Inflammatory sensitization of nociceptors depends on activation of NMDA receptors in DRG satellite cells.

The present study evaluated the role of N-methyl-D-aspartate receptors (NMDARs) expressed in the dorsal root ganglia (DRG) in the inflammatory sensitization of peripheral nociceptor terminals to mechanical stimulation. Injection of NMDA into the fifth lumbar (L5)-DRG induced hyperalgesia in the rat hind paw with a profile similar to that of intraplantar injection of prostaglandin E2 (PGE2), which was significantly attenuated by injection of the NMDAR antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP-5) in the L5-DRG. Moreover, blockade of DRG AMPA receptors by the antagonist 6,7-dinitroquinoxaline-2,3-dione had no effect in the PGE2-induced hyperalgesia in the paw, showing specific involvement of NMDARs in this modulatory effect and suggesting that activation of NMDAR in the DRG plays an important role in the peripheral inflammatory hyperalgesia. In following experiments we observed attenuation of PGE2-induced hyperalgesia in the paw by the knockdown of NMDAR subunits NR1, NR2B, NR2D, and NR3A with antisense-oligodeoxynucleotide treatment in the DRG. Also, in vitro experiments showed that the NMDA-induced sensitization of cultured DRG neurons depends on satellite cell activation and on those same NMDAR subunits, suggesting their importance for the PGE2-induced hyperalgesia. In addition, fluorescent calcium imaging experiments in cultures of DRG cells showed induction of calcium transients by glutamate or NMDA only in satellite cells, but not in neurons. Together, the present results suggest that the mechanical inflammatory nociceptor sensitization is dependent on glutamate release at the DRG and subsequent NMDAR activation in satellite glial cells, supporting the idea that the peripheral hyperalgesia is an event modulated by a glutamatergic system in the DRG.

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion.

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 µL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1ß (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1ß. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1ß in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1(+) cells of the DRG, significantly increased after carrageenan or IL-1ß administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1ß in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

Cytokine inhibition and time-related influence of inflammatory stimuli on the hyperalgesia induced by the nucleus pulposus.

INTRODUCTION: The symptoms of lumbar disc herniation, such as low back pain and sciatica, have been associated with local release of cytokines following the inflammatory process induced by the contact of the nucleus pulposus (NP) with the spinal nerve. MATERIAL AND METHODS: Using an animal experimental model of intervertebral disc herniation and behavioral tests to evaluate mechanical (electronic von Frey test) and thermal (Hargreaves Plantar test) hyperalgesia in the hind paw of rats submitted to the surgical model, this study aimed to detect in normal intervertebral disc the cytokines known to be involved in the mechanisms of inflammatory hyperalgesia, to observe if previous exposure of the intervertebral disc tissue to specific antibodies could affect the pain behavior (mechanical and thermal hyperalgesia) induced by the NP, and to observe the influence of the time of contact of the NP with the fifth lumbar dorsal root ganglion (L5-DRG) in the mechanical and thermal hyperalgesia. RESULTS: The cytokines present at highest concentrations in the rat NP were TNF-α, IL-1ß and CINC-1. Rats submitted to the disc herniation experimental model, in which a NP from the sacrococcygeal region is deposited over the right L5-DRG, showed increased mechanical and thermal hyperalgesia that lasted at least 7 weeks. When the autologous NP was treated with antibodies against the three cytokines found at highest concentrations in the NP (TNF-α, IL-1ß and CINC-1), there was decrease in both mechanical and thermal hyperalgesia in different time points, suggesting that each cytokine may be important for the hyperalgesia in different steps of the inflammatory process. The surgical remotion of the NP from herniated rats 1 week after the implantation reduced the hyperalgesia to the level similar to the control group. This reduction in the hyperalgesia was also observed in the group that had the NP removed 3 weeks after the implantation, although the intensity of the hyperalgesia did not decreased totally. The removal of the NP after 5 weeks did not changed the hyperalgesia observed in the hind paw, which suggests that the longer the contact of the NP with the DRG, the greater is the possibility of development of chronic pain. CONCLUSION: Together our results indicate that specific cytokines released during the inflammatory process induced by the herniated intervertebral disc play fundamental role in the development of the two modalities of hyperalgesia (mechanical and thermal) and that the maintenance of this inflammation may be the most important point for the chronification of the pain.

