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PubMed1999

Possible involvement of matrix metalloproteinase-9 in Langerhans cell migration and maturation.

Epidermal Langerhans cells (LC) are potent dendritic cells in the induction of primary T cell-mediated immune responses in the skin. They capture foreign Ags and migrate to regional lymph nodes to carry and present these Ags to naive T cells. We investigated the role of matrix metalloproteinase-9 (MMP-9) in LC migration using an anti-MMP-9 mAb. Intradermal injection of anti-MMP-9 mAb before rhodamine B or oxazolone painting markedly inhibited these hapten-induced decreases in LC number in the epidermis and the accumulation of dendritic cells in the regional lymph nodes, indicating that MMP-9 plays some important roles in LC migration in the induction phase of contact sensitization. Treatment with anti-MMP-9 mAb also blocked the increase in cell size, dendrite development, and the enhanced expression of MHC class II Ags in LC induced by hapten painting. In addition, intradermal injection of purified MMP-9 induced marked increases in cell size, dendrite extension, and enhanced expression of MHC class II Ags in LC. These results strongly suggested that MMP-9 is involved not only in LC migration, but also in their morphological and phenotypic maturation in the skin.

Animals↗
PubMed2004

Serum antibody responses after intradermal vaccination against influenza.

BACKGROUND: If found to be safe and immunogenic, reduced doses of influenza vaccine given by the intradermal route could increase the number of available doses of vaccine. METHODS: In an open-label study, we randomly assigned 119 subjects to receive an intradermal injection of trivalent inactivated influenza vaccine, containing 6 mug of hemagglutinin for each antigen (40 percent of the usual dose), and 119 to receive an intramuscular injection of the standard dose of 15 mug of hemagglutinin for each antigen. The two groups were subdivided according to age (18 to 60 years and older than 60 years). RESULTS: Among subjects who were 18 to 60 years of age, serum antibody responses were vigorous and did not differ significantly between the intradermal and intramuscular groups, and all subjects had hemagglutination-inhibition (HAI) titers of at least 1:40. Although the subjects who were older than 60 years of age also had a vigorous antibody response, there was a trend toward a better response in the intramuscular route, but this finding was significant only for antigen to the H3N2 strain. Nevertheless, 100 percent of older subjects in the intramuscular group and 93 percent of such subjects in the intradermal group had an HAI antibody titer to the H3N2 strain of more than 1:40, and 100 percent in each group had a titer of this level for both the H1N1 and B strains. Local pain was significantly more common in the intramuscular group than in the intradermal group among subjects who were 18 to 60 years of age but not among subjects who were over 60 years old. Signs of local inflammation were significantly more common among subjects in the intradermal group than among those in the intramuscular group, in both age groups. CONCLUSIONS: As compared with an intramuscular injection of full-dose influenza vaccine, an intradermal injection of a reduced dose resulted in similarly vigorous antibody responses among persons 18 to 60 years of age but not among those over the age of 60 years.

Adolescent↗
PubMed2002

Tumour necrosis factor-alpha-induced migration of human Langerhans cells: the influence of ageing.

