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Dynamic of migration of HSV-1 from a medullary pronociceptive centre: Antinociception by overexpression of the preproenkephalin transgene
Abstract
Herpes Simplex Virus type 1 (HSV-1) vectors are known to inhibit nociceptive transmission at the spinal cord after peripheral applications. Similar approaches may also be useful when applied at the supraspinal pain control system as the system includes pronociceptive (facilitatory) components. We performed a study aimed to analyse the migration of HSV-1 along with the inhibition of pronociception from the medullary dorsal reticular nucleus (DRt), a major facilitatory component of the supraspinal pain control system. To study the migration, a HSV-1 vector expressing lacZ under control of the human cytomegalovirus (hCMV) promoter was injected in the DRt and the expression of beta-galactosidase (beta-gal) was detected at 2, 4, 7, 10 and 14 days. Numerous beta-gal-immunoreactive neurons were observed at the injection site until day 4, and at some of the brain areas projecting to the DRt until day 7. To block the pronociceptive effects of the DRt, a HSV-1 vector expressing the preproenkephalin transgene, under the control of hCMV promoter, was injected into the DRt. Behavioural evaluation was performed at the time-points referred above, using the paw withdrawal latency test to evaluate thermal nociceptive responses. Anti-hyperalgesic effects persisted during 4 days, decreasing after that time-point. The present study demonstrates that selective migration of HSV-1 should be considered in gene therapy strategies based on HSV-1 injections into the brain. The study also shows that it is possible to decrease pain facilitation from the brain using opioidergic inhibition of pronociceptive supraspinal areas.
... Opioid peptides were shown to inhibit DRt pain facilitation as shown by HSV-1-mediated overexpression of enkephalin at the DRt, which produced anti-hyperalgesia in a model of acute pain (Martins et al., 2008). Opioids likely act through direct inhibition of DRt spinally projecting neurons since these neurons express µ-opioid receptors (Pinto et al., 2008a,b). ...
... Opioids also act the DRt through additional inhibitory mechanisms, likely by disinhibiting enkephalinergic interneurons which receive input from GABAergic interneurons expressing µ-opioid receptors and being presynaptically inhibited by δ-opioid expressingfibers (Pinto et al., 2008a). Opioid peptides responsible for the anti-hyperalgesic action found in our studies are mostly released from local interneurons but also from DRt afferent sources namely the RVM, the A 5 noradrenergic cell group and the hypothalamus (Figure 2; Martins et al., 2008). ...
... 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.
... Since DMH neurones do not project directly to the spinal cord [9,10], a potential pronociceptive pathway mediating the descending action of GAL in the DMH is the dorsal reticular nucleus (DRt) [11], a structure receiving strong afferent projections from the DMH [9,10]. Neurones in the DRt respond exclusively or mainly to noxious stimulation [12,13]. ...
... Since DMH neurones do not project directly to the spinal cord [9,10], a potential pronociceptive pathway mediating the descending action of GAL in the DMH is the dorsal reticular nucleus (DRt) [11], a structure receiving strong afferent projections from the DMH [9,10]. Neurones in the DRt respond exclusively or mainly to noxious stimulation [12,13]. ...
... The pronociceptive effect of GAL in the DMH is at least partly mediated by the DRt. Baseline PWL evoked by noxious heating of the ipsilateral hind paw (i.e., distal to the inflamed knee joint in the ARTH group) was not significantly different between SHAM and ARTH animals (t 10 To study the phasic and tonic action of the DRt upon nociceptive modulation, PWLs on both contralateral and ipsilateral hind paws were evaluated after injecting GLU, LIDO or SAL in the right (ipsilateral to the inflamed knee joint in the ARTH group) DRt. In the contralateral (left) paw, drug injection in the DRt significantly altered PWL (main effect of drug: F 2,20 = 7.388, P = 0.004), although post hoc tests did not indicate any difference between treatment groups (Fig 2A). ...
Activation of the dorsomedial nucleus of the hypothalamus (DMH) by galanin (GAL) induces behavioural hyperalgesia. Since DMH neurones do not project directly to the spinal cord, we hypothesized that the medullary dorsal reticular nucleus (DRt), a pronociceptive region projecting to the spinal dorsal horn (SDH) and/or the serotoninergic raphe-spinal pathway acting on the spinal 5-HT3 receptor (5HT3R) could relay descending nociceptive facilitation induced by GAL in the DMH. Heat-evoked paw-withdrawal latency (PWL) and activity of SDH neurones were assessed in monoarthritic (ARTH) and control (SHAM) animals after pharmacological manipulations of the DMH, DRt and spinal cord. The results showed that GAL in the DMH and glutamate in the DRt lead to behavioural hyperalgesia in both SHAM and ARTH animals, which is accompanied particularly by an increase in heat-evoked responses of wide-dynamic range neurons, a group of nociceptive SDH neurones. Facilitation of pain behaviour induced by GAL in the DMH was reversed by lidocaine in the DRt and by ondansetron, a 5HT3R antagonist, in the spinal cord. However, the hyperalgesia induced by glutamate in the DRt was not blocked by spinal ondansetron. In addition, in ARTH but not SHAM animals PWL was increased after lidocaine in the DRt and ondansetron in the spinal cord. Our data demonstrate that GAL in the DMH activates two independent descending facilitatory pathways: (i) one relays in the DRt and (ii) the other one involves 5-HT neurones acting on spinal 5HT3Rs. In experimental ARTH, the tonic pain-facilitatory action is increased in both of these descending pathways.
... Pain models Gene product Delivery References Acute pain Proenkephalin Medullary dorsal reticular nucleus (DRt) [55] Neuropathic Tyrosine Hydroxylase antisense Medullary dorsal reticular nucleus (DRt) [34] ...
... These areas were elected based on the extensive neurobiological knowl‐ edge of their role in pain modulation [53] [54]. Overexpression of opioid precursors in the DRt and VLM induced analgesia in acute pain tests and models of sustained or chronic inflamma‐ tory pain [19] [20] [55]. Brain areas involved in pain control and which are of easier neurosurgical access are the amygdala and the rostral agranular insular cortex. ...
... Our research group has pioneer work in studying the dynamics of HSV-1 migration in the brain after injections of the vector in pain control centres of the medulla oblongata, namely the caudal medulla oblongata (VLM) and the dorsal reticular nucleus (DRt). After injections of a HSV-1 vector expressing the lacZ reporter gene, under control of the human cytomegalovirus promoter (hCMV), in pain control centres of the medulla oblongata, migration in VLM and DRt afferents was detected [19] [55] (Fig. 2). However, not all the brain afferents of the VLM and DRt exhibited β-galactosidase (β-gal), the product of lacZ expression. ...
This chapter provides an overview of the main current applications of gene therapy for chronic pain in what concerns animal studies and putative clinical applications. The value of gene therapy in unravelling neuronal brain circuits involved in pain modulation is also analyzed. After alerting to the huge socioeconomic impact of chronic pain in modern societies and justifying the need to develop new avenues in pain management, we review the most common animal studies using gene therapy, which consisted on deliveries of replication-defective viral vectors at the periphery with the aim to block nociceptive transmission at the spinal cord. Departing from the data of these animal studies, we present the latest results of clinical trials using gene therapy for pain management in cancer patients. The animal studies dealing with gene delivery in pain control centres of the brain are analyzed in what concerns their complexity and interest in unravelling the neurobiological mechanisms of descending pain modulation. The chapter will finish by analysing possible futures of gene therapy for chronic pain management based on the development of vectors which are safer and more specific for the different types of chronic pain.
... Opioids act through direct inhibition of DRt spinally projecting neurons and also through disinhibition of enkephalinergic interneurons which receive input from GABAergic interneurons expressing MOR (Pinto et al., 2008a). We have previously shown that opioids inhibit DRt descending facilitation (Martins et al., 2008) and that in a model of chronic inflammatory pain there is a loss of inhibitory opioidergic tone, likely produced by decreased MOR expression, which results in enhanced descending pain facilitation (Pinto et al., 2008a). The impact of neuropathic pain on the opioidergic modulation of the DRt has never been explored. ...
... The present study shows that during SNI there is increased release of the endogenous opioid peptides Met-and Leu-enkephalin at the DRt. These peptides are likely released from local enkephalinergic interneurons and also from DRt afferent sources namely the RVM, the A 5 noradrenergic cell group and the hypothalamus (Martins et al., 2008). Because these peptides were not detected in sham-animals we were not able to quantify the magnitude of this increase. ...
... Opioids act through direct inhibition of DRt spinally projecting neurons and also through disinhibition of enkephalinergic interneurons which receive input from GABAergic interneurons expressing MOR (Pinto et al., 2008a). We have previously shown that opioids inhibit DRt descending facilitation (Martins et al., 2008) and that in a model of chronic inflammatory pain there is a loss of inhibitory opioidergic tone, likely produced by decreased MOR expression, which results in enhanced descending pain facilitation (Pinto et al., 2008a). The impact of neuropathic pain on the opioidergic modulation of the DRt has never been explored. ...
... The present study shows that during SNI there is increased release of the endogenous opioid peptides Met-and Leu-enkephalin at the DRt. These peptides are likely released from local enkephalinergic interneurons and also from DRt afferent sources namely the RVM, the A 5 noradrenergic cell group and the hypothalamus (Martins et al., 2008). Because these peptides were not detected in sham-animals we were not able to quantify the magnitude of this increase. ...
Opioids play a major role at descending pain modulation but the effects of neuropathic pain on the brain opioidergic system remain understudied. Since descending facilitation is enhanced during neuropathic pain, we studied the opioidergic modulation of the dorsal reticular nucleus (DRt), a medullary pain facilitatory area, in the spared nerve injury (SNI) model of neuropathic pain. We first performed a series of behavioral experiments in naïve-animals to establish the role of μ-opioid receptor (MOR) in the effects of endogenous and exogenous opioids at the DRt. Specifically, we showed that lentiviral-mediated MOR-knockdown at the DRt increased sensitivity to thermal and mechanical stimuli while the MOR agonist DAMGO induced the opposite effects. Additionally, we showed that MOR-knockdown and the pharmacological blockade of MOR by CTAP at the DRt decreased and inhibited, respectively, the analgesic effects of systemic morphine. Then, we performed in vivo microdialysis to measure enkephalin peptides in the DRt and evaluated MOR expression in the DRt at mRNA, protein and phosphorylated form levels by quantitative real-time PCR and immunohistochemistry, respectively. SNI-animals, compared to sham control, showed higher levels of enkephalin peptides, lower MOR-labeled cells without alterations in MOR mRNA levels, and higher phosphorylated MOR-labeled cells. Finally, we performed behavioral studies in SNI animals to determine the potency of systemic morphine and the effects of the pharmacologic and genetic manipulation of MOR at the DRt. We showed a reduced potency of the antiallodynic effects of systemic morphine in SNI-animals compared to the antinociceptive effects in sham animals. Increasing MOR-cells at the DRt of SNI-animals by lentiviral-mediated MOR-overexpression produced no effects on mechanical allodynia. DAMGO induced anti-allodynia only after MOR-overexpression. These results show that MOR inhibits DRt pain facilitatory actions and that this action contributes to the analgesic effects of systemic opioids. We further show that the inhibitory function of MOR is impaired during neuropathic pain. This is likely due to desensitization and degradation of MOR which are adaptations of the receptor that can be triggered by MOR phosphorylation. Skipping counter-regulatory pathways involved in MOR adaptations might restore the opioidergic inhibition at pain facilitatory areas.
