Dermal nerve fibre and mast cell density, and proximity of mast cells to nerve fibres in the skin of patients with complex regional pain syndrome

An interaction between cutaneous nerves and mast cells may contribute to pain in complex regional pain syndrome (CRPS). To explore this, we investigated the density of dermal nerve fibres, and the density and proximity of mast cells to nerve fibres, in skin biopsies obtained from the affected and unaffected limbs of 57 patients with CRPS and 28 site-matched healthy controls. The percentage of the dermis stained by the pan-neuronal marker protein gene-product 9.5 was lower in the affected limb of patients than in controls (0.12 ± 0.01% versus 0.22 ± 0.04%, P \u3c 0.05), indicating a reduction in dermal nerve fibre density. This parameter did not correlate with CRPS duration. However, it was lower in the affected than unaffected limb of patients with warm CRPS. Dermal mast cell numbers were similar in patients and controls, but the percentage of mast cells less than 5 μm from nerve fibres was significantly lower in the affected and unaffected limbs of patients than in controls (16.8 ±1.7%, 16.5 ± 1.7% and 31.4 ± 2.3% respectively, P \u3c 0.05). We confirm previous findings of a mild neuropathy in CRPS. Our findings suggest that this either develops very early after injury or precedes CRPS onset. Loss of dermal nerve fibres in CRPS might result in loss of chemotactic signals, thus halting mast cell migration towards surviving nerve fibres. Failure of normal nerve fibre-mast cell interactions could contribute to the pathophysiology of CRPS

Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species

Exposure to man-made electromagnetic fields (EMFs), which increasingly pollute our environment, have consequences for human health about which there is continuing ignorance and debate. Whereas there is considerable ongoing concern about their harmful effects, magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine, oncology, orthopedics, and neurology. This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified. Here, we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields (PEMFs) stimulates rapid accumulation of reactive oxygen species (ROS), a potentially toxic metabolite with multiple roles in stress response and cellular ageing. Following exposure to PEMF, cell growth is slowed, and ROS-responsive genes are induced. These effects require the presence of cryptochrome, a putative magnetosensor that synthesizes ROS. We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs, which can account for either therapeutic or pathological effects depending on exposure. Clinically, our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS

Burn injury has a systemic effect on reinnervation of skin and restoration of nociceptive function

Burn injury can lead to abnormal sensory function at both the injury and at distant uninjured sites. Here, we used a mouse model to investigate return of nociceptive function and reinnervation of the skin at the wound and uninjured distant sites following a 3% total burn surface area full-thickness burn injury. We have previously shown that topical application of zinc-metallothionein-IIA (Zn7-MT-IIA) accelerates healing following burn injury, and here, we investigated the potential of Zn7-MT-IIA to enhance reinnervation and sensory recovery. In all burn-injured animals, there was a significant reduction in nociceptive responses (Semmes–Weinstein filaments) at locations near and distant to the wound up to 8 weeks following injury. Cutaneous nerve reinnervation (assessed using protein gene product 9.5 immunohistochemistry) of the wound center was slow in the epidermis but rapid in the dermis. In the dermis, nerves subsequently degenerated both at the wound center and in distant uninjured areas. In contrast, epidermal nerve densities in the distant uninjured areas returned to normal, uninjured levels. Zn7-MT-IIA did not influence return of nociceptive function nor reinnervation. We conclude that burn injury compromises nociceptive function and nerve regeneration both at the injury site and systemically; thus, therapies in addition to Zn7-MT-IIA should be explored to return normal sensory function

Node/paranode complexes in ventral ON after partial transection.

<p>(a) Representative images of Caspr^+ve paranodes (green) flanking the β-III tubulin^+ve (red) paranodal gap, and β-III tubulin^+ve areas (red) colocalised with Na_v1.6^+ve nodes (blue) in normal ventral ON and at 1 day post injury; colocalised areas are yellow and purple respectively (examples indicated by arrows), scale  = 20 μm. Mean ± SEM length of the paranodal gap (b), paranode length (c) and the ratio of node to paranode lengths (e) in ventral ON of normal animals and 1, 3, 7 days, 1 and 3 months after injury; representative images (d), scale bar  = 1 μm. Orthogonal projection of a representative z stack illustrating a large atypical node/paranode complex well within the stack of images (f, boxed), scale bar  = 5 μm; Caspr^+ve paranodes are green, β-III tubulin^+ve axons are red (note: only projections in the z plane of the identified node/paranode complex are shown in the panels adjacent to the main image). Mean ± SEM percentages of node/paranode complexes that were atypical (hemi – nodes, single paranodes) (g), * significantly different from normal for each complex type (p≤0.05) (n = 6 animals/time point)</p

