When an intradermal injection is correctly administered Which of the following should appear?

Adjuvant arthritis

Intradermal injection of heat-killed M. tuberculosis in rats induces a disease similar in many respects to rheumatoid arthritis. An autoimmune process involving T lymphocytes is responsible for the generation of the disease, and transfer of T cells from arthritic rats to irradiated recipients can cause a transient form of the disease in the latter. One arthritogenic T cell clone was shown to recognize a mycobacterial hsp60 epitope. Also, immunization of mice with mycobacterial hsp60 in immunostimulating complexes (ISCOMS) will induce CD8+ αβ CTLs which cross-react with murine hsp60 and are therefore autoreactive.

8.3.2.4 Intradermal Injection

Intradermal injections are delivered into the outer layers of the dermis, underneath the upper skin layer (the epidermis). This procedure requires that the animal remain very still; anesthesia is often needed so that animals are sufficiently restrained.

Most intradermal injections are aqueous-based compounds that are physiologically buffered to have a neutral pH. If the solution is not buffered, tissue necrosis can occur at the injection site. The dose range per injection site is 50–100 μL. Injections exceeding this range can cause tissue necrosis at the injection site or leakage of the compound out of the site due to pressure. The needle size range is typically 25–30 gauge.

The injection site is prepared by clipping the hair at the injection site. The skin is stretched taut between the technician's thumb and index finger, providing stability to the skin when positioning the needle. The needle is placed, bevel up, on the skin and is gently inserted into the skin between the epidermis and the dermis layers. The needle is advanced just beyond the bevel and the substance is injected slowly. A small bleb or blister will form in the skin, indicating that the needle was properly placed. When removing the needle, allow the skin to stretch over the bleb to prevent loss of the injected material. Blotting or wiping the area should be avoided as that can cause the compound to leak from the injection sites.

Adjuvant Disease

Intradermal injection of complete Freund adjuvant (mineral oil, detergent, and Mycobacterium butyricum) into the footpad of susceptible strains of rats results in the development of a chronic polyarthritis, sometimes accompanied by lesions of the skin and auricular cartilages and an anterior uveitis.91,219,220 This disease is adoptively transferable by T lymphocytes, and the model may be manipulated by T-cell clones capable of inducing, preventing, or ameliorating the disease.221,222 Antibody appears to have no significant role in pathogenesis. The course of adjuvant arthritis may be intermittent or chronic, resulting in tissue destruction with calcification and ankylosis of joints. The arthritogenic component of the adjuvant is a peptidoglycan dimer, muramyl dipeptide.223

Intradermal and subcutaneous injection

Intradermal or subcutaneous injection avoids the barrier presented by the stratum corneum, and entry into the general circulation is limited mainly by the rate of blood flow to the site of injection. However, these sites generally only allow the administration of small volumes of drugs and tend to be used mostly for local effects, such as local anaesthesia, or to deliberately limit the rate of drug absorption (e.g. insulin; see Chapter 40). Subdermal implants are increasingly used for long-term hormonal contraception; the implants are flexible polymer rods or tubes inserted under the skin of the upper arm that slowly release the hormone for up to 3 years, with contraception being reversible by removal of the implant (see Chapter 45).

EPR and LPR in the skin

Intradermal injection of allergen induces a characteristic ‘triple response’ characterized by an almost immediate reddening of the skin (histamine-mediated arteriolar vasodilatation) at the site of allergen injection, which is followed within 5–10 minutes by the development of an area of oedema, or wheal (histamine-mediated increased permeability) (Fig. 1.32). The third component of the triple response is an area of erythema, or flare, around the wheal. This is initiated by the stimulation of histamine receptors on afferent non-myelinated nerves, which results in the release of neuropeptides with consequent vasodilatation and skin erythema. Histamine-induced nerve stimulation also results in itch. The size of the flare is again dose dependent and may measure several centimetres across. The wheal-and-flare generally resolves within about 30 minutes. However, in up to 50% of subjects challenged intradermally with a high dose of allergen the immediate reaction evolves into a late phase reaction characterized by an indurated erythematous inflammatory reaction. The latter reaches a peak at about 6–8 hours and often persists for 24 hours. The reduction in the size of the LPR to intradermal allergen challenge correlates well with the clinical response to subcutaneous allergen immunotherapy in patients with allergic rhinitis.

