A possible role for inducible arginase isoform (AI) in the pathogenesis of chronic venous leg ulcer.

Chronic venous ulcer (CVU) is a major cause of chronic wounds of lower extremities and presents a significant financial and resource burden to health care systems worldwide. Defects in the vasculature, matrix deposition, and re-epithelialization are the main histopathological changes believed to impede healing. Supplementation of the amino acid arginine that plays a crucial role in the interactions that occur during inflammation and wound healing was proven clinically to improve acute wound healing probably through enhancing activity of inducible arginase (AI) locally in the wounds. However, the possible mechanism of arginine action and the potential beneficial effects of AI/arginine in human chronic wounds remain unclear. In the present study, using biopsies, taken under local anesthesia, from adult patients (n = 12, mean age 55 years old) with CVUs in lower extremities, we investigated the correlation between AI distribution in CVUs and the histopathological changes, mainly proliferative and vascular changes. Our results show a distinct spatial distribution of AI along the ulcer in the epidermis and in the dermis with the highest level of expression being at the ulcer edge and the least expression towards the ulcer base. The AI cellular immunoreactivity, enzymatic activity, and protein levels were significantly increased towards the ulcer edge. Interestingly, a similar pattern of expression was encountered in the proliferative and the vascular changes with strong correlations between AI and the proliferative activity and vascular changes. Furthermore, AI cellular distribution was associated with increased proliferative activity, inflammation, and vascular changes. Our findings of differential expression of AI along the CVU base, edge, and nearby surrounding skin and its associations with increased proliferative activity and vascular changes provide further support to the AI implication in CVU pathogenesis. The presence of high levels of AI in the epidermis of chronic wounds may serve as a molecular marker of impaired healing and may provide future targets for therapeutic intervention.

Chronic inflammatory pain is associated with increased excitability and hyperpolarization-activated current (Ih) in C- but not Aδ-nociceptors.

Inflammatory pain hypersensitivity results partly from hyperexcitability of nociceptive (damage-sensing) dorsal root ganglion (DRG) neurons innervating inflamed tissue. However, most of the evidence for this is derived from experiments using acute inflammatory states. Herein, we used several approaches to examine the impact of chronic or persistent inflammation on the excitability of nociceptive DRG neurons and on their expression of I(h) and the underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which regulate neuronal excitability. Using in vivo intracellular recordings of somatic action potentials from L4/L5 DRG neurons in normal rats and rats with hindlimb inflammation induced by complete Freund's adjuvant (CFA), we demonstrate increased excitability of C- but not Aδ-nociceptors, 5 to 7 days after CFA. This included an afterdischarge response to noxious pinch, which may contribute to inflammatory mechanohyperalgesia, and increased incidence of spontaneous activity (SA) and decreased electrical thresholds, which are likely to contribute to spontaneous pain and nociceptor sensitization, respectively. We also show, using voltage clamp in vivo, immunohistochemistry and behavioral assays that (1) the inflammation-induced nociceptor hyperexcitability is associated, in C- but not Aδ-nociceptors, with increases in the mean I(h) amplitude/density and in the proportion of I(h) expressing neurons, (2) increased proportion of small DRG neurons (mainly IB4-negative) expressing HCN2 but not HCN1 or HCN3 channel protein, (3) increased HCN2- immunoreactivity in the spinal dorsal horn, and (4) attenuation of inflammatory mechanoallodynia with the selective I(h) antagonist, ZD7288. Taken together, the findings suggest that C- but not Aδ-nociceptors sustain chronic inflammatory pain and that I(h)/HCN2 channels contribute to inflammation-induced C-nociceptor hyperexcitability.

Leak K⁺ channel mRNAs in dorsal root ganglia: relation to inflammation and spontaneous pain behaviour.

Two pore domain potassium (K2P) channels (KCNKx.x) cause K⁺ leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1 day and 4 days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1 day and 4 day rats <1h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n=4-6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order: Kv1.4>TRESK(KCNK18)>TRAAK(KCNK4)>TREK2(KCNK10)=TWIK2(KCNK6)>TREK1 (KCNK2)=THIK2(KCNK12)>TASK1(KCNK3)>TASK2(KCNK5)>THIK1(KCNK13)=TASK3(KCNK9). During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1 day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4 days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4 days. Ipsilateral mRNA levels at 4days compared with 1 day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.