BACKGROUND: Langerhans cells (LCs) play essential roles in the initiation and regulation of cutaneous immune responses mediated through their successful migration from the epidermis to draining lymph nodes while carrying antigen. Tumour necrosis factor (TNF)-alpha, a keratinocyte-derived cytokine, has recently been shown to play an important role in the mobilization of LCs from human epidermis. Although it is known that with age the immune system changes, the influence of increasing age on the function of human LCs has not been defined clearly. OBJECTIVES: To examine the influence of age on the ability of TNF-alpha to induce LC migration. METHODS: Ten elderly (six men, four women; mean age 76 years, range 72-79) and 10 young (six men, four women; mean age 23 years, range 18-35) volunteers received intradermal injections of 200 U of human recombinant TNF-alpha diluted in sterile saline, and control injections of sterile saline alone, at each of two paired sites identified on photoprotected buttock skin. Two hours later, paired injection sites were excised by punch biopsy. One set of paired biopsies was processed for assessment of the frequency and morphology of epidermal LCs, following preparation of epidermal sheets and immunofluorescence staining for the LC marker CD1a. The remaining paired biopsies were processed in formalin and the inflammatory response to TNF-alpha was assessed by standard histological examination. RESULTS: Mean +/- SEM baseline values for LC frequency within epidermal sheets were significantly different between young (1156.3 +/- 38.5 cells mm(-2)) and elderly subjects (835.7 +/- 48.2 cells mm(-2); P < 0.01). Intradermal injections of 200 U of TNF-alpha caused a significant reduction in the frequency of LCs in both elderly and young subjects (P < 0.01). However, the extent of TNF-alpha-induced LC migration was substantially different between the two groups, with a mean 9% reduction in LC frequency in elderly volunteers compared with a mean 23% decrease in young subjects. Exposure to TNF-alpha was associated with a perivascular polymorphonuclear infiltrate at 2 h in all young subjects; in contrast, only 50% of the elderly individuals showed evidence of such a response. CONCLUSIONS: There are significant differences between young and old skin with respect to both resting LC numbers and their response to TNF-alpha. These age-related changes in LC frequency and function may contribute to the altered cutaneous immune function observed in the elderly.

Adolescent↗
PubMed1985

Relationship between delayed hypersensitivity response and acquired cell-mediated immunity in C57BL/6J mice infected with Leishmania donovani.

The relationship between the development of acquired cell-mediated immunity and the concomitant level of specific delayed hypersensitivity (DH) in C57BL/6J mice infected with the intracellular protozoan parasite Leishmania donovani was studied. It was determined that the intradermal injection of Leishmania antigen (heat- or phenol-killed flagellated forms of L. donovani) could elicit a DH response as early as day 10 and as late as day 120 postinfection in mice infected by the intravenous route. Dose-response studies showed that there is an inverse relationship between the size of the infecting dose of parasites and the magnitude of the DH response. Thus, increasing the degree of infection depressed the DH response. This depression, however, could be abrogated by treatment of mice with cyclophosphamide (201 mg/kg) before intradermal injection of antigen.

Animals↗
PubMed2006

Pigment epithelial growth factor suppresses inflammation by modulating macrophage activation.

PURPOSE: To study the contribution of murine retinal pigment epithelial (RPE) cells to the innate immune-privilege status of the subretinal space as determined by the ability of pigment epithelial-derived factor (PEDF) and somatostatin (SOM), produced by RPE, to regulate macrophage-mediated inflammation. METHODS: Serum-free medium was added to RPE eyecups (a healthy monolayer of RPE resting on choroid and sclera) and the supernatants were removed after 24 hours (RPE SN). The RPE SN was assayed for the presence of PEDF and SOM and for its ability to regulate interleukin (IL)-12, IL-10, and nitric oxide (NO) production by resting and activated macrophages. A group of mice received intradermal injection of lipopolysaccharide (LPS) and PEDF in one ear and LPS alone in the other ear. Ear thickness was measured before- and 24 hours after ear injections. RESULTS: Soluble factors present in the RPE SN inhibited IL-12 production and substantially increased IL-10 while having minimal effects on NO production by activated macrophages. The message for PEDF, SOM, and IL-10 was detected in RPE cells, and the protein for these factors was found in the RPE SN. The stimulation of IL-10 and suppression of IL-12 production by RPE-SN-treated macrophages was neutralized by anti-PEDF antibodies. Neutralization of SOM in the RPE SN, suppressed NO production by activated macrophages. Intradermal injection of PEDF substantially inhibited LPS-induced inflammatory response. CONCLUSIONS: PEDF inhibits LPS-driven macrophage activation in vitro and in vivo. By producing PEDF, the RPE contributes to innate immune privilege of the eye.

