets for impeding pain chronification are being developed using the manipulation of the cross talk between brain and dorsal horn, at both sites of the loop.Section editors:Frank Porreca – University of Arizona, Tucson, USAMichael Ossipov – University of Arizona, Tucson, USA
Increases in neuronal activity in response to tissue injury lead to changes in gene expression and prolonged changes in the nervous system. These functional changes appear to contribute to the hyperalgesia and spontaneous pain associated with tissue injury. This activity-dependent plasticity involves neuropeptides, such as dynorphin, substance P and calcitonin gene-related peptide, and excitatory amino acids, such as NMDA, which are chemical mediators involved in nociceptive processing. Unilateral inflammation in the hindpaw of the rat results in an increase in the expression of preprodynorphin and preproenkephalin mRNA in the spinal cord, which parallels the behavioral hyperalgesia associated with the inflammation. Cellular intermediate-early genes, such as c-fos, are also expressed in spinal cord neurons following inflammation and activation of nociceptors. Peripheral inflammation results in an enlargement of the receptive fields of many of these neurons. Dynorphin applied to the spinal cord also induces an enlargement of receptive fields. NMDA antagonists block the hyperexcitability produced by inflammation. A model has been proposed in which dynorphin, substance P and calcitonin gene-related peptide enhance excitability at NMDA receptor sites, leading first to dorsal horn hyperexcitability and then to excessive depolarization and excitotoxicity.
C-fos is a proto-oncogene that is expressed within some neurons following depolarization. The protein product, fos, has been proposed as an anatomical marker for neuronal activity following noxious peripheral stimulation. However, the literature on noxious-stimulus induced fos expression contains several puzzling observations on the time course and laminar distribution of neuronal labeling within the spinal cord. This study has analyzed the effect of stimulus duration on the expression of fos-like immunoreactivity (FLI) within the spinal cord of anesthetized rats. In order to examine the time course of fos expression following brief periods of stimulation, we required a type of stimulus that was intense enough to activate nociceptors but that did not produce tissue damage. We have therefore employed pulsed, high intensity electrical stimulation, with stimulus durations ranging from 3 s to 24 h. The results indicate that stimulus duration has a profound effect upon the number of labeled cells, the intensity of neuronal labeling, the laminar pattern of FLI, and the time course of fos expression. Brief stimulation periods induce relatively few and relatively lightly labeled neurons, located predominantly within the most superficial laminae of the dorsal horn. Maximal immunoreactivity appears approximately 2 h after stimulation has ceased, and disappears within hours. Continuous stimulation produces many more labeled cells, darker labeling, and FLI within both dorsal and ventral laminar regions. Maximal FLI is seen after approximately 4.5 h of continuous stimulation, with reduction in the number of labeled cells thereafter. These data indicate that the results of any study employing c-fos as a marker for neuronal activity may be affected by the duration of the exciting stimulus.
Increased levels of preproenkephalin mRNA in spinal cord neurons induced by peripheral tissue inflammation were examined usingin 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 doublelabeled 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.
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)
The mechanism by which Ca2+ mediates gene induction in response to membrane depolarization was investigated. The adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) was shown to function as a Ca(2+)-regulated transcription factor and as a substrate for depolarization-activated Ca(2+)-calmodulin-dependent protein kinases (CaM kinases) I and II. CREB residue Ser133 was the major site of phosphorylation by the CaM kinases in vitro and of phosphorylation after membrane depolarization in vivo. Mutation of Ser133 impaired the ability of CREB to respond to Ca2+. These results suggest that CaM kinases may transduce electrical signals to the nucleus and that CREB functions to integrate Ca2+ and cAMP signals.
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.
Freund's adjuvant induced polyarthritis in rats has been used extensively to study pain processes of long duration. There are limitations of this model for chronic studies of pain/arthritis since the severe systemic changes provoke ethical concerns and also affect behaviour, physiology and biochemistry. Attempts to limit adjuvant-induced arthritis by plantar injection of the inoculum have been made. In this model, however, the process evolved to produce widespread polyarthritis if followed for the 6-plus-weeks necessary for chronic studies. Therefore, although it offers the researcher a reliable limited model of inflammation and nociception at the outset, for longer studies it may have all the disadvantages of the polyarthritic rat. The purpose of the present study was to produce a limited arthritic process in rats, stable over 6 weeks and suitable for behavioural and neurochemical studies of various chronic pain treatment methods. Injection (0.05 ml) of complete adjuvant containing 300 micrograms Mycobacterium butyricum in the tibio-tarsal joint produces a predictable monoarthritis, stable clinically and behaviourly from weeks 2 through 6 post injection. As revealed by clinical observations and X-ray examinations, the arthritis produced was limited anatomically, pronounced, prolonged and stable. A marked increase in sensitivity to paw pressure was seen in the affected limb. Animals gained weight and remained active, indicating little systemic disturbance as opposed to polyarthritic rats. We propose this limited model of arthritis as a suitable alternative to the polyarthritic rat for prolonged studies.
