Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons
Introduction
Neuronal damage is generally associated with recruitment of immune cells to the site of injury, thus resulting in neuroinflammation. During the acute phase of neuroinflammation, circulating polymorphonuclear leukocytes (PMN or neutrophils) are among the first leukocytes to detect inflammatory signals that trigger PMN rolling along blood vessels, firm adhesion and transendothelial migration into endoneurial space (Nathan, 2006). Subsequently, activated PMN release several mediators of cellular toxicity to kill invading pathogens. As such, activated PMN come into close proximity with neurons in a plethora of neurological conditions, including neurodegenerative (multiple sclerosis, Parkinson's, Alzheimer's) and neurotraumatic disorders (spinal cord injury, ischemia, painful neuropathy). Although the inflammatory response is a critical component of healing and repair, abundant evidence in rodent models suggest that limiting PMN migration can positively influence the outcome of neuronal injury (Ryu et al., 2007, Yamasaki et al., 1997, Prestigiacomo et al., 1999, Connolly et al., 1996, Huang et al., 1999, Taoka et al., 1997).
Blocking PMN migration mediated by cytokine-induced chemoattractant-1 can ameliorate the neurological outcome following stroke (Yamasaki et al., 1997). Neutropenia or deficiency in the intercellular adhesion molecule ICAM-1 that mediates PMN migration confers neuronal protection after experimental cerebral ischemia and stroke (Connolly et al., 1996, Huang et al., 1999, Prestigiacomo et al., 1999, see also Dirnagl et al., 1999, Lo et al., 2003). Administration of anti-P-selectin antibody, which disrupts interaction between activated PMN and endothelial cells, attenuates tissue injury and improves motor performance following spinal cord compression (Taoka et al., 1997).
Neuropathic pain is a neurological disorder caused by nerve lesion whereby patients face unsatisfactory treatment options (Dworkin et al., 2003). In different animal models of sciatic neuropathy, PMN accumulate at the site of nerve lesion (Perkins and Tracey, 2000), as well as proximally in lumbar dorsal root ganglia (DRG) (Morin et al., 2007). Notably, neuropathic pain is associated with hyperexcitability of lumbar DRG neurons (Amir et al., 2005, Djouhri et al., 2006, Devor, 1999). Interestingly, neuropathic pain is attenuated by blocking PMN migration after 5-hydoxytryptamine (5-HT)-induced subcutaneous inflammation (Oliveira et al., 2007) or by PMN depletion preemptive to sciatic transection (Perkins and Tracey 2000), raising the possibility that PMN targeting of peripheral neurons can lead to hyperexcitability of ‘pain-sensing’ DRG neurons (nociceptors).
Recently, Nguyen et al. (2007) reported that a PMN-conditioned medium is sufficient to cause rapid (within 2 h) and significant loss of dissociated DRG neurons in culture, whereas Dinkel et al. (2004) demonstrated a delayed reduction in the viability of primary hippocampal neurons 3 days after co-culture with PMN in a cell contact dependant manner. However, the functional effects of PMN on neuronal activity and/or excitability remain unexplored. Therefore, this study was undertaken to characterize the activation state of PMN exposed to DRG cells, to determine the range of PMN titers that significantly reduce DRG neuronal count in culture, and to examine the effect of PMN on DRG neuronal excitability.
Our results indicate that co-incubation of PMN with DRG cells triggers activation of PMN which is detrimental to the viability of neurons, induces neuronal calcium transients, and increases the excitability of nociceptors.
Section snippets
Materials and methods
Neuronal and PMN cells were obtained from adult (200–250 g) male Sprague–Dawley rats that were used according to procedures approved by the Institutional Animal Care and Use Committee at Rhode Island Hospital.
Statistical analysis
Two-tailed analyses were performed using parametric tests at the alpha significance level of 0.05. Statistical significance was computed using one-way ANOVA or three-way ANOVA for analysis of variation in firing frequency with time at increasing current intensities. Tests of factors including pair-wise comparisons were performed with either unpaired or paired Student's t-test. In each cell loss experiment, PMN and DRG cells were obtained from 2 rats and co-incubated in at least 3 wells per
PMN activation
Within 5 min of addition of PMN (200 μl, 106 cells/ml) to a microwell coated with poly-D-lysine, laminin and collagen, prepared similarly as used for plating DRG cells and containing DMEM-F12 (2 ml), less than 5% of cells were fluorescently labeled with CM-DCF (data not shown), indicating a very weak intracellular ROS activity and therefore, predominantly resting PMN. However, more than 90% of cells manifested a clear fluorescent signal 2 min after addition of PMA, indicating their viability
Discussion
This study shows functional consequences of activated blood PMN on the viability, activity and excitability of DRG neurons. Using dissociated DRG neurons in culture, we demonstrate a significant decrease in neuronal count after co-incubation with activated PMN at a threshold E/T ratio between 0.2:1 and 0.4:1. Neuronal loss is probably unrelated to neuronal firing because lidocaine fails to abrogate this effect. The detrimental effect of PMN on neuronal viability does not necessarily require PMN
Acknowledgments
This study is supported by funds from Rhode Island Hospital and Rhode Island Foundation (CYS), and by NCRR/NIH P20 RR018728 and Rhode Island Foundation (SKS). The authors thank Derek M. Kozikowski for conceptualizing and writing the software used to analyze calcium transients and Zachary Jaffa for rodent handling.
References (54)
- et al.
Transient loss of terminals from non-peptidergic nociceptive fibers in the substantia gelatinosa of spinal cord following chronic constriction injury of the sciatic nerve
Neuroscience
(2006) - et al.
Nerve growth factor induced hyperalgesia in the rat hind paw is dependent on circulating neutrophils
Pain
(1998) - et al.
Control of inflammatory pain by chemokine-mediated recruitment of opioid-containing polymorphonuclear cells
Pain
(2004) Unexplained peculiarities of the dorsal root ganglion
Pain, Suppl.
(1999)- et al.
Pathobiology of ischaemic stroke: an integrated view
Trends Neurosci.
(1999) - et al.
Assessment of the neurotoxicity of styrene, styrene oxide, and styrene glycol in primary cultures of motor and sensory neurons
Toxicol. Lett.
(1995) - et al.
Neutrophil infiltration is implicated in the sustained thermal hyperalgesic response evoked by allergen provocation in actively sensitized rats
Pain
(2006) - et al.
Loss of dorsal root ganglion cells concomitant with dorsal root axon sprouting following segmental nerve lesions
Neuroscience
(1997) - et al.
Neutrophils invade lumbar dorsal root ganglia after chronic constriction injury of the sciatic nerve
J. Neuroimmunol.
(2007) - et al.
5-HT acts on nociceptive primary afferents through an indirect mechanism to induce hyperalgesia in the subcutaneous tissue
Neuroscience
(2007)