Elsevier

Experimental Neurology

Volume 208, Issue 1, November 2007, Pages 145-158
Experimental Neurology

Modulation of the cAMP signaling pathway after traumatic brain injury

https://doi.org/10.1016/j.expneurol.2007.08.011Get rights and content

Abstract

Traumatic brain injury (TBI) results in both focal and diffuse brain pathologies that are exacerbated by the inflammatory response and progress from hours to days after the initial injury. Using a clinically relevant model of TBI, the parasagittal fluid-percussion brain injury (FPI) model, we found injury-induced impairments in the cyclic AMP (cAMP) signaling pathway. Levels of cAMP were depressed in the ipsilateral parietal cortex and hippocampus, as well as activation of its downstream target, protein kinase A, from 15 min to 48 h after moderate FPI. To determine if preventing hydrolysis of cAMP by administration of a phosphodiesterase (PDE) IV inhibitor would improve outcome after TBI, we treated animals intraperitoneally with rolipram (0.3 or 3.0 mg/kg) 30 min prior to TBI, and then once per day for 3 days. Rolipram treatment restored cAMP to sham levels and significantly reduced cortical contusion volume and improved neuronal cell survival in the parietal cortex and CA3 region of the hippocampus. Traumatic axonal injury, characterized by β-amyloid precursor protein deposits in the external capsule, was also significantly reduced in rolipram-treated animals. Furthermore, levels of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), were significantly decreased with rolipram treatment. These results demonstrate that the cAMP–PKA signaling cascade is downregulated after TBI, and that treatment with a PDE IV inhibitor improves histopathological outcome and decreases inflammation after TBI.

Introduction

Traumatic brain injury (TBI) is a prevalent, debilitating health problem, occurring in 1.4 million people each year and disabling 5 million people in the United States (Langlois et al., 2004). The subsequent progressive injury after brain trauma develops from hours to days after the initiating insult, providing an accessible time window for pharmacological therapies. Despite intense efforts, research in TBI has not yielded a therapy that has passed Phase III clinical trials (Doppenberg et al., 2004).

Brain trauma results in contusion formation, neuronal apoptosis, and axonal tract damage. These pathologies are worsened by the inflammatory cascade set into motion by the initial injury (Morganti-Kossmann et al., 2002, Dietrich et al., 2004). Two pro-inflammatory cytokines released after TBI are tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Numerous studies have documented rapid increases in TNF-α and IL-1β levels after TBI (Taupin et al., 1993, Shohami et al., 1994, Fan et al., 1996, Kinoshita et al., 2002, Vitarbo et al., 2004).

IL-1β synergistically acts with TNF-α to induce cell death after TBI. These pro-inflammatory cytokines stimulate inflammatory cells to release damaging reactive oxygen and nitrogen species, raise glutamate levels to excitotoxic levels, impair the ability of glia cells to buffer extracellular potassium, compromise the blood–brain barrier, and attract more inflammatory cells into the brain (Tanaka et al., 1994, Meda et al., 1995, Soares et al., 1995, Hu et al., 1997, Keeling et al., 2000). Once initiated, the inflammatory cascade becomes a toxic positive-feedback loop, further exacerbating brain pathology.

In other models of CNS injury, several studies have demonstrated that restoration of cyclic AMP (cAMP) levels improves outcome. In spinal cord injury, application of rolipram to inhibit the degradation of cAMP promotes axon sparing and results in locomotor improvements (Nikulina et al., 2004, Pearse et al., 2004). Similarly, in transient global ischemia rolipram improves neuronal survival in the hippocampus and hippocampal-dependent learning (Kato et al., 1995, Block et al., 1997, Imanishi et al., 1997, Block et al., 2001).

The effects of cAMP are short-lived because phosphodiesterases (PDEs) rapidly degrade cAMP (Manganiello et al., 1995). Of the 10 classes of PDEs, two isoforms are highly selective for degrading cAMP, PDE IV and VII. Rolipram, a selective inhibitor of PDE IV, reduces inflammation in a number of diseases including asthma, multiple sclerosis, septic shock, rheumatoid arthritis, and inflammatory bowel disease (Dal Piaz and Giovannoni, 2000, Castro et al., 2005). Consequently, PDE IV inhibitors are widely-utilized by the pharmaceutical industry as anti-inflammatory drugs.

A primary action of cAMP is activation of protein kinase A (PKA). PKA phosphorylates transcription factors, including cAMP-responsive element binding (CREB) protein and nuclear factor-κB (NF-κB) p50 (Montminy and Bilezikjian, 1987, Hou et al., 2003). Phosphorylation of CREB stimulates transcription of cell survival genes (Mayr and Montminy, 2001). Phosphorylation of NF-κB p50 subunit suppresses transcription of genes with IκB elements in their promoters; this includes the pro-inflammatory cytokines TNF-α and IL-1β (Cogswell et al., 1994, Verghese et al., 1995, Hou et al., 2003). Thus, we hypothesized that rolipram treatment may improve TBI outcome by decreasing pro-inflammatory cytokine production.

Section snippets

Traumatic brain injury

All experimental procedures were in compliance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the University of Miami Animal Care and Use Committee. Male Sprague–Dawley rats (270–320 g; Charles River Laboratories, Raleigh, NC, USA) were anesthetized with 3% halothane, 70% N2O, and 30% O2, then intubated endotracheally and mechanically ventilated (Harvard Apparatus, Holliston, MA, USA) with 1.5% halothane, 70% N2O, and 30% O2. To immobilize the animals and

Results

To ascertain if the cAMP–PKA pathway is a potential therapeutic target after TBI, we first determined if the cAMP–PKA pathway is modulated after TBI. At various times after sham or FPI surgery, the ipsilateral parietal cortex, hippocampus, and thalamus were assayed by ELISA for cAMP. Absolute levels of cAMP from cortices of sham animals were similar to levels previously reported in the literature (parietal cortex cAMP levels 184.1 ± 5.6 pmol/ml, n = 6) (Pearse et al., 2004). We found that cAMP

Discussion

The parasagittal FPI model leads to reproducible histopathology in the brain, similar to the pathology typically seen in TBI patients (Dietrich et al., 1994, Gennarelli, 1994, Keane et al., 2001, Thompson et al., 2005). Accordingly, there are consistent, quantifiable focal and diffuse histopathologies that are all potential therapeutic targets (Dietrich et al., 1994, Bramlett et al., 1997, Ciallella et al., 2002, Grady et al., 2003, Suzuki et al., 2003, Suzuki et al., 2004, Witgen et al., 2005

Acknowledgments

This work was supported by NIH grants NS30291 and NS42133 (W.D.D.). We thank the Dietrich and Pearse labs for helpful discussions and Beata Frydel and Jarret Weinrich for technical assistance.

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