Noxious stimulation provokes the activation of genes that are thought to play a crucial role in the phenomena of stress and pain. Among these is the prodynorphin gene. By double-labeling in situ hybridization/immunohistochemistry, we show that increased prodynorphin gene expression is preceded, in the same neurons, by an early induction of c-fos. Inspection of the prodynorphin promoter region revealed the presence of several AP-1-like sequences. We demonstrate that only one of these sites is a functional AP-1 element. It is constituted by the noncanonical TGACAAACA sequence, in which the palindromic structure is partly conserved by the 3' terminal CA dinucleotide. Transfection experiments in NCB20 neuroblastoma cells indicated that this site is a target of Fos/Jun trans-activation. Our results suggest that Fos/Jun oncoproteins may function as third messengers in the signal transduction mechanisms of stress/pain processes.
The mechanism by which the calcium influx signal, triggered by membrane depolarization, is transduced to the nucleus to activate c-fos proto-oncogene transcription has been characterized. A calcium response element (CaRE) that is indistinguishable from a cAMP response element (CRE) mediates transcriptional inducibility by depolarization. Its cognate transcription factor CREB is the target for both calcium and cAMP signals. CREB is rapidly phosphorylated in response to depolarization or cAMP, at a site known to be important for the transcriptional activating function of this protein. The convergent effects of calcium and cAMP on CREB activation are mediated by distinct protein kinase signaling pathways. CREB and its binding site, the Ca/CRE, can thus function as a regulatory element that integrates both calcium and cAMP signals in the control of gene expression.
Fos and Jun form a heterodimeric complex that associates with the nucleotide sequence motif known as the AP-1 binding site. Although this complex has been proposed to function as a transcriptional regulator in neurons, no specific target gene has yet been identified. Proenkephalin mRNA increased in the hippocampus during seizure just after an increase in c-fos and c-jun expression was detected. Fos-Jun complexes bound specifically to a regulatory sequence in the 5' control region of the proenkephalin gene. Furthermore, c-fos and c-jun stimulated transcription from this control region synergistically in transactivation assays. These data suggest that the proenkephalin gene may be a physiological target for Fos and Jun in the hippocampus and indicate that these proto-oncogene transcription factors may play a role in neuronal responses to stimulation.
Peripheral inflammation produces a rapid elevation (within 4 h) in preprodynorphin mRNA in neurons of the dorsal spinal cord and an even more rapid elevation in c-fos proto-oncogene mRNA (within 30 min). During this period a relatively modest increase is also observed in spinal cord preproenkephalin mRNA. Previous anatomical studies have shown that the neurons in which these transcripts increase have overlapping distributions. Assuming that these events occur in the same cells, it suggests the possibility that newly synthesized c-fos protein may participate in transcriptional regulation of opioid genes in spinal cord.
Autoradiographic studies localize [3H]bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. [3H]Bradykinin labeling is also observed over myocardial/coronary visceral afferent fibers. The localization of [3H]bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.
Transganglionic transport of wheatgerm agglutinin conjugated horseradish peroxidase (WGA-HRP) was used to reveal the central distribution of terminals of primary afferent fibers from peripheral nerves innervating the hind leg of the rat. In separate experiments the sizes and locations of cutaneous peripheral receptive fields were determined by electrophysiological recording techniques for each of the nerves that had been labeled with WGA-HRP. By using digital image analysis, the sizes and positions of the peripheral receptive fields were correlated with the areas of superficial dorsal horn occupied by terminals of primary afferents from each of these receptive fields.
The need for rapid histological feedback on neural tissue is ever present. Although there are several stains which can be readily used for staining either cell bodies or fiber tracts, adequate contrasting stains which are both rapid and easy to apply are not generally available. In 1936 Chang presented a technique for whole brains utilizing the metachromatic properties of thionin. Unfortunately this procedure was very time consuming. For the last several years we have worked with several variations of this stain and have found that thionin can be reliably used as a polychrome stain for sections of neural tissue obtained from a freezing microtome.
The properties of the neuronal responses to different types of mechanical peripheral stimulation were studied during electrode penetrations in the first somatosensory cortex of anaesthetized rats with polyarthritis. Very few neurons were driven by light cutaneous stimulation (such as brushing) or by intense mechanical stimulation. Most of the neurons were driven by joint movement and/or moderate pressure on the skin. These neurons could be found in all cortical layers, the majority being located in layer V. These results contrast sharply with the properties and laminar distribution of the different functional categories of cortical neurons, as observed in normal animals.
