A–H) Images from sections of thoracic back skin biopsies processed with polyclonal anti-human CGRPα (A–F) captured at the same camera settings, and monoclonal anti-human CGRPα (G,H) captured at a higher camera sensitivity. Keratinocyte labeling is shown between arrows. Innervation labeling is shown with arrowheads. A, B) Examples of the variations in CGRP-IL expression among keratinocytes as shown in images from the left and right side respectively in a control subject (23-year old male) that had the most intense expression encountered among six control subjects. The CGRP-IL is heterogenous among keratinocytes mostly located in the middle third of the epidermis. C, D) Examples of CGRP-IL labeling among keratinocytes in a subject (29-year-old male) that had the least difference in labeling intensity between the PHN afflicted skin (D, VAS = 6.5) and the nonpainful unafflicted mirror-image contralateral skin (C). E, F) Examples of CGRP-IL labeling in a subject (48 year-old male, VAS = 5.0) that was representative of most of the PHN afflicted skin (F) and nonpainful unafflicted mirror-image contralateral skin (E). As revealed with the polyclonal antibody, nonpainful contralateral skin consistently had less intense and more heterogenous labeling of keratinocytes, whereas PHN skin had more intense and more homogenous labeling. In both the afflicted and unafflicted skin of the PHN subjects, CGRP-IL was expressed among keratinocytes spanning most of the thickness of the epidermis. In the PHN skin, the stratum basalis was usually less intensely labeled than the more superficial strata. CGRP positive innervation (arrowheads) appears to be less in PHN skin than in contralateral skin or control skin. E–H) Comparison of polyclonal (E, F) versus monoclonal (G, H) CGRP-IL in sections from the same biopsies from the same subject. Monoclonal CGRP-IL of keratinocytes was far less intense and more heterogenous than polyclonal labeling, but was still more intense and widespread among keratinocytes in the PHN (H) versus the contralateral skin (G). Because of the higher camera sensitivity setting, innervation labeled with the monoclonal antibody appears more intense than that with the polyclonal antibody (arrowheads). I–L) Examples of polyclonal CGRP-IL among keratinocytes from the hypothenar (I, J) and lateral foot (K, L) of control subjects (I, K) and subjects with severe CRPS type 1 (J, L). CGRP-IL among keratinocytes has a very low intensity in control skin and a moderate to high intensity among CRPS skin. In the CRPS skin, there are fewer and more weakly CGRP-IL detectable axons and endings in the upper dermis and epidermis than in control skin (arrowheads). Scale bar = 65µm in A–H, 50µm in I–L. M, N) Similar to other published reports, PGP-IL of epidermal endings from healthy controls (M) and the PHN patient cohort (N) reveals decreased epidermal nerve fiber density among the active PHN and ipsilateral sites compared with a mirror-image contralateral site, in 4 of the 5 PHN subjects, whereas healthy controls showed no lateral differences.

A–D) CFA hindpaw injection time course, depicting in each vertical panel, representative double label (CGRP/PGP) results from glabrous pad crown (A–D), dermal vasculature (‘), and dermal nerve (‘’), with each separate channel split below or to the left of the double label image. A) Control uninjected rats depicting CGRP-IL of a subset of innervation (PGP-IL, green) among the epidermal endings and subepidermal plexus (yellow arrows). The separate channels are split below the double labeled images. Epidermal keratinocyte CGRP-IL (red chevrons) appeared heterogeneous and weak among the volar pad crown. A’) Neural innervation to dermal vasculature contained a mix of CGRP-IL-positive and CGRP-IL-negative axons among the tunica media and advential layers. A”) Dermal nerves contained a rich contingent of CGRP-IL axons. B) By 2 hrs after CFA injection, increased CGRP expression is apparent among epidermal keratinocytes, with little change among the innervation of the epidermis, subepidermal plexus, dermal vasculature (B’), or in the composition of dermal nerves (B”). C) By 3 days after CFA injection, epidermal CGRP expression is increased among suprabasal keratinocytes, while neural innervation appears depleted (i.e., lack of CGRP or PGP axon staining) from the epidermis and subepidermal plexus. C’) Interestingly, among the dermal vasculature, a dramatic loss of CGRP immunopositive medial and adventitial innervation is observed, although PGP immunopositive axons are still present. Dermal nerves appear distraught with ragged PGP labeling and limited CGRP axons present (C”). D) By 14 days following CFA, epidermal CGRP expression remains elevated among the most suprabasal keratinocytes, and immunopositive axons are absent from the epidermis or subepidermal plexus. D’) Dermal vasculature appeared without CGRP immunopositive innervation among the medial and adventitial layers, although PGP immunopositive axons were still present, and dermal nerves remained distraught in appearance with limited CGRP axons (D”). Magnification bar (A) =50µm for A–D, and =25µm for (‘) and (‘’). Anti-CGRP (guinea pig polyclonal, 1:800; Peninsula Labs, San Carlos, CA) and anti-PGP (rabbit polyclonal, 1:1000; UltraClone, Wellow, UK) were used.

