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BACKGROUND: The intrathecal (IT) dosing route introduces drugs directly into the CSF to bypass the blood-brain barrier and gain direct access to the CNS. We evaluated the use of convective forces acting on the cerebrospinal fluid as a means for increasing rostral delivery of IT dosed radioactive tracer molecules and antisense oligonucleotides (ASO) in the monkey CNS. We also measured the cerebral spinal fluid (CSF) volume in a group of cynomolgus monkeys. METHODS: There are three studies presented, in each of which cynomolgus monkeys were injected into the IT space with radioactive tracer molecules and/or ASO by lumbar puncture in either a low or high volume. The first study used the radioactive tracer 64Cu-DOTA and PET imaging to evaluate the effect of the convective forces. The second study combined the injection of the radioactive tracer 99mTc-DTPA and ASO, then used SPECT imaging and ex vivo tissue analysis of the effects of convective forces to bridge between the tracer and the ASO distributions. The third experiment evaluated the effects of different injection volumes on the distribution of an ASO. In the course of performing these studies we also measured the CSF volume in the subject monkeys by Magnetic Resonance Imaging. RESULTS: It was consistently found that larger bolus dose volumes produced greater rostral distribution along the neuraxis. Thoracic percussive treatment also increased rostral distribution of low volume injections. There was little added benefit on distribution by combining the thoracic percussive treatment with the high-volume injection. The CSF volume of the monkeys was found to be 11.9 ± 1.6 cm3. CONCLUSIONS: These results indicate that increasing convective forces after IT injection increases distribution of molecules up the neuraxis. In particular, the use of high IT injection volumes will be useful to increase rostral CNS distribution of therapeutic ASOs for CNS diseases in the clinic.
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Sistema Nervioso Central , Oligonucleótidos Antisentido , Animales , Barrera Hematoencefálica , Inyecciones Espinales , Macaca fascicularisRESUMEN
p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38ß; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300⯵g/10⯵l) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38ß MAPK ASO. Intrathecal injection of p38α or p38ß ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38ß mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38ß MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions.
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Neuralgia/terapia , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación hacia Abajo , Femenino , Ganglios Espinales/metabolismo , Hiperalgesia/inducido químicamente , Hiperalgesia/terapia , Inyecciones Espinales , Masculino , Ratones , Microglía/metabolismo , Proteína Quinasa 11 Activada por Mitógenos/genética , Proteína Quinasa 11 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/genética , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Oligodesoxirribonucleótidos Antisentido/administración & dosificación , Oligodesoxirribonucleótidos Antisentido/genética , Dimensión del Dolor , Dolor Postoperatorio/terapia , Enfermedades del Sistema Nervioso Periférico/metabolismo , Fosforilación , Isoformas de Proteínas , Médula Espinal/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
Peripheral inflammation initiates changes in spinal nociceptive processing leading to hyperalgesia. Previously, we demonstrated that among 102 lipid species detected by LC-MS/MS analysis in rat spinal cord, the most notable increases that occur after intraplantar carrageenan are metabolites of 12-lipoxygenases (12-LOX), particularly hepoxilins (HXA(3) and HXB(3)). Thus, we examined involvement of spinal LOX enzymes in inflammatory hyperalgesia. In the current work, we found that intrathecal (IT) delivery of the LOX inhibitor nordihydroguaiaretic acid prevented the carrageenan-evoked increase in spinal HXB(3) at doses that attenuated the associated hyperalgesia. Furthermore, IT delivery of inhibitors targeting 12-LOX (CDC, Baicalein), but not 5-LOX (Zileuton) dose-dependently attenuated tactile allodynia. Similarly, IT delivery of 12-LOX metabolites of arachidonic acid 12(S)-HpETE, 12(S)-HETE, HXA(3), or HXB(3) evoked profound, persistent tactile allodynia, but 12(S)-HpETE and HXA(3) produced relatively