Rats rendered polyarthritic by injection of Mycobacterium butyricum into the tail were used as a model for the study of 'chronic pain'. In such rats unitary responses of ventrobasal thalamic neurons to somatic stimulations were dramatically modified by comparison to those described in normal rats investigated in the same anaesthetic conditions. (1) Only the neurons with receptive fields located on inflamed areas (168/194 in 33 rats) have been considered in this study. 27/168 activated only by brushing displayed the classical properties of lemniscal responses; only 20/168 were activated exclusively by intense cutaneous stimuli and 13/168 already activated by light cutaneous stimuli had enhanced discharges when the stimulus intensity was increased. By contrast numerous units (108/168) were excited by mild stimulations applied to the joints or to adjacent cutaneous areas (82 were driven by joint movement and/or mild lateral pressure on the articulation, 26 by brushing the overlapping skin); these responses presented atypical characteristics and displayed unusual patterns with very long afterdischarges of duration several times that of the stimulus. (2) In 20 additional arthritic rats, responses to transcutaneous electrical stimulation (TES) and/or to noxious heat, were obtained for 34 neurones responding to joint stimuli. (a) 16 of 18 neurones tested with transcutaneous electrical stimulation had latencies of 25-100 ms, and thresholds of 1-4 mA (width of shock 2 ms). (b) Neurones activated by joint stimuli frequently responded to noxious heat (radiant or waterbath). Initially, their response thresholds tested in 16 neurones were higher by about 4 degrees C than those of 'noxious' VB neurones in normal rats; however, following sensitization to heat, thresholds were decreased by 4 degrees C. For 8 neurones there was a linear relation between stimulus intensity and responses. (3) Several different factors which could explain the important modification of neuronal responses in VB complex of arthritic rats by comparison with normal are proposed in the discussion.
Peripheral tissue damage or nerve injury often leads to pathological pain processes, such as spontaneous pain, hyperalgesia and allodynia, that persist for years or decades after all possible tissue healing has occurred. Although peripheral neural mechanisms, such as nociceptor sensitization and neuroma formation, contribute to these pathological pain processes, recent evidence indicates that changes in central neural function may also play a significant role. In this review, we examine the clinical and experimental evidence which points to a contribution of central neural plasticity to the development of pathological pain. We also assess the physiological, biochemical, cellular and molecular mechanisms that underlie plasticity induced in the central nervous system (CNS) in response to noxious peripheral stimulation. Finally, we examine theories which have been proposed to explain how injury or noxious stimulation lead to alterations in CNS function which influence subsequent pain experience.
Rats, receiving an intrathecal pretreatment of oligodeoxynucleotide complementary to c-fos mRNA (antisense), showed no increases in Fos protein or preprodynorphin messenger RNA in the outer laminae of the lumbar spinal cord when challenged 4 h later with a 50 microliters intraplantar injection of 5% formalin. Animals pretreated with saline or sense oligodeoxynucleotide showed marked increases in Fos protein (2 h after formalin challenge) and preprodynorphin mRNA (20 h after formalin challenge) in the lumbar region of the cord ipsilateral to the side of the injection. The behavioural consequences of antisense pretreatment were an increase in the formalin-induced licking/biting responses during the tonic, but not the acute phase. These observations could be interpreted as representing a sequence of events beginning with the formalin-induced increase in the transcription factor Fos, which in turn increases the synthesis of preprodynorphin messenger RNA resulting in the production of the dynorphin opioid peptides which then exert a modulatory antinociceptive action.
In the present work, we recorded the neuronal properties of the ventromedial medulla, a brainstem structure involved in the descending spinal control systems related to nociception, in awake, freely moving healthy and polyarthritic rats. These animals were rendered polyarthritic with a subcutaneous administration of the Freund's adjuvant into the tail, and studied at 20 and 30 days post-inoculation. At the ventromedial medulla level, the single-unit activities were recorded by means of a chronically implanted device supporting a 50 microns platinum-iridium wire as the recording electrode. With a total of 308 recorded neurons, we determined that in both healthy rats, i.e. animals having received mineral oil only and arthritic rats, there were ventromedial medulla units with common physiological properties, but also changes. In agreement with the results from anesthetized arthritic rats at spinal and thalamic levels, the systematic analysis of the responses to light touch and mechanical shock revealed that the 'multimodal, multireceptive' units, excited by innocuous and noxious stimuli, were much more responsive to both modalities in arthritic rats. Approximately 7% of these neurons displayed a 'paroxysmal' spontaneous activity, also reported in the literature for other structures. In addition, we recorded a significant number of neurons inhibited or excited-inhibited by innocuous and noxious cutaneous stimulations, and a few with a regular spontaneous activity, also responding, which has never been the case in healthy rats. We conclude that a peripheral chronic inflammation, such as arthritis, can produce changes of the ventromedial medulla neuronal properties, as compared to healthy animals. Consequently, in addition to its classical role in the spinal control of nociception, the ventromedial medulla is able to develop some form of plasticity in the case of persistent pain of peripheral origin.
The influence of stimulus duration on spinal induction of the c-fos proto-oncogene was analysed in the rat by pinching or heating the skin for periods varying from 20 s to 2 h. At stimulation periods shorter than 20 min, c-fos activation occurred in laminae I-IIi following mechanical stimulation and I-IIo following thermal stimulation. Mechanical stimulation produced delayed activation in laminae III-IV, V and VII at 30 min, 60 min and 2 h, respectively, and thermal stimulation in lamina IIi at 50 min. It is suggested that late c-fos activation signals inflammatory pain and is due to sensitization of primary afferent neurones.