EVALUATION OF HYPERALGESIA AND HISTOLOGICAL CHANGES OF DORSAL ROOT GANGLION INDUCED BY NUCLEUS PULPOSUS.

UNLABELLED: To evaluate the hyperalgesia and histological abnormalities induced by contact between the dorsal root ganglion and the nucleus pulposus. METHODS: Twenty Wistar rats were used, divided into two experimental groups. In one of the groups, a fragment of autologous nucleus pulposus was removed from the sacrococcygeal region and deposited on the L5 dorsal root ganglia. In the other group (control), a fragment of adipose tissue was deposited on the L5 dorsal root ganglia. Mechanical and thermal hyperalgesia was evaluated on the third day and the first, third, fifth and seventh weeks after the operation. A L5 dorsal root ganglion was removed in the first, third, fifth and seventh weeks after the operation for histological study using HE staining and histochemical study using specific labeling for iNOS. RESULTS: Higher intensity of mechanical and thermal hyperalgesia was observed in the group of animals in which the nucleus pulposus was placed in contact with the dorsal root ganglion. In this group, the histological study showed abnormalities of the dorsal root ganglion tissue, characterized by an inflammatory process and axonal degeneration. The histopathological abnormalities of the dorsal root ganglion tissue presented increasing intensity with increasing length of observation, and there was a correlation with maintenance of the hyperalgesia observed in the behavioral assessment. Immunohistochemistry using specific labeling for iNOS in the group of animals in which the nucleus pulposus was placed in contact with the dorsal root ganglion showed higher expression of this enzyme in the nuclei of the inflammatory cells (glial cells) surrounding the neurons. CONCLUSION: Contact between the nucleus pulposus and the dorsal root ganglion induced mechanical and thermal hyperalgesia and caused histological abnormalities in the dorsal root ganglion components. These abnormalities were characterized by an inflammatory and degenerative process in the structures of the dorsal root ganglion, and they presented increasing intensity with longer periods of observation.

PHARMACOLOGIC TREATMENT OF HYPERALGESIA EXPERIMENTALLY INDUCED BY NUCLEUS PULPOSUS.

OBJECTIVE: To evaluate the effect of anti-inflammatory drugs (dexamethasone, indomethacin, atenolol and indomethacin plus atenolol) and analgesic drugs (morphine) on hyperalgesia experimentally induced by the nucleus pulposus (NP) in contact with the L5 dorsal root ganglion (DRG). METHODS: Thirty male Wistar rats of weights ranging from 220 to 250 g were used in the study. Hyperalgesia was induced by means of a fragment of NP removed from the sacrococcygeal region that was placed in contact with the L5 dorsal root ganglion. The 30 animals were divided into experimental groups according to the drug used. The drugs were administered for two weeks after the surgical procedure to induce hyperalgesia. Mechanical and thermal hyperalgesia was evaluated using the paw pressure test, von Frey electronic test and Hargreaves test, over a seven-week period. RESULTS: The greatest reduction of hyperalgesia was observed in the group of animals treated with morphine, followed by dexamethasone, indomethacin and atenolol. Reductions in hyperalgesia were observed after drug administration ceased, except for the group of animals treated with morphine, in which there was an increase in hyperalgesia after discontinuation of the treatment. CONCLUSION: Hyperalgesia induced by NP contact with the DRG can be reduced through administration of anti-inflammatory and analgesic drugs, but a greater reduction was observed with the administration of dexamethasone.

Tratamento farmacológico da hiperalgesia experimentalmente induzida pelo núcleo pulposo / Pharmacologic treatment of hyperalgesia experimentally induced by nucleus pulposus

