Wayne State University, School of Medicine, Department of Anatomy and Cell Biology, Detroit, MI 48202, USA.
Exp Neurol. 2012 Nov;238(1):79-88. doi: 10.1016/j.expneurol.2012.08.009. Epub 2012 Aug 19.
Recovery of respiratory activity in an upper cervical hemisection model (C2H) of spinal cord injury (SCI) can be induced by systemic theophylline administration 24-48 h after injury. The objectives in the present study are (1) to identify pro-inflammatory and neurotrophic factors expressed after C2H and (2) molecular signals involved in functional recovery. Four groups of adult female rats classified as (i) sham (SH) controls, (ii) subjected to a left C2 hemisection (C2H) only, (iii) C2H rats administered theophylline for 3 consecutive days 2 days after C2H (C2H-T day 5) and (iv) C2H rats treated with theophylline for 3 consecutive days 2 days after C2H and then weaned for 12 days (C2H-T day 17) prior to assessment of respiratory function and molecular analysis were employed. Corresponding sham controls, C2H untreated (vehicle only controls) and C2H treated (theophylline) rats were sacrificed, C3-C6 spinal cord segments quickly dissected and left (ipsilateral) hemi spinal cord and right (contralateral) hemi spinal cord were separately harvested 2 days post surgery. Sham operated and C2H untreated-controls corresponding to C2H-T day 5 and C2H-T day 17 rats, respectively, were prepared similarly. Messenger RNA levels for pro-inflammatory genes (TXNIP, IL-1β, TNF-α and iNOS) and neurotrophic and survival factors (BDNF, GDNF, and Bcl2) were analyzed by real time quantitative PCR. Gene expression pattern was unaltered in SH rats. TXNIP, iNOS, BDNF, GDNF and Bcl2 mRNA levels were significantly increased in the ipsilateral hemi spinal cord in C2H rats. BDNF, GDNF and Bcl2 levels remained elevated in the ipsilateral hemi spinal cord in C2H-T day 5 rats. In this same group, there was further enhancement in TXNIP and IL-1β while iNOS returned to basal levels. Theophylline increased DNA binding activity of transcription factors - cyclic AMP responsive element (CRE) binding protein (CREB) and pro-inflammatory NF-κB. Messenger RNA levels for all genes returned to basal levels in C2H-T day 17 rats. However, BDNF mRNA levels remained significantly elevated after weaning from the drug. Our results suggest that enhanced resolution of early inflammatory processes and expression of pro-survival factors may underlie theophylline-induced respiratory recovery. The results identify potential targets for gene and drug therapies.
在脊髓损伤（SCI）的上颈椎半切（C2H）模型中，全身性茶碱给药可在损伤后 24-48 小时诱导呼吸活动的恢复。本研究的目的是（1）鉴定 C2H 后表达的促炎和神经营养因子，以及（2）参与功能恢复的分子信号。将成年雌性大鼠分为四组：（i）假手术（SH）对照组，（ii）仅行左侧 C2 半切（C2H），（iii）C2H 大鼠在 C2H 后 2 天连续 3 天给予茶碱（C2H-T 第 5 天），以及（iv）C2H 大鼠在 C2H 后连续 3 天给予茶碱，然后停药 12 天（C2H-T 第 17 天），然后评估呼吸功能并进行分子分析。相应的 SH 对照组、未治疗的 C2H 大鼠（仅用载体对照）和 C2H 治疗（茶碱）大鼠被处死，C3-C6 脊髓段迅速解剖，左侧（同侧）半脊髓和右侧（对侧）半脊髓分别收获于手术后 2 天。分别对应于 C2H-T 第 5 天和 C2H-T 第 17 天大鼠的假手术和未治疗的 C2H 对照大鼠，按照类似的方法进行制备。通过实时定量 PCR 分析促炎基因（TXNIP、IL-1β、TNF-α 和 iNOS）和神经营养和生存因子（BDNF、GDNF 和 Bcl2）的信使 RNA 水平。SH 大鼠的基因表达模式没有改变。在 C2H 大鼠中，同侧半脊髓中 TXNIP、iNOS、BDNF、GDNF 和 Bcl2 mRNA 水平显著升高。在 C2H-T 第 5 天大鼠的同侧半脊髓中，BDNF、GDNF 和 Bcl2 水平仍然升高。在同一组中，TXNIP 和 IL-1β进一步增强，而 iNOS 恢复到基础水平。茶碱增加了转录因子-环磷酸腺苷反应元件（CRE）结合蛋白（CREB）和促炎 NF-κB 的 DNA 结合活性。在 C2H-T 第 17 天大鼠中，所有基因的信使 RNA 水平均恢复到基础水平。然而，在停药后，BDNF mRNA 水平仍显著升高。我们的结果表明，早期炎症过程的增强缓解和促生存因子的表达可能是茶碱诱导呼吸恢复的基础。结果确定了基因和药物治疗的潜在靶点。