We have used the evoked expression of the immediate early gene-encoded proteins (c-Fos, Fos B, Jun B, Jun D, c-Jun and Krox-24) to monitor sensory processing in the hindbrain structures of rats undergoing somatic inflammation. Experiments were performed on freely moving animals that did not experience constraints other than those imposed by the disease itself. Local injections of chemicals were used to cause subcutaneous inflammation of the plantar foot or monoarthritis by intracapsular injection. Labelling was studied at survival times that corresponded either to the time points of maximum labelling in the spinal cord (4 h for the subcutaneous model, 24 h and two weeks for the monoarthritis model) or at survival times that corresponded to the chronic phase of monoarthritis evolution (six, nine and 15 weeks). Controls consisted of freely moving, unstimulated animals. Basal expression was observed for all immediate early genes and in a variety of stuctures, but always remained moderate. All immediate early gene-encoded protein expressions except c-Jun were evoked, but except for c-Fos, and to a lesser extent Jun D, intensities of staining always remained faint. The following results will be mainly based on c-Fos expression, as this protein proved to be the most effective marker for all the survival times studied.
The nociceptive nature of the neurons of the superficial dorsal horn (laminae I-III) which project to the medullary ventrolateral reticular formation is studied in the rat. Medullary injections of Fluoro-Gold showed exclusive retrograde labeling of laminae I-III cells when the tracer filled a zone intermediate between the lateral tip of the lateral reticular nucleus and the spinal trigeminal nucleus, pars caudalis. This zone is here called VLMlat. Following noxious mechanical or thermal stimulation of the skin, double-labeled neurons, which stained retrogradely and were Fos-immunoreactive, prevailed in laminae I and IIo. Double-labeled neurons were few in lamina IIi after thermal stimulation and entirely lacking in lamina III after the two kinds of stimulation. Findings in lamina I confirm previous electrophysiological data (see Menétrey et al., J. Neurophysiol., 52 (1984) 595-611) showing that lamina I cells projecting to the ventrolateral reticular medulla convey noxious messages. The occurrence of numerous double-labeled cells in lamina IIo suggests that this lamina is also involved in nociceptive transmission to the VLMlat.
We have used the evoked expression of both immediate early gene (IEG)‐encoded proteins (Krox‐24, c‐Fos, Fos B, Jun D, Jun B, c‐Jun), and dynorphin to monitor sensory processing in the spinal cords of rats undergoing subacute or chronic somatic inflammation (i.e., subcutaneous inflammation of the plantar foot and monoarthritis, respectively). Behavioral and immunocytochemical approaches were conducted in parallel up to 15 weeks postinjection in order to detect possible relationships between clinical evolution and spatiotemporal pattern of IEG‐encoded protein expression.
Each disease had specific characteristics both in terms of their clinical evolution and pattern of evoked protein expression. All IEG proteins were expressed in both cases. Most of the staining was observed in both the superficial layers of the dorsal horn and deep dorsal horn (laminae V–VII and X). Monoarthritis was distinguished by a high level of total protein expression. Staining was especially dense in the deep dorsal horn. More labelled cells were observed at 1–2 days and at 2 weeks postinjection, corresponding to the initiation and progressive phases of the disease, respectively. Subcutaneous inflammation was characterized by a moderate level of total IEG expression. More labelled cells were observed in the first day following injection. It is the relative degree of expression of each IEG‐encoded protein with regard to the others that characterized the progression of the diseases. Early stages of the diseases coincided with the expression of all Fos and Jun proteins, while late stages showed an increase in Jun D and Fos B involvement; Krox‐24 was induced mostly during the early phases and/or periods of paroxysm of the diseases. Persistent stimulation was characterized by a predominant expression in deep versus superficial layers of the dorsal horn. Evoked expression of c‐Jun in motoneurons was only observed in monoarthritis. The peak of dynorphin expression was late in regard to both the induction of inflammation and period of maximal IEG‐encoded protein expression.
The present work indicates that the neural processing that takes place during progression of these diseases can be monitored well at the spinal cord level by using the expression of an array of IEG‐encoded proteins. Study of long term evolutive diseases and especially those that evolve into chronicity can largely benefit from such an approach. © 1993 Wiley‐Liss, Inc.
The laminar distribution of spinal cord neurones expressing immunoreactivity to the Fos protein was evaluated in the rat following chemical, thermal or mechanical noxious stimulation of the skin for 2 h. After stimulation by 20% or 5% formalin, Fos-immunoreactive neurones prevailed in lamina I where they accounted for 64% and 59%, respectively, of the entire population of Fos-immunoreactive spinal cells. Values in the remaining laminae were low (2-10%). Following thermal stimulation by radiant heat at 65 degrees C or 58 degrees C, Fos cells were concentrated in laminae I and IIo, amounting to 57% and 62%, respectively, in lamina I, and to 26% and 29% in lamina IIo. Values were lower than 10% in the remaining laminae. Following mechanical stimulation by pinching or needle prick, Fos-positive cells were regularly distributed throughout laminae I-V amounting to 25-26% in lamina I, and 10-20% in each of the remaining laminae. These findings suggest that the spinal neuronal groups upon by prolonged noxious stimulation differ according to the nature of the stimulus.