Intradermal injection

Intradermal (ID) injection is not a route that is commonly used for toxicity studies. It may be appropriate to test products intended for ID administration to humans by that same route in mice, and studies of limited duration are technically feasible. The ID route offers the advantage of slow absorption owing to the poor vascular perfusion of the skin relative to tissues in other areas of potential administration. This slow absorption is typically associated with longer time to onset of effects, lower peak plasma levels, but more sustained effects than routes that result in faster absorption. To the extent that the test article may be metabolized by the skin, the ID route would be expected to offer greater opportunity for such metabolism than subcutaneous injection, but less than with topical administration. Injected volume for ID dosing should be limited to about 1 mL/kg per injection site or less, with smaller volumes preferred if repeated doses will be administered. It is acceptable to administer ID doses at multiple sites simultaneously if higher total doses are required. Irritatant formulations of the test article must be avoided, especially if multiple doses will be administered, as ulceration and necrosis of the skin can result.

B Local Application

Intradermal injection in the rat of 10−9 mol of IL-8 or PAF induced emigration of neutrophils from small vessels as early as 10 min after application, whereas 10−13 mol of endotoxin had no effect (Fig. 1). Within 30 min after IL-8 application there was a distinct intraluminar, vessel wall, and perivascular accumulation of neutrophils, especially in and around venules in the lower dermis (Fig. 2a). A striking finding was the presence of massive neutrophil aggregates between the endothelial and smooth muscle layers of the vessel wall. At the same time point, PAF caused a milder and more diffuse perivascular accumulation of neutrophils, whereas endotoxin and PFS did not cause any significant inflammatory reaction (Figs. 1 and 3). One hour after application of IL-8 there was still a high number of neutrophils in the vessel walls (Fig. 2b) and an increasing number in perivascular spaces. The intensity of the neutrophilic infiltrate was much more pronounced after injection of IL-8 than after injection of PAF or endotoxin. Neutrophil infiltration peaked at 4 hr after injection of IL-8 and PAF. Similar to the 1-hr time point, at 4 hr the infiltration score of IL-8 sites was distinctly higher than the scores for PAF or endotoxin sites. At this time point, IL-8 caused a massive neutrophilic infiltration of the upper and lower dermis with areas of diffuse distribution of neutrophils (Fig. 2c) and areas with dense, microabscess-like neutrophil accumulation, mainly around venules in the deep dermis. Focally the inflammatory infiltrate extended into the panniculus carnosus. After 8 hr and especially after 16 hr a decrease in the number of neutrophils in the inflammatory infiltrate could be observed, except for the endotoxin-treated sites, where the number remained comparable to the 4-hr value. The neutrophils in the IL-8-treated sites demonstrated signs of degeneration such as cell swelling, pycnosis, karyorrhexis, and karyolysis (Fig. 2d). The inflammatory score of IL-8-treated sites decreased steadily over the next 12 hr, and was back to the levels of the untreated sites at the end of the observation period (Fig. 3).

We also compared the activity of different doses of IL-8,. NAP-2, and GRO-α 4 hr after intradermal injection into rats. All three substances induced a dose-dependent accumulation of neutrophils. However, the magnitude of the inflammatory reaction was different with the three inflammatory pep-tides tested. GRO-α induced an appreciable intradermal neutrophil accumulation at 10−11 mol/site, whereas at 10−10 and especially 10−9 mol/site it caused an extremely strong inflammatory reaction (Figs. 4b and 5). IL-8 induced a slightly weaker but still substantial inflammatory reaction (Fig. 5), whereas NAP-2 elicited a much lower number of neutrophils than the other two agonists (Fig. 5).