OBJETIVO: Avaliar o efeito de drogas anti-inflamatórias (dexametasona, indometacina, atenolol, indometacina e atenolol) e analgésica (morfina) sobre a hiperalgesia experimentalmente induzida pelo núcleo pulposo em contato com o gânglio da raiz dorsal de L5. MÉTODOS: Trinta ratos Wistar machos com peso de 220 a 250g foram utilizados no estudo. A indução da hiperalgesia foi realizada por meio do contato de fragmento de núcleo pulposo retirado da região sacrococcígea e colocado sobre o gânglio da raiz dorsal de L5. Os 30 animais foram divididos em grupos experimentais de acordo com a droga utilizada. As drogas foram administradas durante duas semanas a partir da realização do procedimento cirúrgico para a indução da hiperalgesia. A hiperalgesia mecânica e térmica foram avaliadas por meio do teste da pressão constante da pata, von Frey eletrônico e Hargraves por um período de sete semanas. RESULTADOS: A maior redução da hiperalgesia foi observada no grupo de animais tratados pela morfina, seguido pela dexametasona, indometacina e atenolol. A redução da hiperalgesia foi observada após a interrupção da administração das drogas, com exceção do grupo de animais tratados com morfina, nos quais ocorreu aumento da hiperalgesia após a interrupção do tratamento. CONCLUSÕES: A hiperalgesia induzida pelo contato do núcleo pulposo com o gânglio da raiz dorsal pode ser reduzida com a administração de anti-inflamatórios e analgésicos, tendo sido observado a maior redução da hiperalgesia com a administração da morfina e dexametasona.

OBJECTIVE: To evaluate the effect of antiinflammatory (dexamethasone, indomethacin, atenolol, indomethacin and atenolol) and analgesic drugs (morphine) on hyperalgesia experimentally induced by nucleus pulposus (NP) contact with the L5- dorsal root ganglion (DRG). METHODS: Thirty male Wistar rats with weights ranging from 220 to 250 g were used in the study. The hyperalgesia was induced by contact of a fragment of NP removed from the sacrococcygeal region and placed on the dorsal root ganglion of L5. The 30 animals were divided into experimental groups according to the drug administered. The drugs were administered during the two weeks after the surgical procedure to induce hyperalgesia. Mechanical and thermal hyperalgesia was evaluated by the paw pressure test, von Frey electronic test, and the Hargraves test, for a period of seven weeks. RESULTS: The greatest reduction of hyperalgesia was observed in animals treated by morphine and dexamethasone, followed by dexamethasone, indomethacin, and atenolol. The reduction of hyperalgesia was observed after drug administration ceased, except for animals treated with morphine, in which there was an increased hyperalgesia after cessation of treatment. CONCLUSION: Hyperalgesia induced by NP contact with L5-DRG can be reduced by administration of antiinflammatory and analgesic drugs, but there was a greater reduction observed with the administration of dexamethasone and indometacin.

Avaliação da hiperalgesia e alterações histológicas do gânglio da raiz dorsal induzidas pelo núcleo pulposo / Evaluation of hyperalgesia and histological changes of dorsal root ganglion induced by nucleus pulposus

OBJETIVO: Avaliar a hiperalgesia e as alterações histológicas induzidas pelo contato do gânglio da raiz dorsal com o núcleo pulposo. MÉTODOS: Foram utilizados 20 ratos Wistar, divididos em dois grupos experimentais. Em um dos grupos um fragmento do NP autólogo retirado da região sacrococcígea foi colocado sobre os gânglios da raiz dorsal de L5, e no outro grupo (controle) um fragmento de tecido adiposo foi colocado sobre o gânglio da raiz dorsal de L5. A hiperalgesia mecânica e térmica foi avaliada no terceiro dia, na primeira, terceira, quinta e sétima semanas de pós-operatório. O gânglio da raiz dorsal de L5 foi retirado na primeira, terceira, quinta e sétima semanas de pós-operatório para estudo histológico por meio da coloração com HE e estudo histoquímico (marcação específica para iNOS). RESULTADOS: Foi observada hiperalgesia térmica e mecânica de maior intensidade no grupo de animais em que o NP foi colocado em contato com o GRD, e nesse grupo o estudo histológico evidenciou alterações dos tecidos do gânglio da raiz dorsal, caracterizadas por processo inflamatório e degeneração axonal. As alterações histopatológicas dos tecidos do gânglio da raiz dorsal apresentaram intensidade crescente com o aumento do período de observação, apresentando correlação com a manutenção da hiperalgesia observada na avaliação comportamental. A imunohistoquímica com marcação específica para iNOS demonstrou, no grupo de animais em que o núcleo pulposo foi colocado em contato com o gânglio da raiz dorsal, aumento da expressão dessa enzima nos núcleos das células inflamatórias (células da glia) ao redor dos neurônios.

