Human Itch Models, with Special Emphasis on Itch in SLS-Inflamed and Normal Skin

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acetylcholine, and other itch inducers have been used to induce itch in AE

patients and healthy volunteers in the same studies (7,10,12). One study

included both AE patients suffering from acute eczema and AE patients

during a symptom-free period (12).

However, comparison between patients and healthy volunteers is problematic when assessing the influence of inflammation on experimentally

induced itch, because the two groups do not match each other. Differences

in itch sensation between the groups could be attributed to factors other than

the inflamed skin (e.g., cerebral mechanisms, or differences in Baseline State).

Typically, itch is scored by using a 100-mm visual analog scale (VAS)

(12–14). Both the time interval until itch is perceived (itch latency) (8,15,16)

and itch threshold (i.e., the lowest substance concentration eliciting itch)

(17,18) are measured. Itch duration (7,8,16), itch magnitude (16), and a

combination of these, namely the Total itch index, Tii (or area under the

curve, AUC), are often quantified.

Scratch intensity has also been used as a more objective measure of itch

sensation (19–23). However, itch is subjective in nature and scratching can

only be a surrogate of self-grading (24).

Associated skin symptoms such as wheal (cutaneous edema) and flare

areas are often quantified in itch studies (25–27). Ultrafiltration from the

postcapillary venules creates the wheal (28), while flare is a vasodilatation

resulting from a local axonal skin reflex (28,29). The C-fiber-mediated

responses (flare and itch) often correlate (28,30), while the exclusively

vascular wheal typically does not correlate with the two others (28,31). The

area of itchy skin can be measured in itch models (32). Alloknesis (or itchy

skin) means another sensation, and itch is induced by touching the surrounding area of, e.g., an insect bite (33–35). The area of alloknesis can be

measured. Changes in skin blood flow have also been quantified in itch

studies by laser Doppler flowmetry (10,12).



II.



THE SLS-INFLAMED SKIN MODEL FOR PRURITUS

IN HUMANS



Instead of mimicking inflamed skin seeking for new itch potentiators in a twomediator system, we aimed to establish an itch model in humans comprising

both normal and experimentally inflamed skin, using volunteers as their own

controls (36).

The skin of five selected test sites on one volar forearm was pretreated

for 24 hr with large Finn Chambers containing 1% sodium lauryl sulfate

(SLS) used as a standard contact irritant to induce inflammation.



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After 24 hr, the skin is inflamed, and pruritogenic mediators can be

intradermally injected into inflamed skin (Fig. 1) and normal skin. Different

pruritic substances can be used in the range 10–20 AL, and histamine and

saline can be used as positive and negative control substances. All substances

are intradermally injected into the inflamed test sites, and also into corresponding nontreated sites on the opposite forearm. After injections, the test

individuals score itch intensity on a visual analog scale (VAS) for 20 min, and

wheal area can then be measured. Flare areas are measured after 5 min (37).

In this design, itch can be induced in both normal and inflamed skin at

the same time in the same volunteer. Because volunteers serve as their own

controls, the influence of skin inflammation on different itch mediators and

antipruritics can be directly assessed. When establishing the model, we could

not demonstrate differences in pruritic potential between mediators injected

in inflamed and normal skin, because itch was neither significantly improved



Figure 1 Injection of pruritic substances in sodium lauryl sulfate–inflamed skin.

Extra large Finn Chambers (containing 150 AL of 1% sodium lauryl sulfate) were

applied to the forearms for 24 h to induce inflammation. Thereafter 10 AL or 20 AL

of a pruritic substance was injected in the center of the inflamed skin.



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nor significantly decreased in inflamed skin compared with normal skin (36).

However, after histamine, the wheal area was much ( p<0.001) larger in the

inflamed skin when compared with the normal skin.

The SLS-inflamed skin is closer to a clinically relevant situation compared with the noninflamed skin, which is used in conventional itch models.

The question of how inflammation affects the itch response is of major

scientific and clinical interest. Potential antipruritic substances might work

differently in the two situations. Furthermore, the perturbed skin induced by

SLS, simulating diseased skin with barrier disruption, may be more sensitive

in testing new topical antipruritics simply as a result of better skin penetration

of test drugs (38). Thus it is also relevant to determine the effect of a

potentially new antipruritic in both inflamed and normal skin, to mimic the

skin pharmacology of diseased skin.

Using this new model, pain should always be scored together with itch.

Pain can be scored to distinguish this sensation from itch. However, pain is

furthermore able to modify itch sensation, and the inclusion of pain scores is

becoming increasingly important in experimental itch studies.

This new itch model in humans is important for testing of itch involving

both normal and inflamed skin. The model has a potential in the evaluation of

new topical and systemic treatments of itch.