Oxidative stress indicators in ON after partial transection.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in homogenates of ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration, from normal animals and 1, 3, 7 days, 1 and 3 months after injury, or (b) from ventral ON only from normal animals and 1 or 7 days after injury (6 animals pooled per time point, assayed in duplicate). (c) Semi – quantification of mean ± SEM intensity of CML immunoreactivity in olig1^+ve oligodendrocytes in ventral ON, assessed by tracing identified cells in single images in the z axis; representative images at 1 day (d), scale bar  = 10 μm, (n = 4–5 animals/time point). Similarly, semi – quantification of mean ± SEM intensity of DHE staining in olig1^+ve (e) or CC1^+ve (f) oligodendrocytes in ventral ON; * significantly different from normal (p≤0.05).</p

Effects of 670 nm light treatment on oxidative stress indicators.

<p>(a) Mean ± SEM ROS/RNS assessed as DCF fluorescence in pooled homogenates from dorsal or ventral ON from PT handled or PT 670 nm treated animals, 1 day after injury (6 animals pooled per time point, assayed in duplicate). Semi – quantification of maximum (b) or mean (c) ± SEM intensity of DHE staining in CC1^+ve oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury, assessed by tracing identified cells in single images in the z axis; with (d) representative images, example of identified cells indicated, scale bar  = 10 μm. Similarly, mean ± SEM intensity of CML immunoreactivity in CC1^+ve (e) or olig1^+ve (f) oligodendrocytes in ventral ON of PT handled or PT 670 nm treated animals, 1 day after injury. PT is partial ON transection injury.</p

Representative TEM images from normal ventral ON (a) and from day 1 after injury (b–d).

<p>Note the disorganisation, lack of definition (arrow head) and multi – layering (arrows) in the paranodal loops from ON vulnerable to secondary degeneration (b, c) and the complete breakdown in structure of one paranode in a node/paranode complex (d), scale bar  = 0.5 μm.</p

Effects of 670 nm light on node/paranode complexes of ventral ON.

<p>Mean ± SEM paranode length (a) and length of the paranodal gap (b) in ventral ON of 670 nm treated and control animals, 1 day after injury; representative images (c). Mean ± SEM percentage of single paranodes in the same groups (d); Caspr^+ve paranodes are green, β-III tubulin^+ve paranodal gaps are red, scale bar  = 5 μm, * significant differences indicated (p≤0.05), PT is partial ON transection injury. Representative TEM images of PT injured (e) and PT 670 nm light treated (f) node/paranode complexes in ventral ON 1 day after injury. Note the increased definition of the paranodal loops in 670 nm light treated animals, but continued disorganisation (arrows). Representative example of a putative hemi – node in ventral ON from 670 nm light treated animal, with one half of the node/paranode complex clearly defined and the other disorganised (g), scale bars for TEM images  = 0.5 μm.</p

Effects of 670 nm light on RGC numbers and visual function.

<p>Mean ± SEM retrograde labelled RGC numbers (central or ventral retinal regions) (a) and responses in the optokinetic nystagmus test of visual function (smooth pursuits or fast resets) (b), in 670 nm treated or control animals 3 months after injury, * significant differences indicated (p≤0.05) (n = 4–5 animals / group), PT is partial ON transection injury.</p

Cytochrome<i>c</i> oxidase activity after injury, +/− 670 nm light.

<p>Semi – quantification of mean ± SEM cytochrome <i>c</i> oxidase activity in ON including both the dorsal injury site and the ventral region vulnerable to secondary degeneration (a) or in ventral ON only (b) from normal animals and 1, 3, 7 days, 1 and 3 months after injury. Similarly, mean ± SEM cytochrome <i>c</i> oxidase activity in handled normal, PT handled or PT 670 nm treated animals, encompassing the dorsal injury site and ventral ON (c), or in ventral ON only, 1 day after injury (d). Mean ± SEM cytochrome <i>c</i> oxidase activity encompassing the dorsal injury site and ventral ON (defined by region enclosed in dotted lines in F) in PT handled or PT 670 nm treated ON compared to handled normal, 3 months after injury (e); representative images of cytochrome <i>c</i> oxidase activity histochemistry at 3 months (f), scale bar  = 100 μm (n = 4–5 animals/group), * significant differences indicated (p≤0.05), PT is partial ON transection injury.</p