The aim of this study was to determine the discharge and receptive field properties of spinal cord neurons with ankle input in spinal segments L4-6 in the rat, both under control conditions and during the course of an adjuvant-induced unilateral inflammation in the ankle. The extent of receptive fields in the skin and deep tissue was assessed using brush, pinch and compression stimuli. Neurons were categorized as nociceptive-specific or wide-dynamic-range neurons on the basis of their response thresholds and responses to suprathreshold stimuli. At all stages of inflammation (2, 6, 13 and 20 days post inoculation) the population of neurons with ankle input showed differences from the population of neurons with ankle input in control rats. There was a reduction in the number of neurons that appeared as nociceptive specific and a concomitant increase in the number of neurons showing a wide-dynamic-range response profile. The receptive fields of the neurons with ankle input were markedly larger in rats with inflammation in the ankle region and mainly spread proximally on the ipsilateral hindlimb and also to the abdomen and tail in some cases. There was also an increase in the number of neurons with contralateral excitatory inputs. The mechanical thresholds at the ankle joint and proximal parts of the ipsilateral hindlimb were less in arthritic rats than in controls. The proportion of spontaneously active neurons was also increased in rats during the initial and later stages of inflammation, although there was no significant increase in the mean spontaneous discharge frequency. These data show that there are long-term changes in the receptive field and response properties of neurons in intact rats with chronic unilateral adjuvant-induced inflammation similar to those described previously in spinal cats with acute inflammation (Neugebauer and Schaible 1990). It is presumed that similar afferent and spinal mechanisms are at work under acute and chronic inflammatory conditions which produce hyperexcitability in spinal neurons with joint input.
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.
Expression of the inducible transcription factor c-Fos has been examined in the lumbar spinal cord following noxious chemical stimulation (injection of 2% formalin) of the ankles or the ventral skin of the hindpaws of either normal rats, or monoarthritic rats during the chronic phase of the disease. In normal animals the basal expression of c-Fos was low. One day after induction of monoarthritis by an intra-articular injection of killed Mycobacterium butyricum (in complete Freund's adjuvant) there were numerous c-Fos labelled cells in the ipsilateral dorsal horn, and bilaterally in lamina VIII and in other areas of the ventral horn. Four weeks after induction of the arthritis, although marked inflammation of the ankle was still present, all the expression of c-Fos had returned to the basal levels. One hour after formalin stimulation of the ankle or hindpaw skin of normal rats expression of c-Fos was observed throughout the ipsilateral, but not contralateral dorsal horn. Formalin stimulation of the inflamed ankle in four-week arthritic rats induced a 3-to-6 fold increase in c-Fos expression in the ipsilateral dorsal horn compared to formalin stimulation of the ankle in normal rats. In addition, c-Fos expression was induced in the contralateral deep, but not superficial laminae, at a density similar to that produced ipsilaterally by formalin stimulation of the ankle of normal rats. Formalin stimulation of the contralateral ankle in monoarthritic rats (i.e. the non-inflamed ankle) induced an ipsilateral expression of c-Fos which was similar to that observed after stimulation of the arthritic ankle. This stimulation of the normal ankle also resulted in an expression of c-Fos in the contralateral deep, but not superficial laminae, that was similar to that induced ipsilaterally by stimulation of the arthritic ankle. Finally, formalin stimulation of the hindpaw skin (which was not inflamed) of the arthritic limb induced the same number of c-Fos labelled cells in the superficial laminae as did formalin stimulation of the skin of normal rats; but in the deep laminae there was a 1.6-fold increase in the number of labelled cells. These different observations show that the down-regulation of c-Fos expression observed in chronic monoarthritis is in fact associated with a sensitization and an extension of the field of its expression in response to an acute nociceptive stimulation.
Increases in the expression of immediate early genes have been shown to occur in the lumbar spinal cord dorsal horn after peripheral inflammation. Given that the pontine parabrachial nucleus has been implicated in nociceptive as well as antinociceptive processes and is reciprocally connected with the spinal cord dorsal horn, it seems likely that peripheral inflammation will cause alterations in immediate early gene expression in this nucleus. To test this hypothesis we examined cFos-like immunoreactivity in a rodent complete Freund's adjuvant-induced peripheral inflammatory model of persistent nociception. Unilateral hind paw injections of complete Freund's adjuvant produced inflammation, hyperalgesia of the affected limb, and alterations in open field behaviors. Immunocytochemical analysis demonstrated a bilateral increase in cFos-like immunoreactivity in the lateral and Kolliker-Fuse subdivisions of the parabrachial nucleus at 6 and 24 hours postinjection and an ipsilateral decrease below basal levels in the Kolliker-Fuse subdivision at 96 hours postinjection when compared to saline controls. Taken together, these results suggest that select parabrachial neurons are activated by noxious somatic inflammation. These active parabrachial neurons are likely to participate in ascending nociceptive and/or descending antinociceptive pathways.