III.



EXAMPLE OF DRUG TESTING BY USING

THE SLS-INFLAMED SKIN MODEL FOR

PRURITUS IN HUMANS



In 1997, Yosipovitch et al. (13) reported that topically applied aspirin (acetylsalicylic acid) rapidly decreases histamine-induced itch in humans.

We wanted to confirm this observation, and further study the antipruritic ability of topical aspirin in inflamed skin by using the abovedescribed model (37).

After the inducement of inflamed skin with SLS, aspirin 10%, aspirin

1%, mepyramine 5%, and vehicle were applied to the inflamed and corresponding noninflamed areas 20 min before itch induction with intradermal

histamine injection. Itch and pain were scored on a visual analog scale at

regular intervals. Wheal and flare areas were measured.

No difference in itch intensities was found after the application of test

substances. Similar to Yosipovitch et al., we found that itch duration was

shorter after aspirin application compared to vehicle. However, more itch was

induced when aspirin was applied to inflamed skin compared with normal

skin. In both skin types, wheals and flares were smaller after application of



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143



aspirin. We concluded that despite a significant skin penetration, as measured

by the influence on wheal and flare reactions, topically applied aspirin did not

decrease histamine-induced itch in the model used. Thus our results are both

in accordance, and also in contrast, with the results obtained by Yosipovitch

et al. (13).



REFERENCES

1.

2.



3.

4.

5.



6.

7.

8.

9.



10.



11.

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13.

14.



15.



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1973; 3:608–609.

Fjellner B, Hagermark O. Potentiation of histamine-induced itch and flare

responses in human skin by the enkephalin analogue FK-33-824, beta-endorphin

and morphine. Arch Dermatol Res 1982; 274:29–37.

Wallengren J. The pathophysiology of itch. Eur J Dermatol 1993; 3:643–647.

Lovell CR, et al. Prostaglandins and pruritus. Br J Dermatol 1976; 94:273–275.

¨

Hagermark O, Strandberg K, Hamberg M. Potentiation of itch and flare

¨

responses in human skin by prostaglandins E2 and H2 and a prostaglandin

endoperoxide analog. J Invest Dermatol 1977; 69:527–530.

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Fjellner B, Hagermark O. Pruritus in polycythemia vera: treatment with aspirin

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and possibility of platelet involvement. Acta Derm-Venereol 1979; 59:505–512.

Hagermark O, Rajka G, Bergvist U. Experimental itch in human skin elicited by

rat mast cell chymase. Acta Derm-Venereol 1972; 52:125–128.

Rajka G. Experimental pruritus in the unaffected skin of patients with different

itching dermatoses. Acta Derm-Venereol 1970; 50:270–272.

Hagermark O, Strandberg K. Comparison of the antihistaminic effects in skin of

a tertiary (promethazine) and a quarternary phenothiazine (N-hydroxyethylpromethazine). Acta Allergol 1974; 29:462–468.

Heyer G, Hornstein OP, Handwerker HO. Skin reactions and itch sensation

induced by epicutaneous histamine application in atopic dermatitis and controls.

J Invest Dermatol 1989; 93:492–496.

Groene D, Martus P, Heyer G. Doxepin affects acetylcholine induced cutaneous

reactions in atopic eczema. Exp Dermatol 2001; 10:110–117.

Rukwied R, Heyer G. Cutaneous reactions and sensations after intracutaneous

injection of vasoactive intestinal polypeptide and acetylcholine in atopic eczema

patients and healthy controls. Arch Dermatol Res 1998; 290:198–204.

Yosipovitch G, et al. Topically applied aspirin rapidly decreases histamineinduced itch. Acta Derm-Venereol 1997; 77:46–48.

Darsow U, et al. Skin testing of the pruritogenic activity of histamine and

cytokines (interleukin-2 and tumour necrosis factor-alpha) at the dermalepidermal junction. Br J Dermatol 1997; 137:415–417.

¨

Hagermark O. Influence of antihistamines, sedatives, and aspirin on exper¨

imental itch. Acta Derm-Venereol 1973; 53:363–368.



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16. Simone DA, Alreja M, LaMotte RH. Psychophysical studies of the itch sensation

and itchy skin (alloknesis) produced by intracutaneous injection of histamine.

Somatosens Motor Res 1991; 8:271–279.

17. Shuttleworth D, et al. Relief of experimentally induced pruritus with a novel

eutectic mixture of local anaesthetic agents. Br J Dermatol 1988; 119:535–540.

18. Davies MG, et al. The efficacy of histamine antagonists as antipruritics in

experimentally induced pruritus. Arch Dermatol Res 1979; 266:117–120.