... Neuroanatomic tract tracing techniques showed that the LC and the A 5 noradrenergic cell group are the main noradrenergic brain areas projecting to the DRt, 13 which was confirmed by the retrograde transport from the DRt of a herpes simplex virus-1 viral vector. 14,15,30 In this study, the high percentages of noradrenergic neurons expressing pCREB at the LC and the A 5 noradrenergic cell group of SNI animals suggests that neuropathic pain potentiates the activity of these areas. This agrees with reports showing increased brainstem noradrenergic activation in several neuropathic pain models. ...
... Clonidine was infused at the DRt by reverse dialysis 3 weeks after sham or SNI surgery. Data are expressed as mean percentage of baseline levels across four measurements at each dose (15,30,50, and 60 min after administration), with the respective 95% CIs (n = 5/group). DRt = dorsal reticular nucleus; SNI = spared nerve injury. ...
Noradrenaline reuptake inhibitors are known to produce analgesia through a spinal action but they also act in the brain. However, the action of noradrenaline on supraspinal pain control regions is understudied. The authors addressed the noradrenergic modulation of the dorsal reticular nucleus (DRt), a medullary pronociceptive area, in the spared nerve injury (SNI) model of neuropathic pain.
The expression of the phosphorylated cAMP response element-binding protein (pCREB), a marker of neuronal activation, was evaluated in the locus coeruleus and A5 noradrenergic neurons (n = 6 rats/group). pCREB was studied in noradrenergic DRt-projecting neurons retrogradely labeled in SNI animals (n = 3). In vivo microdialysis was used to measure noradrenaline release in the DRt on nociceptive stimulation or after DRt infusion of clonidine (n = 5 to 6 per group). Pharmacology, immunohistochemistry, and western blot were used to study α-adrenoreceptors in the DRt (n = 4 to 6 per group).
pCREB expression significantly increased in the locus coeruleus and A5 of SNI animals, and most noradrenergic DRt-projecting neurons expressed pCREB. In SNI animals, noradrenaline levels significantly increased on pinprick (mean ± SD, 126 ± 14%; P = 0.025 vs. baseline) and acetone stimulation (mean ± SD, 151 ± 12%; P < 0.001 vs. baseline), and clonidine infusion showed decreased α2-mediated inhibitory function. α1-adrenoreceptor blockade decreased nociceptive behavioral responses in SNI animals. α2-adrenoreceptor expression was not altered.
Chronic pain induces brainstem noradrenergic activation that enhances descending facilitation from the DRt. This suggests that antidepressants inhibiting noradrenaline reuptake may enhance pain facilitation from the brain, counteracting their analgesic effects at the spinal cord.
... The brainstems were removed, immersed in fixative for 4 h followed by 30% sucrose in 0.1 M PBS, overnight, at 4 C, and sliced at 40 mm in coronal orientation in a freezing microtome. Double immunodetection of b-galactosidase (b-gal) and TH was performed using an adaptation of our previous protocol (Martins et al., 2008). The sections were first incubated with a mouse monoclonal antibody against b-gal (Santa Cruz, USA), at 1:1000, overnight, followed by a horse biotinylated anti-mouse serum (Dako, Denmark) and an Alexa Streptavidin 594 (Molecular Probes, Netherlands). ...
... The manipulation of noradrenaline release by the retrogradely transported HSV-1 vector shows that the main input to the DRt originates from the locus coeruleus and the A 5 noradrenergic cell group. Our previous time course studies of HSV-1-mediated gene transfer to DRt noradrenergic afferents shows that along the time most of the transduced neurons occur in the locus coeruleus (Martins et al., 2008). Together these data suggest that noradrenaline released at the DRt is mainly derived from the locus coeruleus. ...
Antidepressants that inhibit the recapture of noradrenaline have variable effects in chronic pain which may be related to the complex role of noradrenaline in pain modulation. Whereas at the spinal cord noradrenaline blocks nociceptive transmission, both antinociception and pronociception were reported after noradrenaline release in the brain. To study the role of noradrenaline in pain modulatory areas of the brain, we elected the dorsal reticular nucleus (DRt), a key pain facilitatory area located at the medulla oblogata. Three studies were performed. First, we show that the infusion in the DRt of nomifensine, which increases local extracellular levels of noradrenaline as shown by in vivo microdialysis, also enhances pain behavioral responses during both phases of the formalin test, a classic inflammatory pain model. Then, we demonstrate that the formalin test triggers the release of noradrenaline in the DRt in a biphasic pattern that matches the two phases of the test. Finally, we show that reducing noradrenaline release into the DRt, using an HSV-1 vector which decreases the expression of tyrosine hydroxylase in noradrenergic DRt-projecting neurons, attenuates pain behavioral responses in both phases of the formalin test. The increased noradrenaline levels induced by the infusion of nomifensine at the DRt, along with the hyperalgesic effects of noradrenaline released at the DRt upon noxious stimulation, indicates that noradrenaline may enhance pain facilitation from the brain. It is important to evaluate if antidepressants that inhibit noradrenaline recapture enhance pain facilitation from the brain herein attenuating their analgesic effects.
... The DRt is also a relay for descending noradrenergic facilitation of pain from the LC [75,76,78,82]. The opioidergic system can directly and indirectly modulate the spinal-DRt-spinal circuitry [83][84][85]. This opioidergic modulation at the DRt is clinically relevant [74,86,87] and is involved in DNIC [4,5,14,59,60,65]. ...
Diffuse noxious inhibitory control (DNIC), also known as conditioned pain modulation (CPM) in humans, is a paradigm wherein the heterotopic application of a noxious stimulus results in the attenuation of another spatially distant noxious input. The pre-clinical and clinical studies show the involvement of several neurochemical systems in DNIC/CPM and point to a major contribution of the noradrenergic, serotonergic, and opioidergic systems. Here, we thoroughly review the latest data on the monoaminergic and opioidergic studies, focusing particularly on pre-clinical models of chronic pain. We also conduct an in-depth analysis of these systems by integrating the available data with the descending pain modulatory circuits and the neurochemical systems therein to bring light to the mechanisms involved in the regulation of DNIC. The most recent data suggest that DNIC may have a dual outcome encompassing not only analgesic effects but also hyperalgesic effects. This duality might be explained by the underlying circuitry and the receptor subtypes involved therein. Acknowledging this duality might contribute to validating the prognostic nature of the paradigm. Additionally, DNIC/CPM may serve as a robust paradigm with predictive value for guiding pain treatment through more effective targeting of descending pain modulation.
... These GABAergic interneurons are also presynaptically inhibited by DORexpressing fibers (102). Local overexpression of opioid peptides, namely ENK, was shown to inhibit DRt pain facilitation (104). The activation of MOR at the DRt plays a fundamental inhibitory role at the DRt and was recently shown to account for the analgesic effects of systemic opioids (105). ...
The treatment of neuropathic pain remains a clinical challenge. Analgesic drugs and antidepressants are frequently ineffective, and opioids may induce side effects, including hyperalgesia. Recent results on brainstem pain modulatory circuits may explain those clinical challenges. The dual action of noradrenergic (NA) modulation was demonstrated in animal models of neuropathic pain. Besides the well-established antinociception due to spinal effects, the NA system may induce pronociception by directly acting on brainstem pain modulatory circuits, namely, at the locus coeruleus (LC) and medullary dorsal reticular nucleus (DRt). The serotoninergic system also has a dual action depending on the targeted spinal receptor, with an exacerbated activity of the excitatory 5-hydroxytryptamine 3 (5-HT3) receptors in neuropathic pain models. Opioids are involved in the modulation of descending modulatory circuits. During neuropathic pain, the opioidergic modulation of brainstem pain control areas is altered, with the release of enhanced local opioids along with reduced expression and desensitization of μ-opioid receptors (MOR). In the DRt, the installation of neuropathic pain increases the levels of enkephalins (ENKs) and induces desensitization of MOR, which may enhance descending facilitation (DF) from the DRt and impact the efficacy of exogenous opioids. On the whole, the data discussed in this review indicate the high plasticity of brainstem pain control circuits involving monoaminergic and opioidergic control. The data from studies of these neurochemical systems in neuropathic models indicate the importance of designing drugs that target multiple neurochemical systems, namely, maximizing the antinociceptive effects of antidepressants that inhibit the reuptake of serotonin and noradrenaline and preventing desensitization and tolerance of MOR at the brainstem.
... 20,21 Opioids play a key role at the dorsal reticular nucleus. µ-opioid receptor is expressed in dorsal reticular nucleus-spinally and nonspinally projecting neurons, 22 and its activation plays a fundamental inhibitory role at the dorsal reticular nucleus, 23 which accounts for the analgesic effects of systemic opioids. 24 However, the effects of sustained opioid treatment on µ-opioid receptor signaling at the dorsal reticular nucleus has never been studied. ...
Background:
Increased descending pain facilitation accounts for opioid-induced hyperalgesia, but the underlying mechanisms remain elusive. Given the role of µ-opioid receptors in opioid-induced hyperalgesia in animals, the authors hypothesized that the dorsal reticular nucleus, a medullary pain facilitatory area, is involved in opioid-induced hyperalgesia through altered µ-opioid receptor signaling.
Methods:
The authors used male Wistar rats (n = 5 to 8 per group), chronically infused with morphine, to evaluate in the dorsal reticular nucleus the expressions of the µ-opioid receptor and phosphorylated cAMP response element-binding, a downstream marker of excitatory µ-opioid receptor signaling. The authors used pharmacologic and gene-mediated approaches. Nociceptive behaviors were evaluated by the von Frey and hot-plates tests.
Results:
Lidocaine fully reversed mechanical and thermal hypersensitivity induced by chronic morphine. Morphine-infusion increased µ-opioid receptor, without concomitant messenger RNA changes, and phosphorylated cAMP response element-binding levels at the dorsal reticular nucleus. µ-opioid receptor knockdown in morphine-infused animals attenuated the decrease of mechanical thresholds and heat-evoked withdrawal latencies compared with the control vector (von Frey [mean ± SD]: -17 ± 8% vs. -40 ± 9.0%; P < 0.001; hot-plate: -10 ± 5% vs. -32 ± 10%; P = 0.001). µ-opioid receptor knockdown in control animals induced the opposite (von Frey: -31 ± 8% vs. -17 ± 8%; P = 0.053; hotplate: -24 ± 6% vs. -3 ± 10%; P = 0.001). The µ-opioid receptor agonist (D-ALA2,N-ME-PHE4,GLY5-OL)-enkephalin acetate (DAMGO) decreased mechanical thresholds and did not affect heat-evoked withdrawal latencies in morphine-infused animals. In control animals, DAMGO increased both mechanical thresholds and heat-evoked withdrawal latencies. Ultra-low-dose naloxone, which prevents the excitatory signaling of the µ-opioid receptor, administered alone, attenuated mechanical and thermal hypersensitivities, and coadministered with DAMGO, restored DAMGO analgesic effects and decreased phosphorylated cAMP response element-binding levels.