Animals↗
PubMedDate not supplied

A skin test for autonomic neuropathy.

Intradermal injection of 0.5 microgram histamine produced equal skin reactions in normal individuals (n = 15) and in diabetic patients with (n = 10) or without (n = 9) evidence of autonomic neuropathy as well as in patients after lumbar sympathectomy (n = 5). Addition of noradrenaline (0.1 microgram) resulted in a significantly smaller skin reaction (mean +/- SEM) in normal (101.9 +/- 10.1 vs. 136.3 +/- 6.5 mm2, p less than 0.005) and in diabetic patients without autonomic neuropathy (101.8 +/- 8.4 vs. 123.9 +/- 8.7 mm2, P less than 0.05), but remained unchanged in diabetic patients with autonomic neuropathy (131.0 +/- 9.0 vs, 130.7 +/- 8.2 mm2, n.s.) and after lumbar sympathectomy (135.2 +/- 17.5 vs 130.2 +/- 19.6 mm2, n.s.) when compared with the reaction to histamine alone. Addition of terbutaline produced similar results as observed with noradrenaline. These findings suggest a defect at the adrenoceptor level in diabetic patients with autonomic neuropathy and in patients with lumbar sympathectomy. Thus, the combined intradermal injection of histamine and the adrenoceptor agonists noradrenaline or terbutaline represents a simple and useful test for identifying patients with impaired adrenergic function.

Adult↗
PubMed2006

A canine model of cutaneous late-phase reactions: prednisolone inhibition of cellular and cytokine responses.

Immunoglobulin E (IgE)-mediated late-phase reactions can be induced in atopic humans by intradermal injection of relevant allergens or anti-IgE antibodies. The histology of these reactions resembles that of naturally occurring atopic dermatitis. Strikingly similar responses can be induced in dogs, suggesting that a canine model could prove valuable for preclinical investigation of drugs targeting late-phase reactions. This study was designed to characterize the cellular, cytokine and chemokine responses after intradermal anti-IgE injection in untreated and prednisolone-treated dogs. Normal beagles were untreated or treated with prednisolone before intradermal injection of polyclonal rabbit anti-canine IgE or normal rabbit IgG. Biopsies were taken before injection and 6, 24 and 48 hr after injection. Samples were evaluated by histological and immunohistochemical staining, as well as by real-time quantitative polymerase chain reaction analysis. Dermal eosinophil and neutrophil numbers increased dramatically within 6 hr after injection of rabbit anti-canine IgE, and remained moderately elevated at 48 hr. The numbers of CD1c(+) and CD3(+) mononuclear cells were also increased at 6 hr. The real-time quantitative polymerase chain reaction demonstrated marked increases in mRNA expression for interleukin-13 (IL-13), CCL2, CCL5 and CCL17. Levels of mRNA for IL-2, IL-4, IL-6 and IFN-gamma did not change within the limits of detection. Prednisolone administration suppressed the influx of neutrophils, eosinophils, CD1c(+) and CD3(+) cells, as well as expression of IL-13, CCL2, CCL5 and CCL17. These data document the cytokine and chemokine responses to anti-IgE injection in canine skin, and they demonstrate the ability of the model to characterize the anti-inflammatory effects of a known therapeutic agent.

Animals↗
PubMed1975

[Isolation and study of the properties of the Meningococcus allergen].

Meningococcus allergen was obtained from meningococcus cultures grown on serum-free media by the method of alkaline extraction and subsequent acid precipitation. In intradermal injection to guinea pigs this allergen indicated a specific sensitization with menigococci in 80% of cases. In the nonsensitized animals and also in guinea pigs sensitized with N. catarrhalis and N. gonorrhoeae positive skin reactions were seen in 7.6% of cases only. In detection of meningococcus sensitization there was found no relationship to their group specificity. The preparation was harmless in diagnostic dose, produced no sensitizing action, by chemical nature served as a glyconucleoproteid devoid of lipid, possessed molecular and electrophoretic heterogeneity. The allergen contained precipitating antigens not associated with its capacity to detect the state of sensitization in intradermal injection.