modest, transient heat hyperalgesia. The pronociceptive effect of HXA(3) correlated with enhanced release of Substance P from primary sensory afferents. Importantly, HXA(3) triggered sustained mobilization of calcium in cells stably overexpressing TRPV1 or TRPA1 receptors and in acutely dissociated rodent sensory neurons. Constitutive deletion or antagonists of TRPV1 (AMG9810) or TRPA1 (HC030031) attenuated this action. Furthermore, pretreatment with antihyperalgesic doses of AMG9810 or HC030031 reduced spinal HXA(3)-evoked allodynia. These data indicate that spinal HXA(3) is increased by peripheral inflammation and promotes initiation of facilitated nociceptive processing through direct activation of TRPV1 and TRPA1 at central terminals.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Araquidonato 12-Lipooxigenasa/metabolismo , Hiperalgesia/fisiopatología , Inflamación/fisiopatología , Médula Espinal/metabolismo , Canales Catiónicos TRPV/fisiología , Canales de Potencial de Receptor Transitorio/fisiología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Ratones , Médula Espinal/enzimología , Canal Catiónico TRPA1RESUMEN
Previously, we observed significant increases in spinal 12-lipoxygenase (LOX) metabolites, in particular, hepoxilins, which contribute to peripheral inflammation-induced tactile allodynia. However, the enzymatic sources of hepoxilin synthase (HXS) activity in rats remain elusive. Therefore, we overexpressed each of the 6 rat 12/15-LOX enzymes in HEK-293T cells and measured by LC-MS/MS the formation of HXB3, 12-HETE, 8-HETE, and 15-HETE from arachidonic acid (AA) at baseline and in the presence of LOX inhibitors (NDGA, AA-861, CDC, baicalein, and PD146176) vs. vehicle-treated and mock-transfected controls. We detected the following primary intrinsic activities: 12-LOX (Alox12, Alox15), 15-LOX (Alox15b), and HXS (Alox12, Alox15). Similar to human and mouse orthologs, proteins encoded by rat Alox12b and Alox12e possessed minimal 12-LOX activity with AA as substrate, while eLOX3 (encoded by Aloxe3) exhibited HXS without 12-LOX activity when coexpressed with Alox12b or supplemented with 12-HpETE. CDC potently inhibited HXS and 12-LOX activity in vitro (relative IC50s: CDC, ~0.5 and 0.8 µM, respectively) and carrageenan-evoked tactile allodynia in vivo. Notably, peripheral inflammation significantly increased spinal eLOX3; intrathecal pretreatment with either siRNA targeting Aloxe3 or an eLOX3-selective antibody attenuated the associated allodynia. These findings implicate spinal eLOX3-mediated hepoxilin synthesis in inflammatory hyperesthesia and underscore the importance of developing more selective 12-LOX/HXS inhibitors.
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Araquidonato 12-Lipooxigenasa/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Hiperalgesia/etiología , Oxidorreductasas Intramoleculares/metabolismo , Animales , Araquidonato 12-Lipooxigenasa/efectos de los fármacos , Araquidonato 15-Lipooxigenasa/efectos de los fármacos , Células HEK293 , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Masculino , RatasRESUMEN
Sensitization of spinal nociceptive circuits plays a crucial role in neuropathic pain. This sensitization depends on new gene expression that is primarily regulated via transcriptional and translational control mechanisms. The relative roles of these mechanisms in regulating gene expression in the clinically relevant chronic phase of neuropathic pain are not well understood. Here, we show that changes in gene expression in the spinal cord during the chronic phase of neuropathic pain are substantially regulated at the translational level. Downregulating spinal translation at the chronic phase alleviated pain hypersensitivity. Cell-type-specific profiling revealed that spinal inhibitory neurons exhibited greater changes in translation after peripheral nerve injury compared to excitatory neurons. Notably, increasing translation selectively in all inhibitory neurons or parvalbumin-positive (PV+) interneurons, but not excitatory neurons, promoted mechanical pain hypersensitivity. Furthermore, increasing translation in PV+ neurons decreased their intrinsic excitability and spiking activity, whereas reducing translation in spinal PV+ neurons prevented the nerve injury-induced decrease in excitability. Thus, translational control mechanisms in the spinal cord, particularly in inhibitory neurons, play a role in mediating neuropathic pain hypersensitivity.
