Activated polymorphonuclear cells promote injury and excitability of dorsal root ganglia neurons

Abstract

Therapies aimed at depleting or blocking the migration of polymorphonuclear leukocytes (PMN or neutrophils) are partially successful in the treatment of neuroinflammatory conditions and in attenuating pain following peripheral nerve injury or subcutaneous inflammation. However, the functional effects of PMN on peripheral sensory neurons such as dorsal root ganglia (DRG) neurons are largely unknown. We hypothesized that PMN are detrimental to neuronal viability in culture and increase neuronal activity and excitability. We demonstrate that isolated peripheral PMN are initially in a relatively resting state but undergo internal oxidative burst and activation by an unknown mechanism within 10 min of co-culture with dissociated DRG cells. Co-culture for 24 h decreases neuronal count at a threshold<0.4:1 PMN:DRG cell ratio and increases the number of injured and apoptotic neurons. Within 3 min of PMN addition, fluorometric calcium imaging reveals intracellular calcium transients in small size (<25 microm diam) and large size (>25 microm diam) neurons, as well as in capsaicin-sensitive neurons. Furthermore, small size isolectin B4-labeled neurons undergo hyperexcitability manifested as decreased current threshold and increased firing frequency. Although co-culture of PMN and DRG cells does not perfectly model neuroinflammatory conditions in vivo, these findings suggest that activated PMN can potentially aggravate neuronal injury and cause functional changes to peripheral sensory neurons. Distinguishing the beneficial from the detrimental effects of PMN on neurons may aid in the development of more effective drug therapies for neurological disorders involving neuroinflammation, including painful neuropathies.

Fig 1

Total neuronal count (mean ±SD) expressed as percentage of control (CT, untreated cultures) after 24 hr co-culture with PMN at different E/T ratios relative to DRG cells or co-culture with peripheral blood mononuclear cells (PBMC, E/T 10:1). TNFα (activator of PMN) or lidocaine (blocker of sodium channels) was added in some cultures concomitantly with PMN (*p<0.05, **p<0.01 relative to CT), or PMN were treated with MMA (iNOS inhibitor) prior to co-culture with DRG cells.

Fig 2

Glial cell count (GFAP-positive cells) expressed as percent control (CT, untreated cultures) and GFAP immunofluorescence per cell relative to background (mean ±SD, *p<0.05). Compared to CT, co-culture of DRG cells with PMN at E/T 10:1 for 24 hr (B) did not change the mean number of GFAP-positive cells (C), but increased GFAP fluorescence (D) suggesting glial activation (For A–B, blue: nuclear DAPI; green: GFAP, horizontal bar= 20 µm).

Fig 3

PMN increase the number of pyknotic and apoptotic neurons in culture. Left panel: Number of pyknotic neurons (mean ±SD) expressed as fold increase compared to control (CT, untreated cultures) after 24 hr co-incubation with PMN at E/T 10:1. Note spherical neuronal soma with clearly delineated cytoplasmic membrane (a, arrow) compared to that of a pyknotic neuron with blebbing (b, arrow), granular cytoplasm and disintegrated nucleus evident by nuclear stain (c, arrow and arrowhead). Right panel: Number of neurons with Annexin V staining expressed as percentage of total neurons after 24 hr co-incubation with PMN E/T 5:1 or 10:1 (*p<0.05, **p<0.01 relative to CT). Photo in ‘d’ shows several DRG cells, same field is shown in ‘e’ with Annexin V fluorescence.

Fig 4

PMN induce calcium responses in different subpopulations of DRG neurons. Total and percent numbers of neurons with calcium response within 15 min of co-incubation with PMN (E/T 10:1). Upper panel: Representative trace showing relative [Ca²⁺]_i in a small size neuron; HBSS and PMN were added as indicated by arrowheads. Note robust and delayed calcium responses 2–3 minutes after addition of PMN. Lower panel: For each treatment, the number of neurons with increase in [Ca²⁺]_i is represented by black histogram superimposed on white histogram showing total number of neurons, or separately (gray histogram) as percentage of total number of neurons.

Fig 5

PMN increase the excitability of DRG neurons. Representative traces from one neuron (a) show membrane potential (resting potential −63 mV) in response to current pulses (pA), with upper trace showing one action potential at 400 pA (current threshold 200 pA) before addition of PMN (pre-PMN), whereas lower trace shows membrane potential (resting potential −56 mV) 4 min after PMN addition (post-PMN) with action potential burst at 150 pA or below baseline current threshold. Histograms (b) represent mean (±SD) current threshold in small diameter IB4+ neurons before and 4 min after addition of PMN (**p<0.01 relative to pre-PMN). In ‘c’, mean (±SD) firing frequency (Hz) of same neurons shown in ‘b’ before and 4 min after PMN versus stimulus intensities of increasing amplitudes (pA, *p<0.05 relative to pre-PMN). In ‘d’, mean (±SD) resting membrane potential (RMP, mV) in same neurons shown in ‘b,c’ before and at 1, 2 and 4 min after PMN addition.