Allergens↗
PubMed1991

Interrelationship between hypersensitivity to soybean proteins and growth performance in early-weaned pigs.

The objective of this growth trial was to determine the interrelationship between immunological criteria, gut morphology, and performance of starter pigs fed soybean proteins processed by different methods. One hundred twenty-five pigs were orally infused with 6 g/d of either dried skim milk, soybean meal (48% CP), soy protein concentrate, extruded soy protein concentrate, or experimental soy protein concentrate from 7 to 11 d of age and then fed a diet containing the same protein sources from weaning (d 21) to 35 d of age. All pigs were fed a corn-soybean meal diet containing 10% dried whey, 1.25% lysine, and 3% soybean oil for the remaining 21 d of the experiment. Xylose absorption and anti-soy immunoglobulin G (IgG) titers were measured on d 6 postweaning, and skin-fold thickness after intradermal injection of protein extracts was measured on d 7 postweaning. A total of 25 pigs (five pigs/treatment) was euthanatized on d 7 postweaning. Villus height and crypt depth from duodenum samples were measured. These measurements were obtained to elucidate a relationship between the hypersensitivity responses to soybean products and growth performance of baby pigs. Pigs fed diets containing soybean meal had a lower (P less than .05) rate of gain (d 0 to 14) and villus height, higher (P less than .01) serum anti-soy IgG titers, and increased skin-fold thickness (d 6 and 7 postweaning) after intradermal injection compared with those fed dried skim milk. Pigs fed other soy proteins also had lower ADG from d 0 to 14 postweaning; however, pigs fed moist extruded soy protein concentrate tended (P less than .09) to have higher ADG and improved feed utilization when compared with pigs fed soybean meal (d 0 to 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗
PubMed1987

Antigen-induced formation of a lymphokine with a possible role as a mediator of the late component of dual allergic reaction.

In a penicillin-allergic patient who showed a strong dual skin reaction (immediate and late components) to an intradermal injection of the antigen, we investigated the possibility that the lymphokine, histamine-releasing factor (HRF), which has been shown to release histamine from human basophils and mast cells, might play a part in the elicitation of the late phase of the 'dual reaction'. In vitro stimulation of this patient's lymphocytes, but not those of a patient with a monophasic (immediate) reaction with the antigen, caused the production of a large amount of HRF, while a moderate lymphoproliferation occurred in both patients. The purified HRF released histamine from the patient's own leucocytes and also caused immediate wheal and flare reaction on intradermal injection.

Antigens↗
PubMed1991

Recruitment of neutrophils during IgE-dependent cutaneous late phase reactions in the mouse is mast cell-dependent. Partial inhibition of the reaction with antiserum against tumor necrosis factor-alpha.

Much of the clinically important pathology associated with IgE-dependent disorders is thought to reflect the actions of the blood-borne leukocytes recruited during these responses. To evaluate the extent to which mast cells are responsible for the leukocyte infiltration associated with IgE-dependent cutaneous reactions, we attempted to elicit these responses in normal mice, genetically mast cell-deficient W/Wv mice, and in W/Wv mice selectively repaired of their mast cell deficiency by the intradermal injection of cultured mast cells derived from the congenic normal (+/+) mice. We found that the tissue swelling associated with IgE-dependent passive cutaneous anaphylaxis reactions developed rapidly and diminished markedly from 2 to 4 h after antigen challenge, but remained detectable for at least 24 h after elicitation of the responses. Infiltration of leukocytes (predominantly neutrophils) also occurred at these sites, but reached maximal levels 6-12 h after antigen challenge, persisted at high levels for 24 h, and largely waned by 48 h. Virtually all of the tissue swelling and leukocyte infiltration associated with IgE-dependent cutaneous reactions was mast cell dependent. Intradermal injection of 40 U of recombinant murine TNF-alpha (rmTNF-alpha) elicited neutrophil infiltration similar in magnitude and kinetics to that observed after IgE-dependent mast cell degranulation. A rabbit anti-rmTNF-alpha (R anti-rmTNF-alpha) antiserum, which was able to inhibit 84% of the neutrophil infiltration observed after i.d. injection of rmTNF-alpha, inhibited IgE-, and mast cell-dependent leukocyte infiltration by 47 +/- 7% in three separate experiments. These findings indicate that TNF-alpha contributes to mast cell-dependent recruitment of leukocytes during IgE-dependent cutaneous late phase reactions, but suggest that other mast cell-associated mediators probably also contribute to this response.