RESUMEN
BACKGROUND: Neuraxial anesthesia is utilized in children of all ages. Local anesthetics produce dose-dependent toxicity in certain adult models, but the developing spinal cord may also be susceptible to drug-induced apoptosis. In postnatal rodents, we examined the effects of intrathecal levobupivacaine on neuropathology and long-term sensorimotor outcomes. METHODS: Postnatal day 3 (P3) or P7 rat pups received intrathecal levobupivacaine 2.5 mg/kg (0.5%) or saline. Mechanical withdrawal thresholds and motor block were assessed. Spinal cord tissue analysis included apoptosis counts (activated caspase-3, Fluoro-Jade C) at 24 h, glial reactivity at 7 days, and histopathology in cord and cauda equina at 24 h and 7 days. Long-term spinal function in young adults (P35) was assessed by hind limb withdrawal thresholds, electromyography responses to suprathreshold stimuli, and gait analysis. RESULTS: Intrathecal levobupivacaine produced spinal anesthesia at P3 and P7. No increase in apoptosis or histopathological change was seen in the cord or cauda equina. In the P3 saline group, activated caspase-3 (mean±SEM per lumbar cord section 6.1±0.3) and Fluoro-Jade C (12.1±1.2) counts were higher than at P7, but were not altered by levobupivacaine (P=0.62 and P=0.11, two-tailed Mann-Whitney test). At P35, mechanical withdrawal thresholds, thermal withdrawal latency, and electromyographic reflex responses did not differ across P3 or P7 levobupivacaine or saline groups (one way ANOVA with Bonferroni comparisons). Intrathecal bupivacaine at P3 did not alter gait. CONCLUSION: Single dose intrathecal levobupivacaine 0.5% did not increase apoptosis or produce spinal toxicity in neonatal rat pups. This study provides preclinical safety data relevant to neonatal use of neuraxial local anesthesia.
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Anestésicos Locales/toxicidad , Enfermedades de la Médula Espinal/inducido químicamente , Anestesia Caudal , Anestésicos Locales/administración & dosificación , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Bupivacaína/administración & dosificación , Bupivacaína/análogos & derivados , Bupivacaína/toxicidad , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/metabolismo , Cauda Equina/patología , Electromiografía , Femenino , Marcha/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Inyecciones Espinales , Levobupivacaína , Proteínas de Microfilamentos/metabolismo , Dimensión del Dolor/efectos de los fármacos , Embarazo , Ratas , Ratas Sprague-Dawley , Médula Espinal/patología , Raíces Nerviosas Espinales/patologíaRESUMEN
Patients with Lafora disease have a mutation in EPM2A or EPM2B, resulting in dysregulation of glycogen metabolism throughout the body and aberrant glycogen molecules that aggregate into Lafora bodies. Lafora bodies are particularly damaging in the brain, where the aggregation drives seizures with increasing severity and frequency, coupled with neurodegeneration. Previous work employed mouse genetic models to reduce glycogen synthesis by approximately 50%, and this strategy significantly reduced Lafora body formation and disease phenotypes. Therefore, an antisense oligonucleotide (ASO) was developed to reduce glycogen synthesis in the brain by targeting glycogen synthase 1 (Gys1). To test the distribution and efficacy of this drug, the Gys1-ASO was administered to Epm2b-/- mice via intracerebroventricular administration at 4, 7, and 10 months. The mice were then sacrificed at 13 months and their brains analyzed for Gys1 expression, glycogen aggregation, and neuronal excitability. The mice treated with Gys1-ASO exhibited decreased Gys1 protein levels, decreased glycogen aggregation, and reduced epileptiform discharges compared to untreated Epm2b-/- mice. This work provides proof of concept that a Gys1-ASO halts disease progression of EPM2B mutations of Lafora disease.