A–E) CGRP-IL is increased among palmar glabrous skin keratinocytes from SIV-infected animals compared with control epidermis. A) Control monkey epidermal primary ridge depicting CGRP-IL among fine delicate innervation of the epidermis, and upper dermal compartments (downward and upward arrows, respectively). B–E) Compared with the control monkey, all monkeys infected with SIV had increased CGRP-IR, although the intensity varied between monkeys. B) As seen in an aged monkey with AIDS and severe SIV encephalitis (SIVE), moderate CGRP-IL is detected among the epidermal keratinocytes. C) This young adult monkey had no AIDS defining illness, but did show abundant systemic viral replication and had severe colitis at the time of sacrifice. Dramatic increases in CGRP-IL of keratinocytes can be seen. D) This represents a similarly young adult monkey with SIV infection, with the most subtle increase in epidermal keratinocyte CGRP-IL observed. In this animal, CGRP-IL among keratinocytes was only slightly higher compared with the control or other SIV animals, and CGRP-IL innervation was observed (arrows). E) This represents another young adult SIV infected animal, demonstrating robust CGRP-IL of epidermal keratinocytes, and CGRP-IL among upper dermal nerves (arrow). Magnification bar (A) =50µm for A–E. Anti-CGRP (guinea pig polyclonal, 1:800; Peninsula Labs, San Carlos, CA) was used.

Utilizing transgenic mice which over-produce noggin or BMP4 driven off of the K14 promoter, hindpaw skin was removed and immunolabeled for CGRP. A,B) Noggin over-expression results in intense CGRP-IL in keratinocytes among the volar pad crown (A) and flat (B) glabrous hindpaw skin, appearing more extensive throughout suprabasal keratinocytes. C,D) BMP4 over-expression results in little CGRP-IL in keratinocytes among the volar pad crown (E) and flat (F) glabrous hindpaw skin. The BMP4 keratinocyte expression appeared heterogeneous and similar to that observed in control rats (see Fig. 2C, I). Note that CGRP immunolabeling on nerve fibers was present in the skin of both noggin and BMP4 over-expressing mice, although a prior study detected differences among subtypes of CGRP-positive fibers. Magnification bar =50µm. Anti-CGRP (rabbit polyclonal, 1:1000; Chemicon Inc, Temecula, CA) was used.

Nocicieptive behaviors were induced in rats by SNL (A) or sciatic CCI (B), and following SNL (C–H) or sciatic CCI (I–P), CGRP-IL increases are evident in hindpaw epidermal keratinocytes (red chevrons), with a reduction in peptidergic cutaneous innervation compared to control skin (yellow and red arrows). A) Graph depicting the extent of chronic neuropathic mechanical allodynia (Von Frey filaments) following SNL, demonstrating persistence out to at least 60 days, at a time corresponding to increased CGRP-IL among the keratinocytes. B) Graph depicting the extent of chronic neuropathic thermal hyperalgesia (Hargreaves test) following CCI, demonstrating persistence out to 14 days, at a time corresponding to increased CGRP-IL among the keratinocytes. C–P) Representative photomicrographs of single or double (merged) label immunocytochemistry for calcitonin-gene related peptide (CGRP, red) or 200kD neurofilament (NF, red) and protein gene product 9.5 (PGP, green). Previous studies have shown that anti-PGP labels all types of innervation, whereas 200kD NF immunolabeling is normally expressed in, and limited to, thick caliber Aβ fibers and thin caliber Aδ fibers. C fibers normally lack NF-IL. CGRP-IL is normally observed in subsets of C and Aδ fibers. C,D) Control, unoperated rats show intense CGRP-IL of axonal innervation (PGP positive, double labeled structures) among the epidermal endings, dermal vasculature endings, and subepidermal and dermal fibers (yellow arrows). Epidermal keratinocyte CGRP-IL (red chevrons) is heterogeneous and moderate among the volar pad crown (C) and flat (D) glabrous hindpaw skin, and remains largely suprabasal. E,F) By 30 days following SNL, a near complete loss of neural innervation is observed (i.e., no PGP nerve labeling; small green arrow in F shows a Langerhans cell, which are known to express PGP), while epidermal keratinocyte CGRP expression appears more extensive throughout suprabasal keratinocytes among the pad crown (E) and flat (F) glabrous hindpaw skin (red chevrons). G, H) By 60 days following SNL, neural innervation has begun to return (PGP axons, green arrows), but largely without a CGRP contingent, whereas the epidermal keratinocyte expression of CGRP remains elevated among pad crown (G) and flat (H) glabrous hindpaw skin (red chevrons). I–L) Sham-operated, non-CCI rats have epidermal keratinocyte expression of CGRP that is heterogeneous and weak among the volar pad crown (I, red chevrons), with intense CGRP immunolabeling of a subset of PGP-positive axonal innervation (I, red arrows; J, PGP positive, red arrows) among the epidermal endings (downward red arrows), and upper dermal fibers (upward red arrows), and within dermal nerves (K, red panel). NF content within the dermal nerves was extensively distributed among mostly the larger caliber axons and some small caliber axons (L, red panel). M–P) CCI rats showed dramatically elevated expression of CGRP among the suprabasal keratinocytes (M, red chevrons), coincident with depleted CGRP-IL epidermal endings and dermal nerve fibers, among an overall depleted innervation (N, PGP positive, red arrows). Interestingly, CGRP content appears to be increased within deep dermal nerves (O, red panel), but was mostly distributed among vacuolated cells affiliated with the larger caliber axons. NF-IL is severely depleted among the large caliber axons within the deep nerves, indicating that these axons may be deteriorating and that the vacuolated cells may be reactive Schwann cells (P, red panel). Magnification bar (D) =50µm for C–H, and I,J,M,N; (K)=25µm for K,L,O,P.