Animals↗
PubMed1996

Cutaneous exposure to the superantigen staphylococcal enterotoxin B elicits a T-cell-dependent inflammatory response.

We analyzed the impact of superantigens secreted by skin-colonizing Staphylococci on the skin and the associated lymphoid tissue following epicutaneous application and intracutaneous injection of small amounts of staphylococcal enterotoxin B (SEB). A single intracutaneous injection of 50 ng of SEB elicited a strong inflammatory response in the skin of BALB/c mice. Three to 6 h later, we observed langerhans cell activation, mast cell degranulation, vasodilation, upregulation of ICAM-1, and induction of VCAM-1 on dermal blood vessels, with vascular adhesion of granulocytes. by 12 to 24 h, cell infiltration of the dermis increased, reaching the epidermis. Among the infiltrating leukocytes, a substantial number of eosinophils was found. After 48 h, the infiltrate was dominated by mononuclear cells. The response to SEB was dose-dependent, and signs of inflammation slowly disappeared over 5 to 7 days. Although the induction of VCAM-1 on dermal blood vessels suggested a role for interleukin-1/tumor necrosis factor-alpha in this reaction, the activation of monocytes/macrophages was not able to substitute for lymphocytes, as severe combined immunodeficiency (SCID) mice (which are lymphocyte-deficient) did not mount an inflammatory skin response to intradermal injection of SEB. The fact that nude mice (T-cell-deficient) also did not mount an inflammatory response to SEB indicated the T-cell dependency of the response. The V beta specificity of the SEB effect was demonstrated by the fact that SJL/J mice, which lack V beta 8+ T cells (the major SEB-reactive T cell population in mice), exhibited much weaker responses. Deletion or tolerization of SEB-reactive V beta T cells was not observed after a single intradermal injection of such minute amounts of SEB.

Animals↗
PubMed1991

Lymphoscintigraphic assessment of leg oedema following arterial reconstruction using a load produced by standing.

Lymphoscintigraphy using a test involving standing from a supine position and performed following an intradermal injection of 99Tcm-human serum albumin (HSA) was developed to evaluate the function of the lymphatic system in the lower extremities of patients who developed lymphoedema following arterial reconstructive surgery. In normal subjects, the load produced by standing tended to increase lymphatic function as indicated by the frequent appearance of a large spiking wave and a rapid stepwise increase in tracer activity and, less often, a phase of decreasing tracer activity. However, there was either no or less activation of lymph flow following standing in the group which developed leg oedema postoperatively. These findings indicate that lymphatic disruption is responsible for the leg oedema seen in these patients. Performance of this test following an intradermal injection of 99Tcm-HSA is technically simple, requires no special apparatus, and can be completed in 30 min. Lymphoscintigraphy using our new method can provide useful information on abnormalities in lymphatic function.

Aged↗
PubMed1992

Leukotriene B4 and platelet-activating factor in human skin.