Fos-immunoreactivity can readily be induced in spinal cord neurones by noxious, but to a much more limited extent, by innocuous peripheral stimuli. The present study has investigated whether low intensity stimuli and electrical stimulation of A beta afferents elicit greater c-fos expression during the behavioural sensory hypersensitivity generated by experimental peripheral inflammation. We have examined the time-course of c-fos expression after inflammation produced by either an intra-plantar injection of the irritant turpentine oil or of complete Freund's adjuvant (CFA). In the former case, a significant initial expression in all dorsal horn laminae was followed by a gradual decrease, whereas after CFA injection, an initial expression limited to the superficial laminae subsequently extended into the deep laminae, with a decrease at 24 h and an increase in labelling at later times. Low intensity touch stimuli repeated for 10 min, when applied at 24 h and 48 h after CFA injection, elicited a significant increase in the number of Fos-immunoreactive neurons in both the superficial and deep laminae of the dorsal horn compared to non-inflamed animals. Electrical stimulation of the sciatic nerve 24 h post-CFA injection, at a strength sufficient only to activate A beta-afferents fibres (100 microA, 50 microseconds, 10 min), also elicited a significant increase in labelling relative to the same stimuli applied in control animals, especially in laminae V-VI. The present results demonstrate that low intensity cutaneous stimuli elicit a significantly greater increase in c-fos expression in dorsal horn neurons during peripheral inflammation and that A beta-afferent input contributes to this, a finding that may relate to the allodynia experienced during inflammation.
The neurokinins, substance P (SP) and neurokinin A (NKA) represent natural, nonspecific ligands of NK1 and NK2 receptors. In our study in conscious rats, we tested the hypothesis that neurokinins, especially SP, are used by neuronal circuits to generate cardiovascular and behavioral responses to stress by using the selective, high-affinity, nonpeptide antagonists of NK1 and NK2 receptors, CP-96, 345, RP 67580 and SR 48968, respectively, Formalin injected s.c. through a chronically implanted catheter in the region of the lower leg was used as a stress stimulus. The antagonists and their inactive enantiomers, RP 68651 and SR 48965, as a control for nonspecific activity, were injected intracerebroventricularly (i.c.v.) 10 min before the s.c. injection of formalin. Formalin (2.5%, 50 microliters, s.c.) induced a marked increase in mean arterial pressure (MAP) and heart rate (HR) as well as hind limb grooming/biting (HG) as the dominant behavioral manifestation. Pretreatment with the NK1 receptor antagonist, CP-96,345 (5 nmol, i.c.v.), significantly attenuated only the HR (-54%; P < .01) but not the MAP response to formalin. The NK1 receptor antagonist, RP 67580, injected i.c.v. at doses of 100, 500 and 2500 pmol significantly reduced both, the MAP and HR responses to formalin by maximally 63% (P < .01) and 52% (P < .01), respectively. In a separate set of experiments, we compared the effect of the individual and simultaneous blockade of central NK1 and NK2 receptors on the cardiovascular and behavioral responses to formalin stress. Pretreatment with RP 67580 (100 pmol, i.c.v.) attenuated the MAP (-30%; P < .05), HR (-40%; P < .01) and HG (P < .05) responses to formalin. The NK2 receptor antagonist, SR 48968 (650 pmol, i.c.v.), affected neither the cardiovascular nor the behavioral responses. i.c.v. pretreatment with both tachykinin receptor antagonists (RP 67580: 100 pmol; SR 48968: 650 pmol) reduced the MAP, HR and HG responses to formalin to the same extent as RP 67580 alone. Pretreatment with the inactive enantiomers, RP 68651 (100 pmol, i.c.v.) and SR 48965 (650 pmol, i.c.v.) did not alter the cardiovascular and behavioral responses to formalin. Our results demonstrate that centrally administered NK1 receptor antagonists inhibit the cardiovascular and behavioral reactions in response to a noxious stimulus. They provide first pharmacological evidence that endogenous SP acts as mediator of stress responses in the brain.
The participation of B1 and B2 types of bradykinin receptors was studied in the rat knee-joint incapacitation test. Five intra-articular successive hourly administrations of bradykinin produced progressive incapacitation, thus indicating that bradykinin induced sensitization to its own nociceptive effect. Four co-injections of bradykinin with the bradykinin B1 receptor antagonist des-Arg9-[Leu8]bradykinin were without nociceptive effect. However, a 5th injection of bradykinin alone produced intense incapacitation. The bradykinin B2 receptor antagonist HOE-140 ([D-Arg)[Hyp3,Thi5,D-Tic7,Oic8]bradykinin), or indomethacin, prevented the bradykinin-induced incapacitation. However, successive co-injections of bradykinin with prostaglandin E2, in contrast to bradykinin alone, did induce incapacitation in animals pretreated with indomethacin or HOE-140. The injection of the bradykinin B1 receptor agonist des-Arg9-bradykinin into prostaglandin E2-treated joints did induce incapacitation, although administration of the bradykinin B1 receptor agonist or prostaglandin E2 alone did not induce incapacitation. In conclusion, in ongoing articular inflammation, it is suggested that the bradykinin B1 receptor is particularly involved with nociceptor activation, while the bradykinin B2 receptor is related to nociceptor sensitization.