Conclusions:
Chronic morphine shifted µ-opioid receptor signaling from inhibitory to excitatory at the dorsal reticular nucleus, likely enhancing descending facilitation during opioid-induced hyperalgesia in the rat. : WHAT WE ALREADY KNOW ABOUT THIS TOPIC: The phenomenon of opioid-induced hyperalgesia is supported by descending pain facilitation from brainstem nucleiThe dorsal reticular nucleus is modulated by opioids and mediates descending pain facilitation in some settings WHAT THIS ARTICLE TELLS US THAT IS NEW: Using a rat model of opioid-induced hyperalgesia, it was shown that reducing dorsal reticular activity with lidocaine blocked nociceptive sensitization from opioid infusionKnockdown of µ-opioid receptors or prevention of excitatory signaling using naloxone in the dorsal reticular nucleus prevented and reversed opioid-induced hyperalgesia.
... This is a solid procedure which allows that the response of each animal is analyzed and compared with the respective baseline values along the time. 10,17,39 Data are represented as the group means ± SEM. The significance of differences in each group was analyzed with two-way ANOVA followed by Tukey's post hoc test (subection 5.6). ...
Kyotorphin (KTP) is an endogenous peptide with analgesic properties when administered into the central nervous system (CNS). Its amidated form (L-Tyr-L-Arg-NH2; KTP-NH2) has improved analgesic efficacy after systemic administration, suggesting blood-brain barrier (BBB) crossing. KTP-NH2 also has anti-inflammatory action impacting on microcirculation. In this work, selected derivatives of KTP-NH2 were synthesized to improve lipophilicity and resistance to enzymatic degradation while introducing only minor changes in the chemical structure: N-terminal methylation and/or use of D amino acid residues. Intravital microscopy data show that KTP-NH2 having a D-Tyr residue, KTP-NH2-DL, efficiently decreases the number of leukocyte rolling in a murine model of inflammation induced by bacterial lipopolysaccharide (LPS): down to 46% after 30 min with 96 mM KTP-NH2-DL. The same molecule has lower ability to permeate membranes (relative permeability of 0.38) and no significant activity in a behavioral test which evaluates thermal nociception (hot-plate test). On the contrary, methylated isomers at 96 mM increase leukocyte rolling up to nearly 5-fold after 30 min suggesting a pro-inflammatory activity. They have maximal ability to permeate membranes (relative permeability of 0.8) and induce long-lasting antinociception.
... Other recent studies have focused in improving target expression to specific neuronal populations [81] and, since there is considerably neuronal heterogeneity in both the PNS and CNS, it would be naive to presume that the behaviour of viral vectors will be the same for all neuronal populations in different regions of the nervous system. Because of this intrinsic complexity, it is more feasible that neural targeting may be achieved through the use of specific promoters to drive expression of the therapeutic gene, such as promoters of genes encoding neuropeptides or enzymes involved in the synthesis of neurotransmitters, which are attractive candidates for cell targeting because many types of neurons are defined by their neurotransmitters [82,83]. ...
The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges.
... Immunohistochemistry Verification of injection site. To assess the position of the injection site of the control vector, b-gal immunostaining was performed, as described previously (Martins et al., 2008). Briefly, the sections were immunoreacted using the avidin-biotin-peroxidase complex (ABC) method with an antib-gal antiserum (BG-02; Santa Cruz Biotechnology, Santa Cruz, CA), diluted at 1:5,000, and a biotinylated swine antirabbit antiserum (Dako, Glostrup, Denmark) and ABC (Vector, Burlingame, CA), both at 1:200. ...
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.
... 16 In this study, the feasibility of administering a replicationselective HSV-1 vector by CED into normal brain was therefore examined in detail. In spite of the large number of preclinical studies that have involved the direct intracranial administration of HSV-based vectors, 17,18,[20][21][22][23][24][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41] remarkably this represents the first published study to evaluate the distribution properties of an HSV-1 vector using appropriate infusion parameters in both gray and white matter, as well as evaluation of strategies to improve vector distribution. ...
The direct intraparenchymal administration of oncolytic viral vectors by convection-enhanced delivery (CED) represents a promising new treatment strategy for malignant gliomas. However, there is no evidence to suggest that oncolytic viruses as large as herpes simplex virus-1 (HSV-1) can be administered by CED, as this has not been systematically examined in an animal model. In this study, the administration of a herpes simplex viral vector, HSV1, has been evaluated in detail in the gray and white matter of both rat and pig models, using high flow-rate infusions, co-infusing heparin or preinfusing the tissue with an isotonic albumin solution. Rat HSV-1 infusions at both slow (0.5 μl min(-1)) and high infusion rates (2.5 μl min(-1)) led to extensive tissue damage and negligible cell transduction. Co-infusion with heparin led to extensive hemorrhage. Preinfusion of tissue with an isotonic albumin solution facilitated widespread vector distribution and cell transduction in white matter only. Using this approach in pig brain led to widespread vector distribution with extensive transduction of astrocytes and activated microglia. In rat brain, enhanced green fluorescent protein expression peaked 48 h after vector administration and was associated with a vigorous immune response. These findings indicate that direct infusions of HSV-1-based viral vectors into the brain lead to minimal vector distribution, negligible cell transduction and extensive damage. Tissue preinfusion with an isotonic solution prior to vector administration represents an effective technique for achieving widespread HSV-1 distribution.
... Thus, in this study, we examined the transduced cells proximal to the injection site. Other studies, using other HSV-1 vector systems, have reported significant levels of retrograde transport of vectors to brain areas distant from the injection site (Martins et al., 2008). The amount of retrograde transport depends upon the strain of HSV-1, properties of the vector system, injection parameters, properties of the brain area receiving the injection, and other variables. ...
Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported approximately 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ss-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in approximately 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an approximately 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex.
Background
The dorsal reticular nucleus is a pain facilitatory area involved in the diffuse noxious inhibitory controls (DNIC), through opioidergic mechanisms that are poorly understood. We hypothesized that signaling of µ-opioid receptors is altered in this area at prolonged chronic inflammatory pain and that this accounts for the loss of DNIC occurring in this condition.
Methods
Monoarthritis was induced in male Wistar rats (n=5-9/group) by tibiotarsal injection of complete Freund’s adjuvant. We quantified the immunolabeling of µ-opioid receptors and the phosphorylated forms of µ-opioid receptors and cAMP response element-binding protein. Pharmacological manipulation of µ-opioid receptors at the dorsal reticular nucleus was assessed in DNIC, through the Randall-Selitto test.
Results
At 42 days of monoarthritis, µ-opioid receptor labeling decreased at the dorsal reticular nucleus, while its phosphorylated form and the phosphorylated cAMP response element-binding protein increased. D-ALA2,N-ME-PHE4,GLY5-OL)-enkephalin acetate (DAMGO) enhanced DNIC analgesia in normal animals ([Mean ± SD]: pre-DNIC: 126.9 ± 7.0g; DNIC - DAMGO: 147.5 ± 8.0g vs. DNIC + DAMGO: 198.1 ± 19.3g, p < 0.001), whereas it produced hyperalgesia in monoarthritis (pre-DNIC: 67.8 ± 7.5g; DNIC - DAMGO: 70.6 ± 7.7g vs. DNIC + DAMGO: 32.2 ± 2.6g, p < 0.001). An ultra-low dose of naloxone, which prevents the excitatory signaling of the µ-opioid receptor, restored DNIC analgesia in monoarthritis (DNIC - Naloxone: 60.0 ± 6.1g vs. DNIC + Naloxone: 98.0 ± 13.5g, p < 0.001), compared to saline (DNIC - Saline: 62.5 ± 5.2g vs. DNIC + Saline: 64.2 ± 3.8g). When injected prior to DAMGO, it restored DNIC analgesia and decreased the phosphorylated cAMP response element-binding protein in monoarthritis.
Conclusions
The dorsal reticular nucleus is likely involved in a facilitatory pathway responsible for DNIC hyperalgesia. The shift of µ-opioid receptor signaling to excitatory in this pathway likely accounts for the loss of DNIC analgesia in monoarthritis.
Brainstem areas involved in descending pain modulation are crucial for the analgesic actions of opioids. However, the role of opioids in these areas during tolerance, opioid-induced hyperalgesia (OIH), and in chronic pain settings remains underappreciated. We conducted a revision of the recent studies performed in the main brainstem areas devoted to descending pain modulation with a special focus on the medullary dorsal reticular nucleus (DRt), as a distinctive pain facilitatory area and a key player in the diffuse noxious inhibitory control paradigm. We show that maladaptive processes within the signaling of the µ-opioid receptor (MOR), which entail desensitization and a switch to excitatory signaling, occur in the brainstem, contributing to tolerance and OIH. In the context of chronic pain, the alterations found are complex and depend on the area and model of chronic pain. For example, the downregulation of MOR and δ-opioid receptor (DOR) in some areas, including the DRt, during neuropathic pain likely contributes to the inefficacy of opioids. However, the upregulation of MOR and DOR, at the rostral ventromedial medulla, in inflammatory pain models, suggests therapeutic avenues to explore. Mechanistically, the rationale for the diversity and complexity of alterations in the brainstem is likely provided by the alternative splicing of opioid receptors and the heteromerization of MOR. In conclusion, this review emphasizes how important it is to consider the effects of opioids at these circuits when using opioids for the treatment of chronic pain and for the development of safer and effective opioids.
Herpes simplex virus type 1 (HSV-1) is a neurotropic virus displaying several outstanding adaptations to the nervous system, and each of them can be rationally exploited in the design of gene therapy vectors with regard to neurological applications. Replication-competent attenuated vectors are becoming a suitable and powerful tool to eradicate brain tumors, such as malignant gliomas, due to their ability to replicate and spread only within the tumor mass, and have reached Phase II clinical trials in some cases. Replication- incompetent recombinant vectors are nontoxic gene transfer tools that preserve most of the neurotropic features of HSV- 1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A first clinical trial, conceived to treat cancerrelated pain, is currently being developed. Helper-dependent amplicon vectors take advantage of the capacity of the virus particle to accommodate up to 150 kbp of foreign DNA, enabling these vectors to deliver complete genomic loci to the nucleus of mammalian cells and making amplicons particularly useful in protocols where stable and physiological transgene expression is required. However, difficulties in obtaining large stocks of helper-free amplicons are hampering the use of these vectors in the clinic. This chapter summarizes current applications of HSV-1-based vectors with particular emphasis in amplicon vectors.
Chronic pain and epilepsy together affect hundreds of millions of people worldwide. While traditional pharmacotherapy provides essential relief to the majority of patients, a large proportion remains resistant, and surgical intervention is only possible for a select few. As both disorders are characterised by neuronal hyperexcitability, manipulating the expression of the most direct modulators of excitability - ion channels - represents an attractive common treatment strategy. A number of viral gene therapy approaches have been explored to achieve this. These range from the up- or down-regulation of channels that control excitability endogenously, to the delivery of exogenous channels that permit manipulation of excitability via optical or chemical means. In this review we highlight the key experimental successes of each approach and discuss the challenges facing their clinical translation.
Copyright © 2015. Published by Elsevier B.V.