19. Ebata T, et al. The characteristics of nocturnal scratching in adults with atopic

dermatitis. Br J Dermatol 1999; 141:82–86.

20. Endo K, et al. Evaluation of scratch movements by a new scratch-monitor to

analyze nocturnal itching in atopic dermatitis. Acta Derm-Venereol 1997; 77:432–

435.

21. Ebata T, et al. Use of a wrist activity monitor for the measurement of nocturnal

scratching in patients with atopic dermatitis. Br J Dermatol 2001; 144:305–

309.

22. Rees JL, Laidlaw A. Pruritus: more scratch than itch. Clin Exp Dermatol 1999;

24:490–493.

23. Daly BM, Shuster S. Effect of aspirin on pruritus. Br Med J (Clin Res Ed) 1986;

293:907.

24. Wahlgren CF. Measurement of itch. Semin Dermatol 1995; 14:277–284.

25. Weisshaar E, et al. Effect of topical capsaicin on the cutaneous reactions and

itching to histamine in atopic eczema compared to healthy skin. Arch Dermatol

Res 1998; 290:306–311.

26. Jorizzo JL, et al. Vascular responses of human skin to injection of substance P

and mechanism of action. Eur J Pharmacol 1983; 87:67–76.

27. Fuller RW, et al. Sensory neuropeptide effects in human skin. Br J Pharmacol

1987; 92:781–788.

28. Darsow U, et al. Correlations between histamine-induced wheal, flare and itch.

Arch Dermatol Res 1996; 288:436–441.

29. Schmelz M, Petersen LJ. Neurogenic inflammation in human and rodent skin.

News Physiol Sci 2001; 16:33–37.

30. Bromm B, et al. Effects of menthol and cold on histamine-induced itch and skin

reactions in man. Neurosci Lett 1995; 187:157–160.

31. Lischetzki G, et al. Nociceptor activation and protein extravasation induced by

inflammatory mediators in human skin. Eur J Pain 2001; 5:49–57.

32. Heyer G, et al. Histamine-induced itch and alloknesis (itchy skin) in atopic

eczema patients and controls. Acta Derm-Venereol 1995; 75:348–352.

33. Weisshaar E, et al. Experimentally induced pruritus and cutaneous reactions

with topical antihistamine and local analgesics in atopic eczema. Skin Pharmacol

1997; 10:183–190.

34. Weisshaar E, Ziethen B, Gollnick H. Can a serotonin type 3 (5-HT3) receptor

antagonist reduce experimentally-induced itch? Inflamm Res 1997; 46:412–416.

35. Heyer G, et al. Opiate and H1 antagonist effects on histamine induced pruritus

and alloknesis. Pain 1997; 73:239–243.

36. Thomsen JS, et al. Experimental itch in sodium lauryl sulphate-inflamed and



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normal skin in humans: a randomized, double-blind, placebo-controlled study of

histamine and other inducers of itch. Br J Dermatol 2002; 146:792–800.

´

37. Thomsen JS, Jensen SB, Benfeldt E, Serup J, Menne T. Topically applied aspirin

decreases histamine-induced wheal and flare reactions in SLS inflamed and normal skin, but does not decrease itch. A randomized, double-blind and placebocontrolled human study. Acta Derm Venereol 2002; 82:30–35.

38. Benfeldt E, Serup J, Menne T. Effect of barrier perturbation on cutaneous

salicylic acid penetration in human skin: in vivo pharmacokinetics using

microdialysis and non-invasive quantification of barrier function. Br J Dermatol

1999; 140:739–748.



15

Microdialysis in Itch Research

Martin Schmelz

University of Heidelberg, Mannheim, Germany



Intradermal microdialysis has been used successfully as an elegant tool to

study the interaction of mediators, nociceptors, inflammatory cells, and vasculature in human skin in vivo. The main advantage of this technique is given

by the combination of atraumatic delivery of exogenous mediators and

analysis of released endogenous mediators with noninvasive techniques to

assess the vascular responses and psychophysical methods to measure quality and intensity of sensation.



I.



TECHNIQUE



Microdialysis is a minimally invasive technique that was originally developed for use in the central nervous system (1), but has also been adapted for

dermal use (2). A semipermeable capillary is inserted into the tissue of interest and is perfused at a constant flow rate of a few microliters per minute.

According to the concentration gradient, mediators will diffuse from the

tissue into the lumen of the capillary and can then be analyzed in the dialysate, which is collected after the tissue passage. If the perfusing medium

contains substances at a concentration exceeding the tissue concentration,

they will be delivered into the tissue by diffusion. The molecular cutoff of the

capillaries is chosen according to the size of the molecules of interest. For

small molecules like histamine, a molecular cutoff of 5 kDa has been

147



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Schmelz



successfully used, whereas for large proteins like mast cell tryptase, a

molecular cutoff of 3 MDa is required.

