Konference: 2011 7. Sympozium a workshop molekulární patologie a histo-cyto-chemie
Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie
Téma: Keynote lectures of invited speakers I
Číslo abstraktu: 001
Autoři: P. Dubový
Peripheral neuropathic pain, manifested as
spontaneous pain, hyperalgesia and allodynia, arises as a result of
many forms of nerve damage. Compelling evidence indicates that
neuropathic pain induced by nerve injury is related to cellular and
molecular changes in the dorsal root ganglia (DRG) containing
bodies of the primary afferent neurons. It is assumed that pro- and
anti-inflammatory cytokines may contribute to both the induction
and maintenance of neuropathic pain. Chronic constriction injury
(CCI) of the rat sciatic nerve is frequently used experimental
model of neuropathic pain.
Unilateral CCI of the rat sciatic nerve performed under aseptic conditions was used to study changes of TNFa, IL-6 and IL-10 proteins and mRNAs in L4-L5 and C7-C8 DRG removed from both sides of naďve (n=16), operated (n=64) and sham-operated (n=64) rats. Neuropathic pain induction was tested by withdrawal threshold of mechanoallodynia and thermal hyperalgesia. Levels of TNFa, IL-6 and IL-10 proteins and their mRNA were analyzed by quantitative immunohistochemistry, ELISA, Western blot, in situ hybridization and RT-PCR in DRG samples from native, CCI- and sham-operated rats for 1, 3, 7 and 14 days.
The native DRG displayed no or very weak immunofluorescence staining for the cytokine proteins in the neuronal bodies with a moderate intensity in small and medium-sized neurons. Unilateral CCI induced bilateral elevation of TNFa, IL-6 and IL-10 immunostaining usually in large-sized neuronal perikarya of both L4-L5 and C7-C8 DRG. ELISA and Western blot confirmed bilateral and temporal increase of TNFa, IL-6 and IL-10 proteins not only in the homonymous lumbar DRG associated with the sciatic nerve, but also in the heteronymous cervical ones non-associated with spinal segments of constricted nerve.
TNFa protein was peaked in L4-L5 DRG at 7 and C7-C8 DRG at 14 days after CCI, while IL-6 protein level was the highest in both lumbar and cervical DRG at 3 days. In contrast, IL-10 protein was significantly increased in lumbar and cervical DRG of both sides at 1 day and 3 days. Elevated levels of TNFa and IL-6 mRNAs were proved in the neuronal bodies of all DRG from CCI rats by in situ hybridization and verified by RT-PCR.
Surprisingly, sham-operated rats displayed bilateral increased staining for IL-6 mRNA in satellite glial cells of both cervical and lumbar DRG, while staining for TNFa mRNA was enhanced in neuronal bodies. These increased levels of mRNAs were confirmed by RT-PCR.
Our results indicate that neuroinflammation expressed by increased production of cytokines is not limited to the primary sensory neurons of injured nerve, but is spread to the contralateral side as well as alongside neuroaxis to DRG neurons of remote segments. In addition, neuroinflammatory reaction of satellite glial cells in sham-operated rats suggests their sensitivity to tissue injury. Both neuroinflammatory reactions influence excitation of the primary sensory neurons and may participate in neuropathic pain induction. Moreover, changes of cytokine proteins in DRG non-associated with damaged nerve could be related with a general neuroinflammatory reaction of the nervous system to injury and thereby promote potential of the DRG neurons for regeneration of their axons following a conditioning lesion.
Supported by MSM0021622404.
Unilateral CCI of the rat sciatic nerve performed under aseptic conditions was used to study changes of TNFa, IL-6 and IL-10 proteins and mRNAs in L4-L5 and C7-C8 DRG removed from both sides of naďve (n=16), operated (n=64) and sham-operated (n=64) rats. Neuropathic pain induction was tested by withdrawal threshold of mechanoallodynia and thermal hyperalgesia. Levels of TNFa, IL-6 and IL-10 proteins and their mRNA were analyzed by quantitative immunohistochemistry, ELISA, Western blot, in situ hybridization and RT-PCR in DRG samples from native, CCI- and sham-operated rats for 1, 3, 7 and 14 days.
The native DRG displayed no or very weak immunofluorescence staining for the cytokine proteins in the neuronal bodies with a moderate intensity in small and medium-sized neurons. Unilateral CCI induced bilateral elevation of TNFa, IL-6 and IL-10 immunostaining usually in large-sized neuronal perikarya of both L4-L5 and C7-C8 DRG. ELISA and Western blot confirmed bilateral and temporal increase of TNFa, IL-6 and IL-10 proteins not only in the homonymous lumbar DRG associated with the sciatic nerve, but also in the heteronymous cervical ones non-associated with spinal segments of constricted nerve.
TNFa protein was peaked in L4-L5 DRG at 7 and C7-C8 DRG at 14 days after CCI, while IL-6 protein level was the highest in both lumbar and cervical DRG at 3 days. In contrast, IL-10 protein was significantly increased in lumbar and cervical DRG of both sides at 1 day and 3 days. Elevated levels of TNFa and IL-6 mRNAs were proved in the neuronal bodies of all DRG from CCI rats by in situ hybridization and verified by RT-PCR.
Surprisingly, sham-operated rats displayed bilateral increased staining for IL-6 mRNA in satellite glial cells of both cervical and lumbar DRG, while staining for TNFa mRNA was enhanced in neuronal bodies. These increased levels of mRNAs were confirmed by RT-PCR.
Our results indicate that neuroinflammation expressed by increased production of cytokines is not limited to the primary sensory neurons of injured nerve, but is spread to the contralateral side as well as alongside neuroaxis to DRG neurons of remote segments. In addition, neuroinflammatory reaction of satellite glial cells in sham-operated rats suggests their sensitivity to tissue injury. Both neuroinflammatory reactions influence excitation of the primary sensory neurons and may participate in neuropathic pain induction. Moreover, changes of cytokine proteins in DRG non-associated with damaged nerve could be related with a general neuroinflammatory reaction of the nervous system to injury and thereby promote potential of the DRG neurons for regeneration of their axons following a conditioning lesion.
Supported by MSM0021622404.
Datum přednesení příspěvku: 29. 4. 2011