DTH REACTION

Intradermal injection of soluble proteins from an intracellular bacterium causes local infiltration of specific T lymphocytes and MP in individuals immune to the same pathogen. The reaction generally reaches its maximum between 40 and 70 hours after antigen application. This DTH reaction has been used successfully for the diagnosis of infections with various intracellular bacteria, particularly M. tuberculosis/M. bovis and M. leprae, because of the difficulty in isolating such microbes from internal organs, where they persist without causing clinical disease: It should be emphasized that a positive DTH response directly reflects the existence of a specific T-cell response and only indirectly indicates microbial existence in the host, and is by far no conclusive indication of a protective immune status. The DTH response is mediated by CD4 αβ T cells, with recent evidence suggesting a vital role for TNF-α in the induction of chemokines. Because soluble antigens are processed inefficiently, if at all, through the MHC class I pathway, CD8 T cells, which are important elements of protective immunity, are not challenged by DTH antigens. Thus, the DTH response reflects an important arm, but not the full armamentarium, of protective immunity against intracellular bacteria. As a further complication, the DTH antigen mixture may miss antigens relevant to protection. According to the stage of disease, different ratios of secreted versus somatic antigens may be required. Whereas secreted antigens may be particularly important during persistent infection, somatic antigens may become more important once bacteria are efficiently destroyed by activated macrophages.

Although DTH responses are generally positive in immune but nondiseased individuals, they are frequently absent during full-blown disease, such as miliary tuberculosis and lepromatous leprosy. The reasons for this anergy are incompletely understood but appear to involve immunosuppression, the absence of appropriate antigens in the DTH reagent, the accumulation of specific T cells at the site of disease manifestation, and their concomitant absence from the periphery.

5.09.5 Alloknesis (Itchy Skin) and Sensitization

Intradermal injection of histamine elicits itch sensation, a wheal at the injection site, and a surrounding flare reaction due to histamine-evoked release of vasodilatory peptides (substance P (SP), calcitonin gene-related peptide). Light stroking in a broad area of normal skin surrounding the histamine injection can elicit itch, a phenomenon called itchy skin or alloknesis (Simone, D. A. et al., 1991). The spread of alloknesis was prevented by local anesthesic block adjacent to the site of histamine injection, indicating that alloknesis is of neurogenic origin (Simone, D. A. et al., 1991). Noxious punctuate stimulation in and around the region of alloknesis also elicits itch (hyperknesis) (Atanassoff, P. G. et al., 1999).

The chronic itch of skin conditions such as atopic dermatitis suggests the possibility of peripheral or central sensitization along the itch-signaling pathway (Ikoma, A. et al., 2004). Moreover, noxious stimuli such as acetylcholine, acid, heat, and pinprick, which normally evoke pain, instead elicit itch in atopic dermatitis patients (Vogelsang, M. et al., 1995; Ikoma, A. et al., 2004). A peripheral sensitization of pruriceptors is suggested by microneurographic recordings from an atopic dermatitis patient, revealing a subset of mechanically insensitive C-fiber afferents that exhibited irregular patterns of spontaneous activity; two of these were activated by pruritic stimulation (Schmelz, M. et al., 2003a). Since mechanically insensitive, histamine-responsive C-fibers normally do not exhibit any spontaneous activity (Schmelz, M. et al., 1997), their increased firing in atopic dermatitis may represent sensitization that contributes to spontaneous itch. Central sensitization is also likely to contribute to pathological itch (Ikoma, A. et al., 2004). Central sensitization of pain is mediated partly via excessive excitation of spinal neurons by nociceptors releasing glutamate and SP, which act at postsynaptic NMDA and neurokinin-1 receptors to initiate Ca2+-dependent intracellular events resulting in enhanced neuronal excitability (Ji, R. R. et al., 2003). Central sensitization is associated with hyperalgesia (increased pain), allodynia (pain from a normally nonpainful stimulus), and expansion of mechanosensitive receptive fields. It is conceivable that conditions associated with chronic itch may induce central sensitization of itch-signaling neurons via mechanisms similar to those for central sensitization of pain (Ikoma, A. et al., 2004).

Sudomotor Axon Reflex Testing

The intradermal injection of acetylcholine (5 to 10 mg) causes local sweating and piloerection if the postganglionic sympathetic fibers are intact. This provides a simple, nonquantitative test of sudomotor and pilomotor functions.

Sweat output in response to an axon reflex can be measured directly and accurately in a specially designed chamber placed on the skin. Iontophoresed acetylcholine activates axon terminals, generating impulses that pass antidromically to a branch point and then are conducted orthodromically down another axon to its terminals, where acetylcholine is released, generating a sweat response. Quantitative sudomotor axon reflex testing (QSART) is a sensitive means of assessing postganglionic sympathetic function and yields reproducible results. It requires sophisticated and expensive equipment, however, so that it is used only in a limited number of centers.