Acute inflammatory reactions are characterized by leukocyte infiltration associated with increases in vascular permeability and in local blood flow. Leukocyte infiltration can be induced by chemotactic factors such as leukotriene B4 (LTB4) and paf-acether (formerly known as platelet-activating factor) that can be generated within inflammatory lesions. Vascular permeability and increase in blood flow are also affected by LTB4 and paf-acether, as well as by several other substances, including histamine and prostaglandins. Derived from arachidonic acid via the 5 lipo-oxygenase pathway, LTB4 is one of the most potent leukocyte chemotactic substances known. Intradermal injections of LTB4 induce dermal neutrophil infiltration in animal models and in humans. Topical application of LTB4 to human skin induces intraepidermal micro-abscesses containing numerous intact neutrophils. LTB4 has been found to be increased in psoriatic lesions, but its synthesis by epidermal cells remains undecided. Like other leukotrienes, LTB4 can stimulate DNA synthesis in cultured human epidermal keratinocytes. However, receptors for LTC4 but not for LTB4 have been found on human keratinocytes in culture. Paf-acether is an ether-linked phospholipid identified as 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine and is considered to be one of the most potent mediators of acute allergic and inflammatory reactions. For instance, intradermal injection of paf-acether induces inflammatory events such as neutrophil infiltration and increase in vascular permeability. Recent data suggest that cutaneous cells, such as fibroblasts and keratinocytes, are capable of producing paf and that paf is released during the development of allergic cutaneous reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermatitis↗
PubMed1982

Mediator studies in skin blister fluid from patients with dual skin reactions after intradermal allergen injection.

Skin blister fluid (SBF) samples obtained after allergen skin testing in a total of 26 patients with late cutaneous reactions (LCRs) were examined for the presence of various vasoactive mediators. Histamine was predominant during the early phase of the wheal and flare reaction(206 +/- 40 ng/ml) and decreased with the development of the LCR toward normal SBF levels (around 20 ng/ml). Kallikrein activity was measurable in low amounts (around 5 ng/ml) in three out of 13 SBF samples taken 30 min after skin testing and in five of 13 SBF samples taken 6 hr after allergen testing. The thromboxane B2 content of SBF showed an increase with the development of LCR (control skin 976 +/- 483 ng/ml; allergen-tested skin 30 min after allergen injection, 1465 +/- 1566 ng/ml; allergen-tested skin 6 hr after antigen injection, 1775 +/- 731 ng/ml). SBF obtained from normal skin as well as from allergen-tested skin showed significant platelet-activating property as measured in an in vitro serotonin-release assay from washed human platelets. It is concluded that during the development of late-phase reactions a complex interaction of various mediator systems takes place.

Adolescent↗
PubMed1992

Interactions of leukocyte integrins with intercellular adhesion molecule 1 in the production of inflammatory vascular injury in vivo. The Shwartzman reaction revisited.

We have investigated the role of leukocyte-endothelial cell interactions in a rabbit model of hemorrhagic vasculitis. Microvascular injury was produced in the skin by intradermal injection of Salmonella typhosa endotoxin followed 20 h later by intravenous zymosan, which activates complement. Hemorrhagic necrosis develops in the "prepared" skin sites which is characterized by microthrombi, neutrophil aggregation, platelet and fibrin deposition, and massive extravasation of erythrocytes. Hemorrhage in these Shwartzman-like lesions was quantitated by 99mTc-labeled autologous erythrocytes. Inhibition of the hemorrhagic response was obtained with mAb reactive with ICAM-1 as well as mAb against the leukocyte CD18 when either was administered intravenously just before intravenous zymosan challenge. This observation suggests that an intravascular event occurring in response to complement activation is required for the development of hemorrhagic vasculitis. We hypothesize that agents which successfully prepare the skin for the Shwartzman response after their intradermal injection do so by promoting increased intercellular adhesion molecule 1 (ICAM-1) expression on the vascular endothelium. Activation of complement then induces CD11/CD18 expression on circulating leukocytes thus producing an intravascular CD11/CD18-ICAM-1 (leukocyte-endothelium) adhesion event. Inhibition of intravascular leukocyte-leukocyte aggregation with mAb against CD11b (Mac-1) showed partial inhibition of hemorrhage, while mAb against CD11a (LFA-1) showed no inhibitory activity. This type of cytokine-primed, neutrophil-dependent vascular damage may be a model of human vasculitic processes where microvascular damage is produced in the absence of immune-complex deposition.

