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Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain.
Neurochem Int. 2004 Jul-Aug; 45(2-3):397-407.NI

Abstract

Activated spinal glial cells have been strongly implicated in the development and maintenance of persistent pain states following a variety of stimuli including traumatic nerve injury. The present study was conducted to characterize the time course of surface markers indicative of microglial and astrocytic activation at the transcriptional level following an L5 nerve transection that results in behavioral hypersensitivity. Male Sprague-Dawley rats were divided into a normal group, a sham surgery group with an L5 spinal nerve exposure and an L5 spinal nerve transected group. Mechanical allodynia (heightened response to a non-noxious stimulus) of the ipsilateral hind paw was assessed throughout the study. Spinal lumbar mRNA levels of glial fibrillary acidic protein (GFAP), integrin alpha M (ITGAM), toll-like receptor 4 (TLR4) and cluster determinant 14 (CD14) were assayed using real-time reverse transcription polymerase chain reaction (RT-PCR) at 4 h, 1, 4, 7, 14 and 28 days post surgery. The spinal lumbar mRNA expression of ITGAM, TLR4, and CD14 was upregulated at 4 h post surgery, CD14 peaked 4 days after spinal nerve transection while ITGAM and TLR4 continued to increase until day 14 and returned to almost normal levels by postoperative day 28. In contrast, spinal GFAP mRNA did not significantly increase until postoperative day 4 and then continued to increase over the duration of the study. Our optimized real-time RT-PCR method was highly sensitive, specific and reproducible at a wide dynamic range. This study demonstrates that peripheral nerve injury induces an early spinal microglial activation that precedes astrocytic activation using mRNA for surface marker expression; the delayed but sustained expression of mRNA coding for GFAP implicates astrocytes in the maintenance phase of persistent pain states. In summary, these data demonstrate a distinct spinal glial response following nerve injury using real-time RT-PCR.

Authors+Show Affiliations

Department of Anesthesiology, Dartmouth-Hitchcock Medical Center, HB 7125, One Medical Center Drive, Lebanon, NH 03756, USA. flobert.y.tanga@dartmouth.eduNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

15145554

Citation

Tanga, F Y., et al. "Quantitative Real-time RT-PCR Assessment of Spinal Microglial and Astrocytic Activation Markers in a Rat Model of Neuropathic Pain." Neurochemistry International, vol. 45, no. 2-3, 2004, pp. 397-407.
Tanga FY, Raghavendra V, DeLeo JA. Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochem Int. 2004;45(2-3):397-407.
Tanga, F. Y., Raghavendra, V., & DeLeo, J. A. (2004). Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. Neurochemistry International, 45(2-3), 397-407.
Tanga FY, Raghavendra V, DeLeo JA. Quantitative Real-time RT-PCR Assessment of Spinal Microglial and Astrocytic Activation Markers in a Rat Model of Neuropathic Pain. Neurochem Int. 2004 Jul-Aug;45(2-3):397-407. PubMed PMID: 15145554.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitative real-time RT-PCR assessment of spinal microglial and astrocytic activation markers in a rat model of neuropathic pain. AU - Tanga,F Y, AU - Raghavendra,V, AU - DeLeo,J A, PY - 2003/03/19/received PY - 2003/06/11/revised PY - 2003/06/11/accepted PY - 2004/5/18/pubmed PY - 2004/7/28/medline PY - 2004/5/18/entrez SP - 397 EP - 407 JF - Neurochemistry international JO - Neurochem Int VL - 45 IS - 2-3 N2 - Activated spinal glial cells have been strongly implicated in the development and maintenance of persistent pain states following a variety of stimuli including traumatic nerve injury. The present study was conducted to characterize the time course of surface markers indicative of microglial and astrocytic activation at the transcriptional level following an L5 nerve transection that results in behavioral hypersensitivity. Male Sprague-Dawley rats were divided into a normal group, a sham surgery group with an L5 spinal nerve exposure and an L5 spinal nerve transected group. Mechanical allodynia (heightened response to a non-noxious stimulus) of the ipsilateral hind paw was assessed throughout the study. Spinal lumbar mRNA levels of glial fibrillary acidic protein (GFAP), integrin alpha M (ITGAM), toll-like receptor 4 (TLR4) and cluster determinant 14 (CD14) were assayed using real-time reverse transcription polymerase chain reaction (RT-PCR) at 4 h, 1, 4, 7, 14 and 28 days post surgery. The spinal lumbar mRNA expression of ITGAM, TLR4, and CD14 was upregulated at 4 h post surgery, CD14 peaked 4 days after spinal nerve transection while ITGAM and TLR4 continued to increase until day 14 and returned to almost normal levels by postoperative day 28. In contrast, spinal GFAP mRNA did not significantly increase until postoperative day 4 and then continued to increase over the duration of the study. Our optimized real-time RT-PCR method was highly sensitive, specific and reproducible at a wide dynamic range. This study demonstrates that peripheral nerve injury induces an early spinal microglial activation that precedes astrocytic activation using mRNA for surface marker expression; the delayed but sustained expression of mRNA coding for GFAP implicates astrocytes in the maintenance phase of persistent pain states. In summary, these data demonstrate a distinct spinal glial response following nerve injury using real-time RT-PCR. SN - 0197-0186 UR - https://www.unboundmedicine.com/medline/citation/15145554/Quantitative_real_time_RT_PCR_assessment_of_spinal_microglial_and_astrocytic_activation_markers_in_a_rat_model_of_neuropathic_pain_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0197018603003024 DB - PRIME DP - Unbound Medicine ER -