The increasing prevalence of chronic pain imposes to search for new therapeutic approaches. Despite the increase in basic and clinical pain research during the last decades, the available analgesic drugs remained considerably unchanged. Gene therapy emerged as an important tool in the pain field. Studies in experimental pain models consisted on blockade of nociceptive transmission at the spinal cord by peripheral delivery of viral vectors (mainly replication-defective forms of herpes simplex virus type 1, HSV-1). Based on these results, clinical trials using gene therapy were successfully implemented in cancer patients with intractable pain.
The studies at the spinal cord opened the possibility to use gene transfer to interfere in a specific and sustained manner with pain control areas of the brain. Our research group departed from detailed morphological and functional characterization of pain control circuits of the brainstem to develop strategies to manipulate those areas using HSV-1 vectors. First, we established the dynamics of migration of HSV-1 upon injection into a unique pain facilitatory area of the medulla oblongata (the dorsal reticular nucleus-DRt). At shorter postinjection timepoints, transduction occurred mainly at the DRt, whereas at longer times a selective migration was detected in DRt brain afferents. Since some of those afferents were noradrenergic and based on the role of noradrenaline in increasing pain facilitation from the DRt, we engineered an HSV-1 vector that contains the tyrosine hydroxylase (TH) promoter and the TH transgene in antisense orientation. This vector decreased neuropathic pain behaviors for about a month. Similar studies addressing other brain areas involved in descending pain modulation are necessary to fully ascertain the full potential of gene therapy for chronic pain control.
Large panel of gene-based techniques is used for many years specifically in the pain research field. From the first identification (cloning) of some "mythic" genes, such as those encoding opioid or capsaicin receptors allowing then the creation of first-generation knockout mice, to the today conditional (time, tissue, cell-type and even pathology-dependent) and regulatable modulation of a gene function, these approaches largely contributed to fundamental leaps forward in our understanding of the function of some proteins and of their interest as possible druggable targets. Perhaps one of the most remarkable evolution in the last years is the passage of these approaches from the bench to the patient; whether it concerns the identification of genes involved in inherited pain insensibility/susceptibility, the search for genetic markers of pain types, the individual pharmacogenomics or even the first gene therapy trials. From many possible variants of gene-grounded techniques used in pain research we focus here on gene knockouts and some recent developments, on viral vectors-based gene transfer and on transgenic models for the tracing of pain pathways. Through these selected examples we attempted to emphasize the immense potential of these approaches and their already well-recognized contribution in both the basic and clinical pain research.
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.
Intuition has always been associated with a hint of the anomalous, with credible examples of intuitive knowledge that appear to arise from the future or from distant locations without sensory mediation. Experimental and anecdotal evidence exists to suggest that a complete understanding of intuition will need to include these anomalous aspects of intuition. To that end it is important to recognize that an understanding of anomalous intuition, though presenting considerable challenges to physics and neuroscience, may not require ‘paranormal’ explanations or entities outside the compass of science. Anomalous intuition has traditionally been studied within the field of parapsychology where is it known as extrasensory perception (ESP). One model of ESP proposes two stages. In the first stage, the process, the anomalous information is ‘received’ and in the second stage, the product, the anomalous information is elaborated into conscious awareness and/or behaviour. The process is a challenge primarily for physics, and physicists both within parapsychology and in the mainstream are addressing it. The product, anomalous intuition, is grounded in normal psychological activity and is a problem for psychology and neuroscience to understand. In order to understand how ESP or anomalous intuition works, it is necessary to understand its purpose within an evolutionary context. Evolution has proven exceptionally effective in enabling species to make use of a wide range of physical phenomena for information gathering and communication. If some yet undiscovered physical process permits retrocausation would we not expect evolution to have capitalized on that process. As a product of evolution, ESP would have to conform to the requirements of evolutionary theory. Suddendorf and Corballis (2007) have elaborated the notion of mental time travel (MTT), the memory-based ability to project oneself into the past as well as the future, and have argued that the “ultimate evolutionary advantage” may lie in the capacity to envisage future events. Their model of MTT provides an ideal context in which to understand an evolutionary path for anomalous intuition. Working through the emotional system, anomalous intuition may operate by influencing the selection of memory images we use to execute our MTT. A growing body of data supports the involvement of the emotional system in anomalous intuition but there is only suggestive evidence for the expected hereditary component. The observed limited effectiveness of anomalous intuition may emerge from the balance achieved through an evolutionarily stable strategy, or result from inherent limitations in capitalizing on the underlying physical process
The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges.
Descending modulation of nociceptive transmission depends on the release of noradrenaline at the spinal cord. The role of noradrenaline in the control of nociceptive transmission at the supraspinal pain control system remains understudied. As chronic pain is associated with enhanced descending facilitation of nociceptive transmission, we sought to determine the role of noradrenaline in pain facilitation from the brain during neuropathic pain. We determined the action of the noradrenergic input to the dorsal reticular nucleus (DRt), a unique pain facilitatory area, using the spared nerve injury model. Injections of the α1-adrenoreceptor agonist phenylephrine into the DRt induced hyperalgesia and allodynia, indicating that α1-adrenoreceptors enhance the facilitatory action of the nucleus. This led us to reduce noradrenaline release at the DRt using a viral vector derived from the Herpes Simplex virus type 1 (HSV-1) which carried the tyrosine hydroxylase (TH) transgene in antisense orientation. The reduction of noradrenaline release, confirmed by microdialysis experiments, induced a long-lasting attenuation of pain responses, which was reverted by the local administration of phenylephrine. The present study indicates that the noradrenergic modulation of a pronociceptive area at the supraspinal pain control system accounts for pain facilitation, through the activation of α1-adrenoreceptors. The study also shows that sustained effects on chronic pain can be achieved by decreasing the release of noradrenaline in a pain facilitatory centre of the brain using gene transfer.
HSV-1 is a neurotropic virus that displays several important adaptations to the nervous system of the host organism, each of which can be rationally exploited in the design of gene therapy vectors for neurological applications. Replication-incompetent (replication-defective) helper-independent recombinant vectors are nontoxic tools for gene transfer that preserve most of the neurotropic features of HSV-1, particularly the ability to express genes after establishing latent infections, and are thus proficient candidates for therapeutic gene transfer in neurons. A clinical trial with the use of a replication-incompetent vector, NP-2 (Diamyd Inc), for the treatment of pain has been initiated. Attenuated replication-competent (oncolytic) vectors are becoming suitable and powerful tools to eradicate brain tumors, such as malignant gliomas, as a result of the ability to replicate and spread only within the tumor mass. Some attenuated replication-competent vectors, such as G-207 and HSV-1716 (Crusade Laboratories Ltd), have been used in clinical trials for the treatment of cancers including recurrent malignant glioma. Helper-dependent amplicon vector technology takes advantage of the capacity of the virus particle to accommodate < or = 150 Kbp of foreign DNA, enabling these vectors to deliver complete genomic loci to the nucleus of mammalian cells, making amplicons particularly useful agents in protocols that require stable and physiological transgene expression. However, difficulties in obtaining large stocks of helper-free amplicons continue to limit the use of these vectors in the clinic.
Molecular Therapy (2005) 11, S63|[ndash]|S64; doi: 10.1016/j.ymthe.2005.06.162
158. Gene Therapy for Chronic Pain Control: Perspectives of HSV-1 Mediated Gene Transfer to Supraspinal Centres|[ast]|
Isabel Martins1, Marta Pinto1, Steven Wilson2, Deolinda Lima1 and Isaura Tavares11Institut of Histology and Embriology, Faculty of Medicine of Porto, Porto, Portugal2Pharmacology, Physiology and Neuroscience, University of South Carolina School of Medicine, Columbia, SC|[ast]|Supported by FCT and FEDER grant POCTI/NSE/38952/2001.
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.
We have previously used a defective herpes simplex virus vector to express a foreign gene in the adult rat brain. One application of this technology would be the in vivo analysis of promoter function in brain after de novo transfer, which would allow the rapid generation of vectors with localized application in a broad range of mammalian species while avoiding influences of other nearby promoters. A 2.7-kb fragment of the rat preproenkephalin promoter was placed upstream of the bacterial lacZ gene in our herpes simplex virus amplicon. A restricted pattern of lacZ expression was observed in vivo, which follows previously observed patterns of endogenous preproenkephalin expression. These results, from the direct gene transfer into an adult animal brain for in vivo promoter analysis, demonstrate that sequence information that influences restricted expression of preproenkephalin is located within 2.7 kb upstream of transcriptional initiation. lacZ expression was also observed in rat brain for 2 months after direct transfer, and PCR analysis confirmed the continued presence of amplicon DNA in lacZ-positive sections. Restricted and long-term expression observed with an endogenous promoter has important implications for gene therapy using viral vectors.
We used retrograde transneuronal transport of herpes simplex virus type 1 to map the origin of cerebellar and basal ganglia "projections" to leg, arm, and face areas of the primary motor cortex (M1). Four to five days after virus injections into M1, we observed many densely labeled neurons in localized regions of the output nuclei of the cerebellum and basal ganglia. The largest numbers of these neurons were found in portions of the dentate nucleus and the internal segment of the globus pallidus (GPi). Smaller numbers of labeled neurons were found in portions of the interpositus nucleus and the substantia nigra pars reticulata. The distribution of neuronal labeling varied with the cortical injection site. For example, within the dentate, neurons labeled from leg M1 were located rostrally, those from face M1 caudally, and those from arm M1 at intermediate levels. In each instance, labeled neurons were confined to approximately the dorsal third of the nucleus. Within GPi, neurons labeled from leg M1 were located in dorsal and medial regions, those from face M1 in ventral and lateral regions, and those from arm M1 in intermediate regions. These results demonstrate that M1 is the target of somatotopically organized outputs from both the cerebellum and basal ganglia. Surprisingly, the projections to M1 originate from only 30% of the volume of the dentate and <15% of GPi. Thus, the majority of the outputs from the cerebellum and basal ganglia are directed to cortical areas other than M1.
To test the utility of gene therapeutic approaches for the treatment of pain, a recombinant herpes simplex virus, type 1, has been engineered to contain the cDNA for an opioid peptide precursor, human preproenkephalin, under control of the human cytomegalovirus promoter. This virus and a similar recombinant containing the Escherichia coli lacZ gene were applied to the abraded skin of the dorsal hindpaw of mice. After infection, the presence of beta-galactosidase in neuronal cell bodies of the relevant spinal ganglia (lacZ-containing virus) and of human proenkephalin (preproenkephalin-encoding virus) in the central terminals of these neurons indicated appropriate gene delivery and expression. Baseline foot withdrawal responses to noxious radiant heat mediated by Adelta and C fibers were similar in animals infected with proenkephalin-encoding and beta-galactosidase-encoding viruses. Sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (Adelta fibers) observed in control animals was reduced or eliminated in animals infected with the proenkephalin-encoding virus for at least 7 weeks postinfection. Hence, preproenkephalin cDNA delivery selectively blocked hyperalgesia without disrupting baseline sensory neurotransmission. This blockade of sensitization was reversed by administration of the opioid antagonist naloxone, apparently acting in the spinal cord. The results demonstrate that the function of sensory neurons can be selectively altered by viral delivery of a transgene. Because hyperalgesic mechanisms may be important in establishing and maintaining neuropathic and other chronic pain states, this approach may be useful for treatment of chronic pain and hyperalgesia in humans.