Several subgroups in the brainstem parabrachial nucleus (PB), which is a major target for nociresponsive neurons in the medullary and spinal dorsal horn, contain large numbers of preproenkephalin (ppENK) mRNA-expressing neurons. To elucidate how noxious stimuli may regulate ppENK transcription in these neurons, we have in the present study investigated whether immunoreactivity for the transcription factors FOS and phosphorylated CREB (pCREB), respectively, is displayed in the ppENK mRNA-expressing neurons after peripheral nociceptive stimulation. Rats received injection of formalin into one hindpaw, and were killed 30-80 min later. With a combination of immunohistochemistry and in situ hybridization, we found that only a small number of ppENK mRNA-expressing neurons in PB displayed FOS-immunoreactivity after nociceptive stimulation. In contrast, large numbers of ppENK mRNA-expressing neurons displayed pCREB-like immunoreactivity after nociceptive stimulation. Most of the ppENK mRNA/pCREB-expressing neurons were found in the Kölliker-Fuse and internal lateral subnuclei, but many double-labeled cells were also seen in the ventral lateral and central lateral subnuclei. In addition, a cluster of ppENK mRNA/pCREB-expressing neurons was found in the medial part of the medial parabrachial nucleus. Our findings suggest that CREB rather than FOS regulates nociceptive-related second messenger activation of ppENK transcription in parabrachial neurons.
Just over a decade has past since Hunt et al. reported that the gene c-fos and its protein product Fos are expressed in the spinal cord of rats subjected to peripheral noxious stimulation. These authors showed that noxious stimulation (application of radiant heat or mustard oil) to the hind paw resulted in a massive increase in the expression of Fos in neurons in the dorsal horn of the lumbar spinal cord. Since then, there has been an explosion of studies in which c-fos has been used to study nociception (pain), and the number of such studies increases each year. The net result has been to establish c-fos expression as a valuable tool in pain research. Moreover, recent studies have provided evidence identifying the role of c-fos expression in spinal nociceptive processes. However, there are several important limitations to the practice of using c-fos to study nociception, and these limitations can be easily overlooked as the practice graduates to the status of an established technique. The increasing use of c-fos to study nociception necessitates a critical review of the practice, identifying the shortcomings as well as the strengths of this tool.
The influence of midthoracic spinalization on thermally and mechanically induced spinal withdrawal reflex responses was studied in the rat. There were three experimental groups of rats: healthy controls, rats with a spinal nerve ligation-induced unilateral neuropathy, and rats with a carrageenan-induced inflammation of one hindpaw. Tail flick response was induced by radiant heat. Hindlimb withdrawal was induced by radiant heat, ice water, and innocuous or noxious mechanical stimulation of the paw. Prior to spinalization, spinal nerve ligated and carrageenan-treated animals had a marked unilateral allodynia and hyperalgesia. Spinalization tended to induce a facilitation of noxious heat-evoked reflexes. This spinalization-induced facilitation was stronger on tail than hindlimb withdrawal. Spinalization-induced skin temperature change did not explain the facilitation of noxious heat-evoked reflexes. In contrast, spinal withdrawal responses induced by noxious cold or mechanical stimulation were significantly suppressed following spinalization. The spinalization-induced facilitatory effects as well as inhibitory ones on spinal reflexes were enhanced in inflamed/neuropathic animals. The results indicate that the tonic descending control of spinal nocifensive responses varies depending on the submodality of the test stimulus, the segmental level of the reflex (tail vs. hindlimb), and on the pathophysiological condition.
The effect of graded inflammatory stimuli (intraplantar-carrageenan, 0.2, 1, and 6 mg/150 microl) on paw edema and c-Fos protein expression at two levels of the spinoparabrachial pathway, the spinal cord and parabrachial area (PB), were studied. The present study, in awake rats, is an extension of previous study (Bester et al. [1997] J. Comp. Neurol. 383:439-458) which evaluated, in anesthetized rats, the effect of graded cutaneous heat stimulation on c-Fos-expression at the same levels. At the spinal level, the c-Fos-protein-like-immunoreactive (c-Fos-LI) neurons were located primarily in superficial laminae ipsilateral to intraplantar carrageenan. The number of c-Fos-LI neurons increased dose dependently (r = 0.973, n = 24) for carrageenan, from a number close to zero for the saline injection. At the PB level, c-Fos was predominantly expressed contralateral to intraplantar carrageenan. c-Fos-LI neurons were located primarily around the pontomesencephalic junction in (i) a restricted pontine area, centered in the lateral crescent, and including an adjacent part of the outer portion of the external lateral subnucleus, and (ii) the mesencephalic superior lateral subnuclei. The number of c-Fos-LI neurons in the PB area was correlated with that in the superficial laminae (r = 0.935, n = 24) and with the paw edema (r = 0.931, n = 24). No significant changes in c-Fos expression were observed in the nucleus of the solitary tract and ventrolateral medulla. The close correlation between c-Fos expression at both the spinal and PB levels and inflammatory edema provides further evidence for the involvement of spinoparabrachial pathway in inflammatory nociceptive processes. The present results are congruent with the existence of electrophysiologically demonstrated spinoparabrachio-amygdaloid and -hypothalamic nociceptive pathways.