OBJECTIVE: To evaluate hyperalgesia and histological changes of dorsal root ganglia induced by nucleus pulposus (NP) contact. METHODS: Twenty Wistar rats were used, divided into two experimental groups. In one of the groups, a fragment of the autologous NP was removed from the sacroccocigeal region and deposited on the L5 dorsal root ganglia. In the control group, the NP was removed from the sacrococcygeal region, L5 dorsal root ganglia were exposed and covered by a piece of adipous fat tissue. Hyperalgesia was evaluated by the von Frey electronic test and Hargreaves test, and histological changes of the dorsal root ganglia by HE staining and immunohistochemistry using iNOS. The evaluation of hyperalgesia and histological changes of the dorsal root ganglia were performed on the third postoperative day and after 1, 3, 5, and 7 weeks. RESULTS: NP induced higher intensity mechanical and thermal hyperalgesia. Dorsal root ganglia in contact with nucleus pulposus presented histological changes and the intensity of these changes were proportional to the length of time in contact. The expression of iNOS was higher in the glial cells in contact with the nucleus pulposus.

Modelo experimental para o estudo da hérnia do disco intervertebral / Experimental model to study intervertebral disc herniation

OBJETIVO: Apresentar um modelo experimental de hérnia de disco e sua validação para estudo da hiperalgesia mecânica e térmica produzidas pelo contato do núcleo pulposo (NP) com as estruturas nervosas envolvidas nessa afecção. MÉTODOS: Foram utilizados ratos Wistar, sendo o NP autólogo retirado da região sacrococcígea e depositado sobre a dura-máter, raiz nervosa ou gânglios das raízes dorsais L4, L5 ou L6. Os experimentos foram divididos em quatro etapas: 1ª) determinação da estrutura nervosa mais sensível ao contato com o NP; 2ª) identificação do melhor nível lombar para a indução da hiperalgesia; 3ª) determinação da ausência de lesão motora; e 4ª) determinação da influência do procedimento cirúrgico no desenvolvimento do processo inflamatório. A hiperalgesia foi avaliada nos testes de von Frey eletrônico e de Hargreaves e a função motora, pelo teste de rota-rod. RESULTADOS: O NP induziu hiperalgesia de maior intensidade na pata quando em contato com o gânglio da raiz dorsal (GRD) do que em contato com a dura-máter ou a raiz nervosa. Quando em contato com o GRD-L5, o NP induziu hiperalgesia ainda maior que a induzida pelo contato com os GRDs L4 e L6. Não foram observadas lesão motora e influência do processo inflamatório cirúrgico sobre a hiperalgesia. CONCLUSÃO: O GRD é a estrutura mais sensível aos componentes do NP para a produção da hiperalgesia, sendo o quinto nível lombar o que apresentou maior alteração nas sensibilidades mecânica e térmica avaliadas na pata dos animais, de acordo com os métodos utilizados.

OBJECTIVE:The purpose of this study is to present an experimental model of disc herniation and to validate such model to study mechanic and thermal hyperalgesia produced by the contact of the nucleus pulposus (NP) with nerve structures involved in this condition. METHODS: The authors used Wistar rats, the autologous NP being removed from the sacrococcygeal region and deposited on the dura mater, nerve root, or L4, L5, or L6 dorsal root ganglia. The experiments were divided into four steps: 1) determining the nerve structure that is the most sensitive to the contact with NP; 2) identifying the best lumbar level to induce hyperalgesia; 3) determining absence of a motor lesion; and 4) determining the impact of the surgical procedure upon the inflammatory process. Hyperalgesia was evaluated by the von Frey electronic test and the Hargreaves test, and the motor function was evaluated by the rota-rod test. RESULTS: NP induced higher intensity hyperalgesia in the paw when it was in contact with the dorsal root ganglion (GRD) than when it was in contact with the dura mater or the nerve root. Contact with GRD-L5, led NP to induce even higher hyperalgesia than that induced in the contact with L4 and L6 GRDs. No motor lesion and impact of the surgical inflammatory process on hyperalgesia were observed. CONCLUSION: GRD is the structure that is most sensitive to NP components to produce hyperalgesia, the fifth lumbar level being that showed the greatest change in the mechanic and thermal sensitivities evaluated in the paws of the animals, under the methods used.