This study examined a mechanism responsible for the enhanced antihyperalgesic and antinociceptive effects of the mu opioid receptor agonist (ORA) [D-Ala(2), NMePhe(4), Gly(5)-ol]enkephalin (DAMGO) microinjected in the rostroventromedial medulla (RVM) of rats with inflammatory injury induced by injection of complete Freund's adjuvant (CFA) in one hindpaw. In rats injected with CFA 4 hr earlier, microinjection of the mu opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) in the RVM antagonized both the marginal enhancement of the potency of DAMGO and its antinociceptive effect. The delta opioid receptor antagonist naltriben (NTB) was without effect. In rats injected with CFA 2 weeks earlier, CTAP antagonized the effects of DAMGO to a lesser extent. However, NTB completely prevented the enhancement of the potency of DAMGO, whereas it did not antagonize DAMGO's antinociceptive effects. Microinjection of NTB alone, but not CTAP in the RVM of CFA-treated rats, enhanced the hyperalgesia present in the ipsilateral hindpaw and induced hyperalgesia in the contralateral, uninjured hindpaw. These results suggest that persistent inflammatory injury increased the release in the RVM of opioid peptides with preferential affinity for the delta opioid receptor, which can interact in a synergistic or additive manner with an exogenously administered mu opioid receptor agonist. Indeed, the levels of [Met(5)]enkephalin and [Leu(5)]enkephalin were increased in the RVM and in other brainstem nuclei in CFA-treated rats. This increase most likely presents a compensatory neuronal response of the CNS of the injured animal to mitigate the full expression of inflammatory pain and to enhance the antinociceptive and antihyperalgesic effects of exogenously administered mu opioid receptor analgesics.
Herpes simplex virus (HSV) has several potential advantages as a vector for delivering genes to the nervous system. The virus naturally infects and remains latent in neurons and has evolved the ability of highly efficient retrograde transport from the site of infection at the periphery to the site of latency in the spinal ganglia. HSV is a large virus, potentially allowing the insertion of multiple or very large transgenes. Furthermore, HSV does not integrate into the host chromosome, removing any potential for insertional activation or inactivation of cellular genes. However, the development of HSV vectors for the central nervous system that exploit these properties has been problematical. This has mainly been due to either vector toxicity or an inability to maintain transgene expression. Here we report the development of highly disabled versions of HSV-1 deleted for ICP27, ICP4, and ICP34.5/open reading frame P and with an inactivating mutation in VP16. These viruses express only minimal levels of any of the immediate-early genes in noncomplementing cells. Transgene expression is maintained for extended periods with promoter systems containing elements from the HSV latency-associated transcript promoter (J. A. Palmer et al., J. Virol. 74:5604-5618, 2000). Unlike less-disabled viruses, these vectors allow highly effective gene delivery both to neurons in culture and to the central nervous system in vivo. Gene delivery in vivo is further enhanced by the retrograde transport capabilities of HSV. Here the vector is efficiently transported from the site of inoculation to connected sites within the nervous system. This is demonstrated by gene delivery to both the striatum and substantia nigra following striatal inoculation; to the spinal cord, spinal ganglia, and brainstem following injection into the spinal cord; and to retinal ganglion neurons following injection into the superior colliculus and thalamus.
Endogenous opiate peptides acting pre- and post-synaptically in the dorsal horn of spinal cord inhibit transmission of nociceptive stimuli. We transfected neurons of the dorsal root ganglion in vivo by footpad inoculation with 30 microl (3 x 10(7) p.f.u.) of a replication-incompetent (ICP4-deleted) herpes simplex virus (HSV) vector with a cassette containing a portion of the human proenkephalin gene coding for 5 met- and 1 leu-enkephalin molecules under the control of the human cytomegalovirus immediate-early promoter (HCMV IEp) inserted in the HSV thymidine kinase (tk) locus. Vector-directed expression of enkephalin produced a significant antinociceptive effect measured by the formalin footpad test, that was most prominent in the delayed ("tonic") phase 20-70 min after the administration of formalin. The magnitude of the antinociceptive effect diminished over 4 weeks after transduction, but reinoculation of the vector reestablished the analgesic effect, without evidence for the development of tolerance. The antinociceptive effect was blocked completely by intrathecal naltrexone. These results suggest that gene therapy with an enkephalin-producing herpes-based vector may prove useful in the treatment of pain.
Rheumatoid arthritis is characterized by erosive inflammation of the joints, new bone proliferation, and ankylosis, leading to severely reduced locomotion and intense chronic pain. In a model of this disease, adjuvant-induced polyarthritis in the rat, neurons involved in pain transmission and control undergo plastic changes, especially at the spinal level. These changes affect notably neurons that contain opioids, such as enkephalins deriving from preproenkephalin A (PA) precursor protein. Using recombinant herpes simplex virus containing rat PA cDNA, we enhanced enkephalin synthesis in sensory neurons of polyarthritic rats. This treatment markedly improved locomotion and reduced hyperalgesia. Furthermore, the progression of bone destruction slowed down, which is the most difficult target to reach in the treatment of patients suffering from arthritis. These data demonstrate the therapeutic efficacy of enkephalin overproduction in a model of systemic inflammatory and painful chronic disorder.
It is known that pain perception can be altered by mood, attention and cognition, or by direct stimulation of the cerebral cortex, but we know little of the neural mechanisms underlying the cortical modulation of pain. One of the few cortical areas consistently activated by painful stimuli is the rostral agranular insular cortex (RAIC) where, as in other parts of the cortex, the neurotransmitter gamma-aminobutyric acid (GABA) robustly inhibits neuronal activity. Here we show that changes in GABA neurotransmission in the RAIC can raise or lower the pain threshold--producing analgesia or hyperalgesia, respectively--in freely moving rats. Locally increasing GABA, by using an enzyme inhibitor or gene transfer mediated by a viral vector, produces lasting analgesia by enhancing the descending inhibition of spinal nociceptive neurons. Selectively activating GABA(B)-receptor-bearing RAIC neurons produces hyperalgesia through projections to the amygdala, an area involved in pain and fear. Whereas most studies focus on the role of the cerebral cortex as the end point of nociceptive processing, we suggest that cerebral cortex activity can change the set-point of pain threshold in a top-down manner.
Neuropathic pain is defined as pain initiated or caused by a primary lesion or dysfunction in the nervous system. It is a devastating and difficult to manage consequence of peripheral nerve injury and has a variety of clinical symptoms. Neuropathic pain is a major health problem. It has been estimated that 70% of patients with advanced cancer and inflammatory pathologies are afflicted by chronic pain. About 95% of patients with spinal cord injuries have neuropathic pain problems. Chronic pain is debilitating and cause of depression and decreasing quality of life. Pharmacological treatment for the symptoms of painful neuropathy is difficult, because there has been limited understanding of the underlying causes and systemic levels that an effective dose can have on multiple side effects. The use of molecular methods, such as gene therapy, stem cell therapy and viral vector for delivery of biologic antinociceptive molecules, has led to a better understanding of the underlying mechanisms of the induction of intractable neuropathic pain.
Trigeminal neuropathic pain represents a real challenge to therapy because commonly used drugs are devoid of real beneficial effect or patients frequently become intolerant or refractory to some of these compounds. In a rat model of trigeminal neuropathic pain, which shares numerous similarities with human trigeminal neuralgia and trigeminal neuropathic pain, we used a genomic herpes simplex virus-derived vector (HSVLatEnk) to examine the possible effect of a local overproduction of proenkephalin A (PA) targeted to the trigeminal primary sensory neurons. Unilateral peripheral inoculation of recombinant vectors on the vibrissal pad territory resulted in an about ninefold increase in proenkephalin A mRNA levels in trigeminal ganglion ipsilateral to the infected side. Transgene-derived met-enkephalin accumulated in numerous nerve cell bodies of trigeminal ganglion and was transported through the sensory nerve fibers located in the infraorbital nerve. Bilateral mechanical hyperresponsiveness, which developed 2 weeks after chronic constrictive injury of the left infraorbital nerve, was significantly attenuated in animals overproducing PA in the trigeminal ganglion ipsilateral to the lesioned infraorbital nerve. This antiallodynic effect was reversed by both the opioid receptor antagonist naloxone and the peripherally acting antagonist naloxone methiodide. Our data demonstrate that the local overproduction of PA-derived peptides in trigeminal ganglion sensory neurons evoked a potent antiallodynic effect through the stimulation of mainly peripherally located opioid receptors and suggest that targeted delivery of endogenous opioids may be of interest for the treatment of some severe forms of neuropathic pain.
Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.
We examined the ability of the herpes simplex virus (HSV) latency-associated promoter (LAP2) to drive biologically relevant prolonged transgene expression in the peripheral nervous system. Rat dorsal root ganglia were transduced in vivo by subcutaneous inoculation of replication-incompetent HSV-based vectors containing nerve growth factor (NGF) or neurotrophin-3 (NT-3) under the control of LAP2 (vectors SLN and QLNT3, respectively) and vector SHN expressing NGF under the control of the human cytomegalovirus immediate early promoter. Twenty-four weeks later a pure sensory neuropathy was induced by overdose of pyridoxine (PDX), and the animals were assessed 6 months after inoculation. Inoculation of SLN, but not SHN, attenuated the nerve damage caused by PDX and protected foot sensory amplitude, H-wave amplitude, and behavioral measures of proprioceptive function. QLNT3 was more effective than SLN in preserving the largest myelinated fibers from degeneration. These results indicate that expression of NGF or NT-3 driven by LAP2 is sufficient to prevent the development of neuropathy 6 months after vector inoculation in rats.
In this study, we evaluated the possible use of lentiviral vectors in the treatment of neuropathic pain. We chose to administer GDNF-expressing vectors because of the known beneficial effect of this trophic factor in alleviation of neuropathic pain in adult rodents. Lentiviral vectors expressing either GDNF or control, green fluorescent protein or beta-galactosidase, were injected unilaterally into the spinal dorsal horn 5 weeks before a spinal nerve ligation was induced (or sham surgery for the controls). We observed that intraspinally administered lentiviral vectors resulted in a large and sustained expression of transgenes in both neurons and glial cells. Injection of GDNF-expressing viral vectors induced a significant reduction of ATF-3 up-regulation and IB4 down-regulation in damaged DRG neurons. In addition, it produced a partial but significant reversal of thermal and mechanical hyperalgesia observed following the spinal nerve ligation. In conclusion, our study suggests that lentiviral vectors are efficient tools to induce a marked and sustained expression of trophic factors in specific areas of the CNS and can, even if with some limitations, be efficient in the treatment of neuropathic pain.