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Enfermedad de Lafora , Humanos , Ratones , Animales , Enfermedad de Lafora/genética , Enfermedad de Lafora/metabolismo , Glucógeno Sintasa/genética , Modelos Animales de Enfermedad , Mutación , Oligonucleótidos Antisentido/uso terapéutico , Glucógeno/metabolismo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Tau plays a key role in Alzheimer's disease (AD) pathophysiology, and accumulating evidence suggests that lowering tau may reduce this pathology. We sought to inhibit MAPT expression with a tau-targeting antisense oligonucleotide (MAPTRx) and reduce tau levels in patients with mild AD. A randomized, double-blind, placebo-controlled, multiple-ascending dose phase 1b trial evaluated the safety, pharmacokinetics and target engagement of MAPTRx. Four ascending dose cohorts were enrolled sequentially and randomized 3:1 to intrathecal bolus administrations of MAPTRx or placebo every 4 or 12 weeks during the 13-week treatment period, followed by a 23 week post-treatment period. The primary endpoint was safety. The secondary endpoint was MAPTRx pharmacokinetics in cerebrospinal fluid (CSF). The prespecified key exploratory outcome was CSF total-tau protein concentration. Forty-six patients enrolled in the trial, of whom 34 were randomized to MAPTRx and 12 to placebo. Adverse events were reported in 94% of MAPTRx-treated patients and 75% of placebo-treated patients; all were mild or moderate. No serious adverse events were reported in MAPTRx-treated patients. Dose-dependent reduction in the CSF total-tau concentration was observed with greater than 50% mean reduction from baseline at 24 weeks post-last dose in the 60 mg (four doses) and 115 mg (two doses) MAPTRx groups. Clinicaltrials.gov registration number: NCT03186989 .
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Enfermedad de Alzheimer , Proteínas tau , Humanos , Proteínas tau/genética , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/líquido cefalorraquídeo , Oligonucleótidos Antisentido/uso terapéutico , Resultado del Tratamiento , Método Doble CiegoRESUMEN
Phosphinositide 3-kinase (PI3K), Akt, and their downstream kinase, mammalian target of rapamycin (mTOR), are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work, we identified the following characteristics of this cascade. (1) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. (2) Increased pAkt and pmTOR were prominent in neurons in laminae I, III, and IV, whereas pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. (3) Intrathecal treatment with inhibitors to PI3K or Akt attenuated Formalin-induced second-phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. (4) Intrathecal rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, intrathecal wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). (5) pAkt and pmTOR are expressed in neurokinin 1 receptor-positive neurons in laminae I-III after peripheral inflammation. Intrathecal injection of Substance P activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by intrathecal wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.
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Hiperalgesia/enzimología , Hiperalgesia/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Médula Espinal/enzimología , Serina-Treonina Quinasas TOR/metabolismo , Androstadienos/farmacología , Androstadienos/uso terapéutico , Animales , Carragenina/efectos adversos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Formaldehído/efectos adversos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Hiperalgesia/tratamiento farmacológico , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Proteínas del Tejido Nervioso/metabolismo , Dimensión del Dolor , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sirolimus/metabolismo , Sirolimus/farmacología , Médula Espinal/patología , Estadísticas no Paramétricas , Sustancia P/farmacología , Factores de Tiempo , WortmaninaRESUMEN
Lipid molecules play an important role in regulating the sensitivity of sensory neurons and enhancing pain perception, and growing evidence indicates that the effect occurs both at the site of injury and in the spinal cord. Using high-throughput mass spectrometry methodology, we sought to determine the contribution of spinal bioactive lipid species to inflammation-induced hyperalgesia in rats. Quantitative analysis of CSF and spinal cord tissue for eicosanoids, ethanolamides and fatty acids revealed the presence of 102 distinct lipid species. After induction of peripheral inflammation by intra-plantar injection of carrageenan to the ipsilateral hind paw, lipid changes in cyclooxygenase (COX) and 12-lipoxygenase (12-LOX) signaling pathways peaked at 4 h in the CSF. In contrast, changes occurred in a temporally disparate manner in the spinal cord with LOX-derived hepoxilins followed by COX-derived prostaglandin E(2), and subsequently the ethanolamine anandamide. Systemic treatment with the mu opioid agonist morphine, the COX inhibitor ketorolac, or the LOX inhibitor nordihydroguaiaretic acid significantly reduced tactile allodynia, while their effects on the lipid metabolites were different. Morphine did not alter the lipid profile in the presence or absence of carrageenan inflammation. Ketorolac caused a global reduction in eicosanoid metabolism in naïve animals that remained suppressed following injection of carrageenan. Nordihydroguaiaretic acid-treated animals also displayed reduced basal levels of COX and 12-LOX metabolites, but only 12-LOX metabolites remained decreased after carrageenan treatment. These findings suggest that both COX and 12-LOX play an important role in the induction of carrageenan-mediated hyperalgesia through these pathways.