Peripheral noxious stimulation evokes functional and biochemical changes in the spinal cord which results in central sensitization and hyperalgesia, but at the same time also induces the activation of inhibitory control systems. The purpose of the present study was to investigate whether the adaptive changes induced by ongoing peripheral inflammation influence the spinal cord expression of c-Fos (a commonly used marker of neuronal activity) following an additional acute noxious stimulus. Therefore, the spinal expression of c-Fos was immunohistochemically investigated following noxious thermal stimulation of a rat monoarthritic hindpaw at various time points (1, 4, 8, 21 days) after induction of monoarthritis. Compared to normal rats, c-Fos expression following ipsilateral noxious thermal stimulation of monoarthritic rats was strongly modified in the deep laminae of the dorsal horn depending on the time course of inflammation. At 1 day of monoarthritis, an enhanced ipsilateral expression (135% and 208% of normal rats in laminae III-VI and VII, respectively) and at 3 weeks a reduced expression (38% and 23% of normal rats in laminae III-VI and VII, respectively) was detected. The amount of c-Fos-positive neurons in the ipsilateral superficial laminae I and II was unchanged at all time points investigated. To assess excitability changes on the contralateral side at an early stage of inflammation, a group of monoarthritic rats received a contralateral noxious stimulus at day 1 of monoarthritis. This resulted in a potentiated expression of c-Fos ipsilateral to the acute noxious stimulus (i.e., contralateral to the monoarthritic hindpaw) restricted to lamina II (137% of normal rats) of the dorsal horn. The data showed that changes in c-Fos expression depended on the time point of noxious heat stimulation (NHS) of monoarthritic rats, and differed in the ipsi- and contralateral side of the spinal cord. In addition to a possible habituation of c-Fos expression, it may be speculated that the time course-dependent changes reflect laminae-specific modulations of excitatory and inhibitory mechanisms during monoarthritis. Further studies are necessary in order to provide more insights into the contribution of these mechanisms on noxious stimulus-evoked c-Fos expression.
Although there is considerable information about the mechanisms through which injury stimuli produce acute pain, recent studies indicate that there are significant long-term consequences of persistent injury. Pain is exacerbated, in part, because of a reorganization of spinal cord circuitry in the setting of persistent injury. This review describes our studies of the contribution of the primary afferent neurotransmitter, substance P (SP), to these changes. By following internalization of the SP receptor in spinal cord dorsal horn neurons, we have identified the stimuli that evoke SP release and the neurons that respond to these stimuli. Importantly, based on the intensities of stimuli required to evoke internalization, we conclude that SP is only released under conditions in which severe pain would be produced, that the release can be evoked by intense stimulation of somatic and visceral tissue, and that multiple stimulus modalities are effective. We also found that the numbers of neurons that are influenced increases dramatically in the setting of inflammation. Using a knockout strategy, we have also raised mice with a deletion of the preprotachykinin-A (PPT-A) gene, which encodes for SP and neurokinin A (NKA), and have identified a specific behavioral phenotype in which the animals do not detect a window of "pain" intensities; this window cuts across stimulus modalities. These results provide an important behavioral correlate of the receptor internalization studies. On the other hand, the allodynia (lowered pain threshold) that occurs in the setting of injury was not altered in these animals. Among the factors that could underlie injury-induced allodynia are the second messenger systems that are activated in dorsal horn neurons. Our studies have recently implicated the gamma isoform of protein kinase C (PKCgamma) in the development of nerve injury-induced neuropathic pain. Specifically, we found that although acute pain responses of mice with a deletion of PKCgamma are not altered, partial injury to the sciatic nerve (which induces a severe thermal and mechanical allodynia in the wild type mouse) is without effect in the knockout. Furthermore, the anatomical/neurochemical reorganization that typically follows sciatic nerve section does not occur in the PKCgamma mutant mice. Because the spinal cord distribution of interneurons that express PKCgamma is concentrated almost exclusively in the inner part of lamina II, we believe that changes in the properties of these neurons are key to the development of nerve injury-induced neuropathic pain conditions. Taken together, these studies emphasize that persistent pain should be considered a disease state of the nervous system, not merely a symptom of some other disease conditions. In the setting of persistent injury, the nervous system undergoes dramatic changes that exacerbate and prolong the pain condition. Our studies underscore the importance of preventing the long-term changes that result from persistent injury.