This study investigated three possible mechanisms by which the antinociceptive effects of the mu-opioid receptor (MOR) agonist [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and the delta-opioid receptor (DOR) agonist [d-Ala(2),Glu(4)]-deltorphin (deltorphin II) (DELT), microinjected into the rostral ventromedial medulla (RVM), are enhanced in rats with persistent inflammatory injury. Radioligand binding determined that neither the B(max) nor the K(d) values of [(3)H]DAMGO differed in RVM membranes from rats that received an intraplantar injection of saline or complete Freund's adjuvant (CFA) in one hindpaw 4 h, 4 days, or 2 weeks earlier. Likewise, neither the EC(50) nor the E(max) value for DAMGO-induced stimulation of guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding differed in the RVM of saline- or CFA-treated rats at any time point. Microinjection of fixed dose combinations of DAMGO and DELT in the RVM of naive rats indicated that these agonists interact synergistically to produce antinociception when DAMGO is present in equal or greater amounts than DELT and, additively, when DELT is the predominant component. Thus, unlike the periphery or spinal cord, potentiation of MOR-mediated antinociception does not entail an increase in MOR number, affinity, or coupling. Rather, the data are concordant with our proposal that potentiation results from a synergistic interaction of exogenous MOR agonist with DOR-preferring enkephalins whose levels are increased in CFA-treated rats (J Neurosci 21:2536-2545, 2001). Virtually no specific [(3)H]DELT binding nor stimulation of [(35)S]GTPgammaS binding by DELT was obtained in RVM membranes from CFA- or saline-treated rats at any time point. The mechanisms responsible for the potentiation of DELT-mediated antinociception remain to be elucidated.
Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin gene-related peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-β-endorphin (ppβEP) led to significant (P < 0.0001) reversal of mechanical allodynia for ≥3 months. The antiallodynic effect could be reversed by the μ-opioid antagonist naloxone 4 months after gene transfer (P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with ppβEP was equally effective (P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.
• adeno-associated virus
• dorsal root ganglion
• gene therapy
• β-endorphin
• IL-10
The use of alpha herpesviruses for defining functionally interconnected populations of neurones has generated considerable interest in recent years (1). Two of the most attractive aspects of using viruses for circuit definition are the fact that they pass transneuronally and replicate in synaptically linked populations of neurones. Thus, the virus is transported through a multi-synaptic pathway and the signal, (i.e. intracellular concentration of virus) progressively increases with time (Figures 1 and 2). These two attributes are clearly superior to those offered by other neuronal tracers which have been shown to pass transneuronally. Wheatgerm agglutinin horseradish peroxidase (WGA-HRP) and the C fragment of tetanus toxin (CTT) are both known to cross synapses, but they do so in such limited amounts that it is at times difficult to identify synaptic transfer with confidence. For these reasons, there has been a dramatic increase in the number of studies which have utilized herpesviruses to characterize functionally distinct populations of neurones.
The opioid peptide enkephalin emerges as a major neuromodulator in the regulation and integration of the physiologic response in stressful conditions. The paraventricular nucleus of the hypothalamus is a coordinating center of neuroendocrine and autonomic functions. However, the detailed distribution of the enkephalin fibers and terminals in the paraventricular nucleus and the sources of enkephalinergic innervation are not well defined. In the present study, we used immunocytochemistry for the proenkephalin-derived octapeptide met-arg6-gly7-leu8 enkephalin to determine the distribution of enkephalin-immunoreactive fibers and somata within paraventricular nucleus. Without colchicine pretreatment, enkephalinergic fibers were prominent mainly in the ventromedial part of the parvicellular subdivision of the paraventricular nucleus, appearing in coronal sections as a dense collection of short segments of enkephalin-immunoreactive fibers. In the periventricular portion of the paraventricular nucleus, enkephalin-immunoreactive fibers produced a moderate plexus of short enkephalin-immunoreactive fibers dorsoventrally oriented. With colchicine treatment, a dense cluster of enkephalin-immunoreactive cell bodies was located in the dorsomedial and the dorsal parts of the parvicellular subdivisions. These enkephalin-immunoreactive neurons were small (< 10 μm) to medium sized (10–15 μm), with round and elongated shapes. Retrograde transport of wheat germ-conjugated gold particles. WGA-apoHRP-Au, from the paraventricular nucleus, combined with immunocytochemistry for enkephalin revealed that the major sources of extrahypothalamic enkephalin afferents to the paraventricular nucleus are provided by enkephalin neurons neurons in the lateral reticular nucleus and the paragigantocellularis reticular nucleus of the medulla (∼20% of retrogradely labeled neurons within this nucleus were double labeled) and in the nucleus solitary tract (∼10% of retrogradely labeled neurons within thisnucleus were double labeled). Retrogradely labeled enkephalin neurons were also observed in the medial preoptic area, median preoptic nucleus, dorsomedial hypothalamic nucleus, lateral septum and hypothalamic arcuate nucleus. These enkephalinergic pathways from the medulla and the forebrain could represent an anatomical substrate underlying the opioid effects on paraventricular neurons during physiological processes, such as a cardiovascular regulation, feeding or stress responses.
This chapter highlights that noxious inputs can be modulated by descending systems that serve to inhibit or facilitate nociception, serving as an endogenous mechanism to regulate pain. These systems are of great clinical significance as it is now apparent that opioids produce their antinociceptive activity, in part, by activating descending pain inhibitory mechanisms. Chronic pain, whether because of peripheral nerve injury or tissue inflammation, is associated with numerous neuroplastic changes that alter the underlying physiology of pain transmission and pain modulatory pathways. The chapter explores many types of chronic pain, which is associated with increased inputs to the central nervous system (CNS), enhanced responsiveness of spinal transmission neurons and, critically, the activation of pain facilitation from supraspinal sites. The chapter concludes that it now seems apparent that ascending/descending loops can be established, which are essential to the maintenance of the chronic pain state and may participate even in more transient pain conditions. Understanding of these mechanisms should increase the likelihood of development of strategies in which the mechanisms maintaining chronic pain states can be disrupted.
Introducing genes into adult neurons in vivo may be a useful experimental tool for studying and modifying neuronal function. In this study two herpes simplex virus type 1 (HSV-1) mutants were used to examine the capability of different types of neostriatal neurons to express a foreign gene introduced through viral infection. In these HSV-1 mutants (7134 and RH105) the Escherichia coli gene, lacZ, under the control of viral promoters active during the early phase of infection, was substituted for viral genes (ICPO and TK, respectively) needed for efficient replication in the nervous system. Adult male rats received unilateral injections of HSV-1 mutant 7134 or RH105 into the neostriatum. Animals survived for 1 to 70 days with no apparent adverse physiological or behavioral effects. At the injection site, both mutant viruses produced focal tissue necrosis and reactive gliosis. Histochemical detection of the lacZ gene product, beta-galactosidase (beta Gal), revealed extensive labeling of neurons with mutant 7134 and relatively limited neuronal labeling with the mutant RH105. Mutant 7134, which is capable of some replication in cells, conferred beta Gal expression in cells over an area that was twofold greater than the necrotic area. In contrast, mutant RH105, which cannot replicate in cells, produced a zone of beta Gal-labeled cells only two-thirds the area of the necrotic core. Both medium- and large-sized neostriatal neurons were positive for beta Gal, and a higher proportion of large cells were labeled as compared to other neuronal populations in the normal striatum. A few glial cells were also beta Gal-positive. Retrograde transport of virus to the substantia nigra pars compacta and to the cortex was minimal and occurred only with mutant 7134. No evidence was seen for anterograde transport. Immunohistochemical localization of beta Gal at the ultrastructural level after inoculation with mutant 7134 revealed that both types of medium-sized neurons (spiny and aspiny types), as well as large neurons, were infected 3 days following inoculation. Immunoreactive neurons ranged from severely pathologic to remarkably healthy. Some of the axon terminals that contacted beta Gal-immunoreactive dendrites and spines were degenerated. These results demonstrate that in the adult rat replication-deficient HSV-1 vectors injected intrastriatally can be used to express a foreign gene in at least three types of neostriatal neurons, while maintaining the long-term survival and general health of the injected animals. The neurotoxicity induced by HSV-1 mutants may still be considerable, however, and ways of minimizing neuropathological effects need to be addressed.
Spinal afferents to the medullary dorsal reticular nucleus were studied using the following retrograde tracers: horseradish peroxidase (diluted in dimethylsulfoxide), wheat germ agglutinin conjugated with horseradish peroxidase, and cholera toxin subunit B. Spinal cord cells projecting to that medullary region were located predominantly in medial lamina I and lamina X. Cell labelling was moderate in the medial part of laminae II-IV and sparse throughout laminae V-VII. Labelling was predominantly ipsilateral in the dorsal horn and bilateral in laminae VII and X. After mechanical lesions of the dorsal white matter which severed most of the ipsilateral cuneate fasciculus, the numbers of superficial dorsal horn cells that were labelled from the dorsal reticular nucleus were considerably decreased caudal to the lesion, which suggests that their axons utilize mostly the cuneate fasciculus. Since the medullary dorsal reticular nucleus of the rat has a predominant population of nociceptive specific neurons, it is suggested that this spino-dorsomedullary reticular pathway is involved in pain processing.
Three mutants of herpes simplex virus type 1 (HSV-1) were used to deliver and express the Escherichia coli lacZ gene in cells of the rat central nervous system. Because the lacZ gene was inserted in place of the genes encoding one of the immediate-early viral proteins ICP0 or ICP4 or the early viral protein thymidine kinase, these mutants were compromised or defective in their ability to replicate. All mutant vectors exhibited reduced pathogenesis in animals as compared to the wild type HSV-1 strain KOS. In all cases lacZ was under the control of immediate-early or early viral promoters that are active in the early phase of infection. Expression of beta-galactosidase was observed in cortical neurons following stereotactic inoculation of mutant viruses into adult rat brains; distinct patterns of expression were observed with each mutant vector. Injection of the ICP0 mutant in the frontal cortex and caudate nucleus resulted in beta-galactosidase expression in a substantial number of cells around the inoculation site and at some distance from it for 14 days, with maximum expression after 3 days. The ICP0 vector appeared to have reached the ipsilateral and contralateral cingulate cortex by retrograde transport. Following inoculations of the ICP4 and thymidine kinase vectors into the same brain regions, only a few cells in areas immediately adjacent to the injection track expressed beta-galactosidase and they did so for only a few days. These herpes virus-derived vectors provide a means for the in situ delivery and expression of specific genes in neurons in the central nervous system with little adverse effect on animals.
A method to measure cutaneous hyperalgesia to thermal stimulation in unrestrained animals is described. The testing paradigm uses an automated detection of the behavioral end-point; repeated testing does not contribute to the development of the observed hyperalgesia. Carrageenan-induced inflammation resulted in significantly shorter paw withdrawal latencies as compared to saline-treated paws and these latency changes corresponded to a decreased thermal nociceptive threshold. Both the thermal method and the Randall-Selitto mechanical method detected dose-related hyperalgesia and its blockade by either morphine or indomethacin. However, the thermal method showed greater bioassay sensitivity and allowed for the measurement of other behavioral parameters in addition to the nociceptive threshold.
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.
A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.
The termination sites in the medullary dorsal reticular nucleus (DRt) of axons arising from superficial or deep dorsal horn cells were analysed by anterograde tracing with biotinylated dextran (BD). Following BD injections in spinal laminae I-III or in these laminae plus the dorsal funiculus, labelled fibres and boutons were located in the dorsal most ipsilateral portion of the DRt. Injections covering laminae III-V or laminae I-V resulted in bilateral labelling of the ventral DRt and ipsilateral labelling of the dorsal DRt. These findings, together with previous data concerning the origin of DRt projections to the superficial or deep dorsal horn, suggest the occurrence of reciprocal anatomical loops connecting the dorsal or the ventral areas of both regions.