Animals↗
PubMed1995

Humoral and cellular responses induced by intradermally administered cytokine and conventional adjuvants.

Serum antibody responses to the model protein antigen avidin were monitored in sheep following intradermal injection of avidin formulated with a range of commercially available and experimental adjuvants, including muramyl dipeptide (MDP), aluminium hydroxide gel (alum), recombinant ovine interleukin1 beta (rovIL-1 beta), rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus. The highest antibody responses were recorded for animals immunised with avidin in rovIL-1 beta + alum, Quil A + alum or Emulsigen Plus, with moderate responses resulting from use of rovIL-1 beta or alum alone as adjuvants. Lower antibody responses to avidin were recorded when avidin was administered alone or with MDP. Delayed-type hypersensitivity (DTH) responses to avidin indicated that the most pronounced cellular response occurred in animals immunised with rovIL-1 beta + alum. Local cellular changes induced after primary and secondary intradermal injections indicated that distinct patterns of cellular recruitment were induced by the different adjuvants. Avidin with MDP resulted in an elevation of CD4+ T cells in the upper dermis while Emulsigen Plus induced an infiltration of large numbers of neutrophils throughout the dermis and reticular layers. CD4+, CD8+ and gamma delta + T cells increased in number and were found evenly distributed throughout these regions. Alum-based adjuvants resulted in the development of distinct cellular accumulations comprising primarily CD4+ T cells and CD45R + B cells arranged in distinct foci in the reticular layer. These cells were strongly class II positive as were the majority of macrophage like cells surrounding the foci. Staining for factor VIII related antigen indicated the presence of endothelial venules in the T and B cell foci and surrounding tissues.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed2004

The lymphatic system in the dorsal skinfold chamber of the Syrian golden hamster in vivo.

The lymphatic network contributes to maintaining tissue homeostasis and immunological function by transporting fluid, plasma protein and cells from peripheral tissue via the lymph nodes into the blood vascular system. In contrast to the blood circulatory system, little is known about the lymphatic system. In particular, suitable animal models are lacking. Therefore, the dorsal skinfold chamber model was used to investigate the existence of a lymphatic system. To analyze the lymphatic network Syrian golden hamsters (n=12) fitted with titanium chambers were used. FITC-dextran of different concentrations (5% or 25%) and different molecular weights (4, 40 or 150 kDa) was used to contrast lymphatic vessels and measure initial lymph flow velocity. Intravital fluorescence microscopy enabled the quantification of diameter, velocity and branching order. Histology and electron microscopy supported the in vivo findings. Immediately after intradermal injection of FITC-dextran the lymphatics including valves were visible. The diameters of the lymphatic vessels (n=189) ranged from 133+/-5.4 microm (branching order 1) to 26+/-4.0 microm (branching order 5). Using different molecular weights of FITC-dextran, no significant differences in velocity were measured (327+/-157 microm/s with 4 kDa, 391+/-126 microm/s with 40 kDa, and 378+/-175 microm/s with 150 kDa). Blood and lymphatic vessels could not be differentiated clearly by H&E staining. However, endothelial cells of vessels with an irregularly shaped lumen containing no erythrocytes in cross section showed a weaker signal for CD31 staining as compared to endothelial cells of vessels containing erythrocytes. Moreover, transmission electron microscopy identified the dye-containing vessels as lymphatics after intradermal injection of Berlin Blue. In conclusion, a lymphatic network was characterized in the dorsal skinfold chamber model of the Syrian golden hamster. Thus, this well-established animal model for intravital microscopy provides the opportunity to elucidate the physiological and pathological function of the lymphatic vascular system.

Animals↗