A.



Measurement of Local Mediator Concentration



Microdialysis has been used to assess local histamine concentrations in

human skin under control conditions (3–5). However, the main results in

the arena of itch research were gained by measuring the histamine release

in response to exogenous mediators. A multitude of studies has been conducted to assess the histamine concentrations in response to various mediators in controls and patients (2,6–21). Studies investigating the role of

neuropeptides and mast cells for itch will be dealt with in detail below.

Histamine release could be assessed in parallel to the itch sensation in type 1

allergy and urticaria (17,22,23). These results mainly confirm the role of

mast cell-derived histamine in these conditions.

The use of high-molecular cutoff membranes has enabled the analysis

of macromolecules with microdialysis. Thereby, cell activation markers like

mast cell tryptase (6), eosinophilic cationic protein (ECP), and myeloperoxidase (24) could be measured. In the later study, cell activation markers

were analyzed during the development of a delayed type 1 reaction following

allergen prick for 7 hr. The increase of ECP matched the histologically observed infiltration by eosinophil granulocytes. Rather than providing data

on single mediators, these studies illustrate that microdialysis can be successfully employed to investigate the complex interactions of various inflammatory cells in vivo.

B.



Simultaneous Assessment of Biological Responses



The use of microdialysis can be largely improved by the combination with

noninvasive techniques to assess the biological response of the tissue. In

Figure 1, the experimental setup of a standard microdialysis study is shown.

As described above, stimulatory mediators or their antagonists can be

delivered via the capillary, while, simultaneously, the released endogenous

mediators are measured in the dialysate. In addition, the vascular response,

which might result directly from a local effect of the applied mediator, from

secondary local release, or from activation of nociceptors and subsequent

release of vasodilatory neuropeptides in their innervation territory (axon

reflex), can be assessed by laser Doppler imaging. It should be noted that

diffusion in the tissue is very limited; thus the local vasodilatation is

restricted to the immediate vicinity of the membrane. Even for the small

and water-soluble histamine, molecule diffusion in the skin is restricted to

less than 3 mm (16). In contrast, the axon reflex erythema can spread for



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149



Figure 1 Schematic illustration of the experimental setup. The muscle relaxant was

delivered by diffusion via plasmapheresis hollow fibers inserted intracutaneously, causing mast cell degranulation. Mediator release, vascular reactions, and sensory effects

were determined.



several centimeters from the stimulation site, as maximum diameters of

chemonociceptors in human skin have been found to be 9 cm (25). As the

mechanisms of local and axon reflex vasodilatation are different, they have

to be analyzed separately. As an example, mast cell degranulation by the

muscle relaxant rocuronium is shown. At lower concentrations, the muscle

relaxant induces local vasodilatation without mast cell degranulation and

nociceptor activation; accordingly, no axon reflex erythema can be assessed.

At higher concentrations, rocuronium directly activates nociceptors and

additionally degranulates mast cells; thus, in addition to local vasodilatation,

an axon reflex erythema is provoked (Fig. 2).

Provocation of an axon reflex erythema indicates the activation of

nociceptors; however, these nociceptors could be involved in pain or in itch

processing. Therefore, psychophysical assessment of quality and intensity of

the induced sensation is necessary for the interpretation. The confirmation

of mast cell degranulation by a mediator does not suffice to prove its pruritic

effect. As shown in Figure 3, the application of two different muscle relaxants may induce either pain or itch, although their mast cell degranulating effect is virtually identical as can be judged from the dose–response curve

for histamine release and protein extravasation (26) (Fig. 3). While rapa-



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Figure 2 Local vasodilatation (upper panels) and axon reflex vasodilatation (lower

panels) as assessed by laser Doppler imaging are shown in response to intraprobe

delivery of different concentrations of rocuronium. Note that the nonneurogenic

vasodilatation is restricted to the stimulatory membrane (upper left panel), whereas

the axon reflex erythema spreads several centimeters (lower left panel). (Modified

from Ref. 26.)



curonium causes mast cell degranulation only, rocuronium also directly

activates nociceptors involved in pain processing (27), and thus the pain

sensation suppresses the itch as discussed in Chapter 3.



II.



NEUROPEPTIDES



Neuropeptides, especially substance P (SP), have been implicated in the

mechanism of itch for decades (28–32). There is no doubt that at high concentrations, SP degranulates mast cells by a nonreceptor-mediated mech-



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Figure 3 Dose–response relations for nociceptor activation (itch or pain; upper

panel), histamine release (center panel), and protein extravasation (lower panel) following intraprobe delivery of two different muscle relaxants via intradermal microdialysis fibers are shown. (Modified from Ref. 27.)