Viral vectors are a means by which genes can be delivered to specific sites in the adult central nervous system. Nevertheless, the interaction between the viral vector and cells of the nervous system, which forms the basis for specific gene transfer, is not well understood. In this study a nonreplicating defective herpes simplex virus type 1 vector, expressing the marker gene lacZ, was stereotaxically injected at varying titers into the rat central nervous system. Three sites were targeted: the caudate nucleus, dentate gyrus, and cerebellar cortex, and the resulting patterns of beta-galactosidase activity were examined. Many cells of neuronal and glial morphology, and of differing neuronal subtypes, expressed beta-galactosidase at each of the injection sites. However, beta-galactosidase activity was also detected in distant secondary brain areas, the neurons of which make afferent connections with the primary sites. This strongly suggested that the retrograde transport of defective virus was the basis for the enzyme activity observed at a distance. Moreover, retrograde transport to secondary sites was found to be highly selective and restricted to certain retrograde neuroanatomical pathways in a specific and titer dependent fashion. The pathways observed were predominantly, but not exclusively, monoaminergic in origin. This finding is supported by reports of specific tropism by HSV for monoaminergic circuits in experimental encephalitis and transneuronal tracing studies. Our observations suggest that certain functional neuronal populations, which are permissive for the retrograde transfer of defective HSV-1 vectors, might be specifically targeted for gene transfer using this approach. Conversely, a knowledge of the pathways permissive for viral uptake, retrograde transfer, and subsequent gene expression will be essential in order to predict the consequences of gene transfer using viral vectors.
We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy.
A defective herpes simplex virus type one (HSV-1) vector that contains a 6.8-kb fragment of the rat tyrosine hydroxylase promoter (pTHlac-7kb) was examined for its capability to target catecholaminergic cell type-specific expression in the CNS. Cell type-specific expression was assessed by comparison with a control vector (pHSVlac) that uses the HSV-1 immediate early 4/5 promoter to support expression in multiple cell types. In initial experiments comparing expression in catecholaminergic and noncatecholaminergic cell lines, pTHlac-7kb supported a seven- to 20-fold increase in reporter gene expression in catecholaminergic cell lines. Four days after stereotactic injection into the midbrain of adult rats, pTHlac-7kb supported a 10-fold targeting of beta-galactosidase expression to tyrosine hydroxylase-expressing neurons in the substantia nigra pars compacta compared with pHSVlac. Expression from pTHlac-7kb was stably maintained for 6 weeks with no significant changes in the pattern of expression. Long-term expression from pTHlac-7kb was confirmed by RNA and DNA analysis. In contrast, reporter gene expression in the midbrain from pHSVlac decreased approximately 30-fold between 4 days and 6 weeks after gene transfer. Thus, within the context of this HSV-1 vector system, the tyrosine hydroxylase promoter enhanced cell type-specific expression and contributed to stable, long-term expression of a recombinant gene product in neurons. The capability to target recombinant gene expression to catecholaminergic neurons in specific brain areas may be useful for studies on the roles of these neurons in brain physiology and behavior.
To evaluate the role of the amygdala in pain modulation and opioid-mediated antinociception, a recombinant, replication-defective herpes virus carrying the human preproenkephalin cDNA was injected bilaterally into the rat amygdala. Four days after gene delivery nociceptive behavior was assessed by the formalin test. Rats infected with the virus expressing preproenkephalin showed a selective, naloxone-reversible abolition of phase 2 flinching behavior compared to rats infected with a control virus. The results implicate the amygdala in the control of pain and in opioid analgesia and demonstrate the use of recombinant herpes viruses as tools for studying gene function in specific neural pathways of the central nervous system.
The alpha herpesviruses, a subfamily of the herpesviruses, are neurotropic pathogens found associated with most mammalian species. The prototypic member of this subfamily is herpes simplex virus type 1, the causative agent of recurrent cold sores in humans. The mild nature of this disease is a testament to the complex and highly regulated life cycle of the alpha herpesviruses. The cellular mechanisms used by these viruses to disseminate infection in the nervous system are beginning to be understood. Here, we overview the life cycle of alpha herpesviruses with an emphasis on assembly and transport of viral particles in neurons.
The entry of herpes simplex virus (HSV)-1 into cells is a complex process mediated in part by the binding of the HSV glycoprotein D (gD) to a specific cellular receptor identified as HveC, or nectin-1. We examined the distribution of HveC in sensory and motor neurons of the peripheral nervous system (PNS) by immunocytochemistry. HveC is expressed at high levels in sensory neurons of dorsal root ganglion and their peripheral axons, at lower levels in motor neurons of spinal cord, and without detectable expression in motor nerve terminals at the neuromuscular junction. These results have implications regarding the tropism of HSV to specific neuronal populations, and for the construction of HSV-based vectors for the peripheral nervous system.
The endogenous pain control system has long been considered as engaged in pain depression through the commitment of multiple descending actions that reduce the response capacity of spinal dorsal horn nociceptive neurones. Such a pure inhibitory antinociceptive nature was lately questioned by the observation of pronociceptive effects from areas classically regarded as antinociceptive. The thereby raised hypothesis of a more versatile functional arrangement that dynamically adjusts the pain modulatory effect to multiple conditions by balancing several excitatory and inhibitory actions found strong support on the recent discovery of a medullary area particularly dedicated to pain facilitation. Lesioning the medullary dorsal reticular nucleus (DRt) depresses nociceptive responses to acute and inflammatory pain, whereas stimulation produces the inverse effect. The decrease in formalin-induced pain behaviour following DRt lesioning is accompanied by a decrease of spinal noxious-evoked c-fos neuronal activation. DRt blocking by lidocaine results in a decrease of the nociceptive activity of spinal dorsal horn neurones, whereas stimulation by glutamate has the opposite effect. A reciprocal disynaptic putative excitatory circuit that links the DRt and the spinal dorsal horn and conveys nociceptive input through the ascending branch was described, indicating that the DRt pain facilitating action is mediated by a reverberating spino-DRt circuit that promotes the enhancement of the response capacity of spinal neurones to noxious stimulation.The demonstration of a primary pronociceptive centre in the endogenous pain control system brings new important data to the emerging concept of pain modulation as a dynamic and flexible process that integrates nociceptive processing by balancing multiple excitatory and inhibitory actions as the way of adapting to the various unsteady pain determinants.
Upon receipt in the dorsal horn (DH) of the spinal cord, nociceptive (pain-signalling) information from the viscera, skin and other organs is subject to extensive processing by a diversity of mechanisms, certain of which enhance, and certain of which inhibit, its transfer to higher centres. In this regard, a network of descending pathways projecting from cerebral structures to the DH plays a complex and crucial role. Specific centrifugal pathways either suppress (descending inhibition) or potentiate (descending facilitation) passage of nociceptive messages to the brain. Engagement of descending inhibition by the opioid analgesic, morphine, fulfils an important role in its pain-relieving properties, while induction of analgesia by the adrenergic agonist, clonidine, reflects actions at alpha(2)-adrenoceptors (alpha(2)-ARs) in the DH normally recruited by descending pathways. However, opioids and adrenergic agents exploit but a tiny fraction of the vast panoply of mechanisms now known to be involved in the induction and/or expression of descending controls. For example, no drug interfering with descending facilitation is currently available for clinical use. The present review focuses on: (1) the organisation of descending pathways and their pathophysiological significance; (2) the role of individual transmitters and specific receptor types in the modulation and expression of mechanisms of descending inhibition and facilitation and (3) the advantages and limitations of established and innovative analgesic strategies which act by manipulation of descending controls. Knowledge of descending pathways has increased exponentially in recent years, so this is an opportune moment to survey their operation and therapeutic relevance to the improved management of pain.
The medullary dorsal reticular nucleus (DRt) was recently shown to belong to the supraspinal pain control system; neurons within this nucleus give origin to a descending projection that increases spinal nociceptive transmission and facilitates pain perception [Almeida et al. (1999), Eur. J. Neurosci., 11, 110-122]. In the present study, the areas of the brain that may modulate the activity of DRt neurons were investigated by using of tract-tracing techniques. Injection of a retrograde tracer into the DRt resulted in labelling in multiple areas of the brain. In the contralateral orbital, prelimbic, infralimbic, insular, motor and somatosensory cortices labelling was prominent, but a smaller ipsilateral projection from these same areas was also detected. Strong labelling was also noted in the central amygdaloid nucleus, bed nucleus of stria terminalis and substantia innominata. Labelled diencephalic areas were mainly confined to the hypothalamus, namely its lateral and posterior areas as well as the paraventricular nucleus. In the mesencephalon, the periaqueductal grey, red nucleus and deep mesencephalic nucleus were strongly labelled, whereas, in the brainstem, the parabrachial nuclei, rostroventromedial medulla, nucleus tractus solitarius, spinal trigeminal nucleus, and the parvocellular, dorsal, lateral and ventral reticular nuclei were the most densely labelled regions. All deep cerebellar nuclei were labelled bilaterally. These data suggest that the DRt integrates information from the somatosensory, antinociceptive, autonomic, limbic, pyramidal and extrapyramidal systems while triggering its descending facilitating action upon the spinal nociceptive transmission.
Neurons in the rat nucleus paragigantocellularis (PGi), located in the ventrolateral medulla, send collateral projections to the locus coeruleus (LC) and to the nucleus of the solitary tract (NTS). The present study examined whether neurons in the PGi that project to both the LC and NTS contain leucine(5)-enkephalin (ENK), and also whether opioid-containing neurons in the PGi are activated following withdrawal from opiates. Retrograde transport of Fluoro-Gold (FG) from the LC and transport of a protein-gold tracer from the NTS was combined with detection of an antibody directed against ENK in the PGi. Using fluorescence and brightfield microscopy, it was established that more than half of the neurons containing both FG and the protein-gold tracer, also exhibited immunolabeling for ENK. The most frequent location of triply labeled neurons was the retrofacial portion of the PGi. In a separate series, rats were chronically implanted with morphine or placebo pellets and, on the fifth day, were subjected to an intraperitoneal injection of naltrexone. Two hours following initiation of withdrawal, rat brains were obtained and processed for detection of c-fos and in situ hybridization labeling of preproenkephalin (PPE) mRNA. Naltrexone injections into morphine-dependent rats caused a dramatic increase in c-fos as compared to control rats. Approximately 66% of the c-fos-labeled neurons exhibited labeling for PPE mRNA. These were also enriched in the retrofacial portion of the PGi. Taken together, the present data indicate that withdrawal from opiates engages opioid neurons in the PGi, some of which may coordinate activity of neurons in both the NTS and the LC.
Carboxypeptidase E (CPE) is involved in the biosynthesis of a number of neuropeptides including opioid peptides. A point mutation in this gene results in a loss of enzyme activity, decrease in mature neuroendocrine peptides, and development of late onset obesity as seen in Cpe(fat)/Cpe(fat) mice. In this study, we examined the processing of peptides derived from prodynorphin and proenkephalin in various brain regions of these mice during development. At 6 to 8 weeks, an age prior to the onset of obesity, levels of dynorphin peptides are decreased in all brain regions, whereas levels of ir-Met-enkephalin are differentially altered. There is an accumulation of C-terminally extended forms of all three opioid peptides in Cpe(fat)/Cpe(fat) mice, consistent with a lack of CPE activity. Thus, it appears that there is no direct correlation between the level of mature opioid peptides and the development of obesity in these mice. Since altered levels of peptides can influence the opioid receptor system, we examined the functional activity of mu and kappa opioid receptors using [35S]guanosine-5'-O-(gamma-thio)-triphosphate binding assays. We find no differences in kappa receptor activity in Cpe(fat)/Cpe(fat) compared with control littermate mice. In contrast, the mu receptor activity is differentially altered in select regions of Cpe(fat)/Cpe(fat) mice in response to a mu-specific ligand. Taken together, these results suggest that the lack of CPE activity leads to alterations in the level of opioid peptides during development and that changes in peptide levels differentially affect opioid receptor activity in vivo.