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Hiperalgesia/metabolismo , Hiperalgesia/patología , Mediadores de Inflamación/fisiología , Lípidos/biosíntesis , Médula Espinal/metabolismo , Médula Espinal/patología , Animales , Araquidonato 12-Lipooxigenasa/líquido cefalorraquídeo , Araquidonato 12-Lipooxigenasa/fisiología , Moduladores de Receptores de Cannabinoides/líquido cefalorraquídeo , Moduladores de Receptores de Cannabinoides/fisiología , Eicosanoides/líquido cefalorraquídeo , Eicosanoides/fisiología , Etanolaminas/líquido cefalorraquídeo , Etanolaminas/farmacología , Ácidos Grasos/líquido cefalorraquídeo , Ácidos Grasos/fisiología , Hiperalgesia/líquido cefalorraquídeo , Mediadores de Inflamación/farmacología , Lípidos/líquido cefalorraquídeo , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Acetaminophen is an analgesic and antipyretic drug believed to exert its effect through interruption of nociceptive processing. In order to determine whether this effect is due to peripheral or central activity, we studied the efficacy of systemic (oral) and intrathecal (IT) application of acetaminophen in preventing the development of hyperalgesia induced through the direct activation of pro-algogenic spinal receptors. Spinal administration of substance P (SP, 30 nmol, IT) in rats produced a decreased thermal threshold, indicating centrally mediated hyperalgesia. Pretreatment of rats with oral acetaminophen (300 mg/kg), but not vehicle, significantly attenuated IT SP-induced hyperalgesia. Acetaminophen given IT also produced a dose-dependent (10-200 microg) antinociceptive effect. In addition, oral acetaminophen suppressed spinal PGE(2) release evoked by IT SP in an in vivo IT dialysis model. The ability of IT as well as oral acetaminophen to reverse this spinally initiated hyperalgesia emphasizes the likely central action and bioavailability of the systemically delivered drug. Jointly, these data argue for an important central antihyperalgesic action of acetaminophen.
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Acetaminofén/administración & dosificación , Analgésicos no Narcóticos/administración & dosificación , Hiperalgesia/prevención & control , Médula Espinal/efectos de los fármacos , Administración Oral , Animales , Dinoprostona/metabolismo , Hiperalgesia/inducido químicamente , Inyecciones Espinales , Masculino , Neurotransmisores/toxicidad , Dolor/tratamiento farmacológico , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Sustancia P/toxicidadRESUMEN
There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.
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Ganglios Espinales/efectos de los fármacos , Nocicepción/fisiología , Oligonucleótidos Antisentido/uso terapéutico , Dolor/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Animales , Modelos Animales de Enfermedad , Ganglios Espinales/fisiopatología , Masculino , Neuronas/efectos de los fármacos , Neuronas/fisiología , Dolor/fisiopatología , Ratas , Ratas Sprague-Dawley , Médula Espinal/fisiopatologíaRESUMEN
Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.
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Antiinflamatorios no Esteroideos/uso terapéutico , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Lipooxigenasas/uso terapéutico , Neuroglía/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Cromatografía Liquida , Inhibidores Enzimáticos/uso terapéutico , Lipopolisacáridos/toxicidad , Masculino , Espectrometría de Masas , Estimulación Física/efectos adversos , ARN Mensajero , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología , Receptor Toll-Like 4/antagonistas & inhibidores , TransfecciónRESUMEN
BACKGROUND: The blood brain barrier (BBB) is an impediment to the development of large and highly charged molecules as therapeutics for diseases and injuries of the central nervous system (CNS). Antisense oligonucleotides (ASOs) are large (6000-8000MW) and highly charged and therefore do not cross the BBB. A method of circumventing the blood brain barrier to test ASOs, and other non-BBB penetrant molecules, as CNS therapeutics is the direct administration of these molecules to the CNS tissue or cerebral spinal fluid. NEW METHOD: We developed a rapid, simple and robust method for the intrathecal catheterization of rats to test putatively therapeutic antisense oligonucleotides. This method utilizes 23-gauge needles, simply constructed ½in. long 19-gauge guide cannulas and 8cm long plastic PE-10 sized catheters. COMPARISON WITH EXISTING METHODS: Unlike the cisterna magna approach, this method uses a lumbar approach for intrathecal catheterization with the catheter residing entirely in the cauda equina space minimizing spinal cord compression. Readily available materials and only a few specialized pieces of equipment, which are easily manufactured, are used for this intrathecal catheterization method. CONCLUSIONS: This method is easy to learn and has been taught to multiple in house surgeons, collaborators and contract laboratories. Greater than 90% catheterization success is routinely achieved with this method and as many as 100 catheters can be placed and test substance administered in one 6-h period. This method has allowed the pre-clinical testing of hundreds of ASOs as therapeutics for CNS indications.