Real time quantitative PCR was performed using primers specific to CGRPα, CGRPβ, BMP4, noggin, and members of the CGRP receptor complex, CRLR, RAMP1, and RCP. A–C) Data is presented as relative mRNA levels, normalized to the reference gene GAPDH. A) Epidermal glabrous skin compartments were obtained by laser capture microdissection from BMP4 or noggin over-expression transgenic mice. Total mRNA was obtained from a pool of 5–7 etched epidermis’ per sample. Transgenic over-expression of noggin results in significantly greater expression of CGRPα and CGRPβ mRNA compared with the BMP4 over-expressing transgenic mice (*). As well, the CGRPβ isoform is expressed in significantly greater amounts (~3.5-fold) than the CGRPα isoform among both lines of transgenic mice (#). B) Normal human epidermal keratinocytes (NHEK) were cultured with standard media (undiff), or calcium differentiated with 2M CaCl₂ (diff), and primary human keratinocytes were grown as a 3D raft culture at the air interface, allowing for stratification and formation of an oragnotypic psudoepidermis (3D raft). Keratinocyte differentiation significantly increases the production of both CGRP mRNA isoforms compared with undifferentiated NHEK cells (*). Importantly, under all three keratinocyte growth conditions, data demonstrates that the CGRPβ isoform is expressed in undifferentiated NHEK (10.4-fold increase), differentiated NHEK (8.0-fold increase), and 3D raft cultures (12.3 fold increase), to a significantly greater degree than the CGRPα isoform (#). C) Under all keratinocyte growth conditions, the complete receptor complex components specific for CGRP were detected. For A–C, * and # p<0.05 by Student’s t-test.

Total protein was isolated from primary and immortalized mouse keratinocytes (MK116), three-dimensional cultured human epidermal keratinocytes (3D raft cultures), cultured mouse fibroblasts (FB), and mouse trigeminal ganglia (TG). Western blot analysis was performed to determine CGRP protein content. A) Duplicate protein samples from primary mouse keratinocytes were loaded at 20, 40, and 60 µg/lane, along with CGRPα positive control lanes, separated, transferred to PDVF nylon membranes and probed with anti-CGRP (Rb polyclonal, 1:2000; Chemicon Inc, Temecula, CA). A low level detectable band was observed from all samples at the predicted weight of CGRP precursor protein (16kDa; arrows). The positive control CGRPα was detected at the processed size (~3.7kD), however, among the in vitro cultures, no mature peptide was detectable. B) Total protein samples from mouse fibroblasts (FB, as negative control), immortalized mouse keratinocytes (MK116), and mouse trigeminal ganglia (TG, as positive control) were loaded (20 µg/lane), separated, transferred to PDVF nylon membranes and probed with anti-CGRP (Rb polyclonal, 1:2000; Chemicon Inc, Temecula, CA). A detectable band corresponding to the predicted molecular weight of the CGRP precursor protein (16kD; arrows) was observed in the keratinocyte and trigeminal ganglia lanes, but was not detectable in the fibroblast lane. C) Total protein samples from organotypic human keratinocytes (3D raft cultures) were loaded, along with CGRPα positive control, separated, transferred to PDVF nylon membranes and probed with anti-CGRP (Rb polyclonal, 1:2000; Chemicon Inc, Temecula, CA). A detectable band corresponding to the predicted molecular weight of the CGRP precursor protein (16kD; arrows) was observed in all lanes. D) Total protein samples from mouse trigeminal ganglia (TG, positive control) and mouse fibroblasts (FB, negative control) were loaded (20 µg/lane), along with CGRPα (1µg) positive control, in duplicate, separated, transferred to PDVF nylon membranes, and the membrane then separated. One half of the membrane (left) was then probed with anti-CGRP (Rb polyclonal, 1:2000; Chemicon Inc, Temecula, CA), while the other half (right) was probed with pre-adsorbed anti-CGRP antibody. Mature CGRP (~3.7kD) and CGRP precursor protein (16kD; arrows) bands were detected on the left membrane, and no bands were detectible on the right membrane after specific primary antibody pre-adsorption.