We examined the pharmacologic characteristics of herpes simplex virus (HSV) vector-mediated expression of proenkephalin in the dorsal root ganglion in a rodent model of neuropathic pain. We found that: (i). vector-mediated enkephalin produced an antiallodynic effect that was reversed by naloxone; (ii). vector-mediated enkephalin production in animals with spinal nerve ligation prevented the induction of c-fos expression in second order sensory neurons in the dorsal horn of spinal cord; (iii). the effect of vector-mediated enkephalin enhanced the effect of morphine, reducing the ED(50) of morphine 10-fold; (iv). animals did not develop tolerance to the continued production of vector-mediated enkephalin over a period of several weeks; and, (v). vector transduction continued to provide an analgesic effect despite the induction of tolerance to morphine. This is the first demonstration of gene transfer to provide an analgesic effect in neuropathic pain. The pharmacologic analysis demonstrates that transgene-mediated expression and local release of opioid peptides produce some effects that are distinct from peptide analogues delivered pharmacologically.
Chronic pain is frequently associated with profound alterations of neuronal systems involved in pain processing and should be considered as an actual disease state of the nervous system. It should not only be relieved, but must really be treated in suffering patients. However, some forms of chronic pain, in particular those of neuropathic origin, are most often not satisfactorily managed with currently available pharmacological agents, some of which, in addition, may be poorly tolerated by some patients. In this context, gene-based approaches may contribute to the search for a better management of chronic pain. The question then arises regarding the most appropriate level for such an intervention using gene-transfer techniques. The first experimental protocols attempted the transfer of opioid precursor genes and their overexpression mainly at the spinal level. They demonstrated the feasibility and the real interest of these approaches by showing that local overproduction of opioid peptides induced antinociceptive effects in animal models of persistent pain, of inflammatory-, neuropathic- and even cancerous origin. Although really tempting data were obtained using gene-based techniques in experimental inflammatory diseases, the possible clinical interest of these approaches in chronic pain has still to be established. Nevertheless, targeting some proinflammatory cytokines, involved not only in inflammation but also in the induction and probably the perpetuation of pain, raises the possibility to block the "development" of chronic pain rather that to "simply" relieve established ongoing pain. Future gene-based protocols will certainly target some of the recently identified molecules involved in pain transduction mechanisms, sensory nerve sensitization or pain perpetuation, and evaluate their potential interest to ideally abolish or, at least, reduce chronic pain.
Spinal application of opiates is the cornerstone of potent analgesia. In the present study, opiate analgesia was investigated after cutaneous application of a recombinant herpes simplex virus type-1 (HSV-1) encoding micro-opioid receptor (microOR) cDNA in reverse orientation with respect to the human cytomegalovirus early enhancer-promoter. Hind paw application of this recombinant vector was used in order to attenuate expression of the microOR in primary afferents and determine whether recombinant vector application would differentially affect the antinociceptive effects of the specific microOR agonist, [D-Ala(2),N-MePhe(4),Gly-ol(5)] enkephalin (DAMGO), on behavioral responses mediated by C- and Adelta-thermonociceptors. The recombinant vector encoding the Escherichia coli lacZ gene marker, KHZ, served as a control virus. Dorsal hind paw surfaces of female Swiss-Webster mice were treated with one of these two viruses (1x10(8)pfu, 10 microl) or vehicle (uninfected). Immunohistochemistry and quantitative image analyses revealed decreased microOR expression in the superficial dorsal horns ipsilateral to hind paws treated with AMOR, but not KHZ. To add, behavioral foot withdrawal latencies of AMOR- and KHZ-treated hind paws demonstrated dose-dependent antinociception after intrathecal DAMGO administration. However, cutaneous application of dorsal hind paw surfaces treated with AMOR, but not KHZ, caused a rightward shift in the C-fiber dose-response, thus, indicating a loss of potency of intrathecal DAMGO. Loss or diminution of DAMGO potency during Adelta-fiber-mediated responses was not observed. These immunohistochemistry and behavioral results of novel, recombinant HSV-1 vector microOR 'knock-down' in nociceptor afferent fibers provide additional evidence for presynaptic localization of microORs on central C-, but not Adelta-terminals.
Broadly defined, the concept of gene therapy involves the transfer of genetic material into a cell, tissue, or whole organ, with the goal of curing a disease or at least improving the clinical status of a patient. A key factor in the success of gene therapy is the development of delivery systems that are capable of efficient gene transfer in a variety of tissues, without causing any associated pathogenic effects. Vectors based upon many different viral systems, including retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses, currently offer the best choice for efficient gene delivery. Their performance and pathogenicity has been evaluated in animal models, and encouraging results form the basis for clinical trials to treat genetic disorders and acquired diseases. Despite some initial success in these trials, vector development remains a seminal concern for improved gene therapy technologies.
The balance between excitatory and inhibitory input exerted upon spinal cord neurones that belong to spinofugal pathways determines the ultimate type of information transmitted to the brain. We compared the relative expression of NK1 and GABAB receptors in two spinomedullary pathways targeting an antinociceptive area and a pronociceptive centre, respectively, the lateral part of the caudal ventrolateral medulla (VLMlat) and the dorsal reticular nucleus (DRt). Spinal cord sections of rats injected in the VLMlat or DRt with the retrograde tracer cholera toxin subunit B were triple-immunoreacted for the tracer, NK1 receptors and GABAB receptors. The dorsal horn neurones labelled from the VLMlat mainly co-localized the two receptors while those labelled from the DRt mainly expressed GABAB receptors, which was particularly evident in neurones of laminae IV-V. The morphological classification of lamina I neurones projecting to the VLMlat showed that fusiform, flattened and pyramidal cells mainly co-localized NK1 and GABAB receptors. As to lamina I neurones projecting to the DRt, multipolar neurones mainly expressed GABAB receptors while the majority of flattened and pyramidal neurones co-localized NK1 and GABAB receptors. The present results suggest that the expression of NK1 and GABAB receptors varies in neurones participating to different spinofugal pathways. The importance of the present findings in the knowledge of the endogenous supraspinal pain control system is discussed.
In the last 15 years a role has been ascribed for the medullary dorsal reticular nucleus as a supraspinal pain modulating area. The medullary dorsal reticular nucleus is reciprocally connected with the spinal dorsal horn, is populated mainly by nociceptive neurons and regulates spinal nociceptive processing. Here we analyze the distribution of brain projections from the medullary dorsal reticular nucleus using the iontophoretic administration of the anterograde tracer biotinylated-dextran amine and the retrograde tracer cholera toxin subunit B.
Peripheral neuropathy is a common medical problem with numerous aetiologies. Unfortunately, for the majority of cases there is no available medical solution for the underlying cause, and the only option is to try to treat the resulting symptoms. Treatment options exist when neuropathy results in positive symptoms such as pain, but there is a significant lack of treatments for negative symptoms such as numbness and weakness. Systemic application of growth factor peptides has shown promise in protecting nerves from neuropathic insults in preclinical animal studies, but translation into human trials has been problematic and disappointing. Significant advancements have been made in the past few years in utilising gene therapy approaches to treat peripheral neuropathy by expressing neuroprotective gene products either systemically or in specific nervous tissues. For example, plasmids expressing vascular endothelial growth factor injected into muscle, or herpes-simplex-virus-based vectors expressing neurotrophin gene products delivered to dorsal root ganglion neurons, have been used to protect peripheral nerve function in animal models of diabetes-associated peripheral neuropathy. Many published studies support the feasibility of this approach, although several questions still need to be addressed as gene therapy to treat peripheral neuropathy moves out of the laboratory and into the clinic.
The endogenous pain modulatory system is a complex network of brain areas that control nociceptive transmission at the spinal cord by inhibitory and facilitatory actions. The balance between these actions ensures effective modulation of acute pain, while during chronic pain the pronociceptive effects appear to prevail. The mechanisms underlying this imbalance were studied as to the role of two medullary components of the pain modulatory system: the dorsal reticular nucleus and the caudal ventrolateral medulla, which function primarily as pronociceptive and antinociceptive centres, respectively. Both areas are connected with the spinal dorsal horn by closed reciprocal loops. In the spino-dorsal reticular nucleus loop, the ascending branch is strongly inhibited by spinal GABAergic neurons, which may act as a buffering system of the dorsal reticular nucleus-centred amplifying effect. In the spino-caudal ventrolateral medulla loop, the ascending branch is under potent excitation of substance P (SP) released from primary afferents, which is likely to trigger the intense descending inhibition detected in acute pain. During chronic pain, the activity in the lateral reticular formation of the caudal ventrolateral medulla changes, so that the action of the caudal ventrolateral medulla upon SP-responsive spinal neurons shifts from inhibitory to excitatory. The mechanisms of this modulatory shift are unknown but probably relate to the decreased expression of micro-opioid, delta-opioid and GABAB receptors. Normalizing receptor expression in the caudal ventrolateral medulla or controlling noci-evoked activity at the dorsal reticular nucleus or caudal ventrolateral medulla by interfering with neurotransmitter release is now possible by the use of gene therapy, an approach that stands out as a unique tool to manipulate the supraspinal endogenous pain control system.
We investigated in different experimental rat models the potential facilitatory contribution of the medullary dorsal reticular nucleus (DRt) descending pathway to the expressions of the sensory spinal neuron sensitization such as increased spontaneous and noxious evoked activities, responsivity to heterotopic afferences stimulation and long lasting afterdischarges (ADs). We carried out experiments by recording from ipsilateral lumbar Wide Dynamic Range (WDR) neurons and by simultaneously monitoring the DRt neuron activity in neuropathic pain rats with chronic constriction injury of one sciatic nerve (CCI), in sham-operated and in "intact" rats. In particular, we recorded the spinal neuron spontaneous activities and the activities evoked by noxious stimulations of ipsi- and contralateral sciatic supplied areas before and during DRt activity blockade. Additionally, in "intact rats" we modulated WDR activity by iontophoretic NMDA to mimic CCI WDR hyperactivity without peripheral damage. We found that during DRt activity blockade in CCI rat neurons and in "intact" rat NMDA-treated neurons, the spontaneous activity was significantly reduced, the responses to contralateral sciatic area stimulation were reduced or suppressed, the responses to ipsilateral sciatic area were poorly affected (slightly reduced or unaffected), except for the poststimulus afterdischarges that were mostly suppressed. In sham-operated rats, the neuronal activity was not affected by DRt blockade. The finding that during the DRt nucleus blockade some expressions of spinal neurons sensitization, seemingly associated to sensory disorders in neuropathic pain, fade or extinguish designates a likely facilitatory role of DRt in the maintenance of neuronal sensitization and thus a contribution to neuropathic pain state.