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Cateterismo/métodos , Modelos Animales , Animales , Cateterismo/efectos adversos , Cateterismo/instrumentación , Catéteres de Permanencia/efectos adversos , Fármacos del Sistema Nervioso Central/administración & dosificación , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Colorantes , Ensayo de Inmunoadsorción Enzimática , Femenino , Hiperalgesia/tratamiento farmacológico , Inmunohistoquímica , Inyecciones Espinales/instrumentación , Inyecciones Espinales/métodos , Vértebras Lumbares , Masculino , Oligonucleótidos Antisentido/administración & dosificación , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores AMPA/metabolismo , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Serotonin (5-HT) derived from bulbo-spinal projection is released by nociceptive input into the spinal dorsal horn. Here we report that formalin injection in the paw produced pain behavior (flinching) and phosphorylation of spinal ERK1/2 (P-ERK1/2, indicating activation) in rats. Depletion of spinal 5-HT by intrathecal (IT) 5,7-DHT, a serotonergic neurotoxin, profoundly reduced formalin evoked flinching and the increase in P-ERK1/2. Ondansetron (a 5-HT3 receptor antagonist) at IT doses that inhibited flinching also attenuated spinal ERK activation. These findings reveal that primary afferent-evoked activation of spinal ERK requires the input from an excitatory 5-HT descending pathway.
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Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Dolor/enzimología , Dolor/fisiopatología , Serotonina/metabolismo , Médula Espinal/enzimología , Animales , Activación Enzimática/efectos de los fármacos , Pie , Formaldehído/farmacología , Hiperalgesia/enzimología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Serotonina/deficiencia , Antagonistas del Receptor de Serotonina 5-HT3RESUMEN
Dynorphin peptide neurotransmitters (neuropeptides) have been implicated in spinal pain processing based on the observations that intrathecal delivery of dynorphin results in proalgesic effects and disruption of extracellular dynorphin activity (by antisera) prevents injury evoked hyperalgesia. However, the cellular source of secreted spinal dynorphin has been unknown. For this reason, this study investigated the expression and secretion of dynorphin-related neuropeptides from spinal astrocytes (rat) in primary culture. Dynorphin A (1-17), dynorphin B, and α-neoendorphin were found to be present in the astrocytes, illustrated by immunofluorescence confocal microscopy, in a discrete punctate pattern of cellular localization. Measurement of astrocyte cellular levels of these dynorphins by radioimmunoassays confirmed the expression of these three dynorphin-related neuropeptides. Notably, BzATP (3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate) and KLA (di[3-deoxy-D-manno-octulosonyl]-lipid A) activation of purinergic and toll-like receptors, respectively, resulted in stimulated secretion of dynorphins A and B. However, α-neoendorphin secretion was not affected by BzATP or KLA. These findings suggest that dynorphins A and B undergo regulated secretion from spinal astrocytes. These findings also suggest that spinal astrocytes may provide secreted dynorphins that participate in spinal pain processing.
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Astrocitos/metabolismo , Dinorfinas/metabolismo , Médula Espinal/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Células Cultivadas , Endorfinas/metabolismo , Espacio Extracelular/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Microscopía Confocal , Neuropéptidos/metabolismo , Dolor/fisiopatología , Embarazo , Precursores de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X/metabolismo , Médula Espinal/citología , Receptor Toll-Like 4/metabolismoRESUMEN
Neurosteroids regulate neuronal excitability though binding sites associated with the ionotropic γ-aminobutyric acid (GABAA) receptor. We sought to characterize the spinal analgesic actions in rats of two 5α-reduced neurosteroids, allopregnanolone and alphaxalone, on nociceptive processing and to determine whether a putative neurosteroid antagonist attenuates this effect: (3α,5α)-17-phenylandrost-16-en-3-ol (17PA). Intrathecal (IT) injection of allopregnanolone (1-30 µg/10 µL in 20% cyclodextrin) delivered through lumbar catheters produced a dose-dependent analgesia in rats as measured by thermal thresholds in the ipsilateral (inflamed by intraplantar carrageenan) and in the contralateral (un-inflamed paws). Similar observations were made with alphaxalone (30-60 µg in 20% cyclodextrin). Effective doses were not associated with suppressive effects on pinnae, blink or placing and stepping reflex. Effects of allopregnanolone (30 µg) on the normal and hyperalgesic paw were completely prevented by IT 17PA (30 µg). Reversal by IT 17PA of an equi-analgesic dose of alphaxalone occurred only at higher antagonist dosing. These results suggest that a spinal neurosteroid-binding site with which 17PA interacts may regulate spinal nociceptive processing in normal and inflamed tissue.
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Androstenoles/administración & dosificación , Hiperalgesia/fisiopatología , Inflamación/fisiopatología , Neurotransmisores/administración & dosificación , Neurotransmisores/antagonistas & inhibidores , Umbral del Dolor/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Calor , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inyecciones Espinales , Masculino , Ratas , Ratas Sprague-Dawley , TactoRESUMEN
We report here in adult rat viral vector mediate-gene knockdown in the primary sensory neurons and the associated cellular and behavior consequences. Self-complementary adeno-associated virus serotype 5 (AAV5) was constructed to express green fluorescent protein (GFP) and a small interfering RNA (siRNA) targeting mammalian target of rapamycin (mTOR). The AAV vectors were injected via an intrathecal catheter. We observed profound GFP expression in lumbar DRG neurons beginning at 2-week post-injection. Of those neurons, over 85% were large to medium-diameter and co-labeled with NF200, a marker for myelinated fibers. Western blotting of mTOR revealed an 80% reduction in the lumbar DRGs (L4-L6) of rats treated with the active siRNA vectors compared to the control siRNA vector. Gene knockdown became apparent as early as 7-day post-injection and lasted for at least 5 weeks. Importantly, mTOR knockdown occurred in large (NF200) and small-diameter neurons (nociceptors). The viral administration induced an increase of Iba1 immunoreactivity in the DRGs, which was likely attributed to the expression of GFP but not siRNA. Rats with mTOR knockdown in DRG neurons showed normal general behavior and unaltered responses to noxious stimuli. In conclusion, intrathecal AAV5 is a highly efficient vehicle to deliver siRNA and generate gene knockdown in DRG neurons. This will be valuable for both basic research and clinic intervention of diseases involving primary sensory neurons.
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Dependovirus/genética , Ganglios Espinales/metabolismo , Ganglios Espinales/virología , Técnicas de Silenciamiento del Gen/métodos , Inyecciones Espinales , Animales , Ganglios Espinales/citología , Vectores Genéticos/genética , Vectores Genéticos/toxicidad , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/virología , Nocicepción , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/toxicidad , Ratas , Ratas Sprague-Dawley , Serina-Treonina Quinasas TOR/deficiencia , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Transducción GenéticaRESUMEN
Pharmacological studies indicate that spinal p38 mitogen-activated protein kinase plays a role in the development of hyperalgesia. We investigated whether either the spinal isoform p38alpha or p38beta is involved in peripheral inflammation evoked pain state and increased expression of spinal COX-2. Using intrathecal antisense oligonucleotides, we show that hyperalgesia is prevented by downregulation of p38beta but not p38alpha, whereas increases in spinal COX-2 protein expression at 8 hours are mediated by both p38alpha and beta isoforms. These data suggest that early activation of spinal p38beta isoform may affect acute facilitatory processing, and both p38beta and alpha isoforms mediate temporally delayed upregulation of spinal COX-2.