2. MATERIAL AND METHODS
2.2 METHODS
2.2.1 Animal experiments
2.2.1.1 Breeding of B6;129S-Tnftm1Gkl/J (TNF-/-) mice
TNF-/- mice were provided by Professor Max Löhning from DRFZ, Berlin. To generate these mice, targeting vector was constructed by replacing TNF gene with MC1neopA cassette (Stratagene) the 438 bp Narl-BglII fragment containing 40 bp of the 5' UTR, all the coding region, including the ATG translation initiation codon, of the first exon and part of the first intron of the mTNF-α gene131. These mice were bred and maintained under pathogen free conditions in animal facility. All experiments were performed according to German animal protection law.
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2.2.1.2 Genotyping of TNF-/- mice
Genomic DNA was isolated from 5 mm2 tail biopsies of TNF-/- mice by using the nucleospin tissue kit, according to manufacturer’s protocol. PCR was performed to identify the genotype of mice. TNF-α gene primer sequences were obtained from the ‘The Jackson laboratory’ site (strain stock no.: 003008) and were synthesized from TIB MOLBIOL, Berlin, Germany and are specified below:
Primer Sequence:
Primer Sequence Primer type (short name) oIMR4182 5’-tagccaggagggagaacaga-3’ Common (GC)
oIMR4183 5’-agtgcctcttctgccagttc-3’ Wild type Reverse (GW) oIMR7297 5’-cgttggctacccgtgatatt-3’ Mutant Reverse (GM)
Reaction component:
Regents Volume (µl) Final concentration
10x GenTherm buffer 1.2 1x
50 mM MgCl2 0.48 2 mM
10 mM deoxyNTPs 0.24 200 nM
10 μM forward primer (GC) 1.2 1 μM
10 μM reverse primer (GW) 1.2 1 μM
10 μM reverse primer (GM) 1.2 1 μM
50 U/μl DNA polymerase 0.075 0.03 U/μl
DNA 2
dH2O (makeup the volume up to 14µl)
The following PCR program was used:
94 °C - 3 min 94 °C - 30 sec
62 °C - 1 min 35 cycles 72 °C - 1 min
72 °C - 2 min
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4 °C - onhold
2 μl of 10x DNA loading dye were added to each PCR products and separated on a 2 % agarose gel. Gels were photographed with a UV light photometer and bands were further analysed to determine the genotype of the mice.
Expected band:
Mice Band size
TNF-/- homozygous 318 bp
TNF-/- heterozygous 183 bp and 318 bp
Wildtype (wt) 183 bp
2.2.1.3 In vivo skin irritation model
Figure 8: Experimental scheme of skin irritation model with different irritants treatment in vivo.
10 week old female C57BL/6 (wt) and TNF-/- mice were gently dry shaved at three different regions and exposed 30 times either to croton oil, 1% SDS or tape stripping using cotton swabs or cello tape. The fourth skin region was shaved 30 times with a help of wet shaver (Fig. 8). The groups of mice were sacrificed after 4
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and 18 hr, and blood was collected for serum. 5 mm2 skin biopsies were collected for immunochemistry and mRNA isolation.
2.2.1.4 Mouse model of AD
Figure 9: Experimental scheme of mouse model of AD with different antibodies/cytokines treatment in vivo
To induce AD, an adapted protocol from Dahten et al 2008 was used132. Briefly, 10 weeks old female wt and TNF-/- mice were sensitized by three subsequent intraperitoneal injections (i.p) with 100 μl of 10 μg ovalbumin (OVA) adsorbed to 1.5 mg Al(OH)3 (alum) on days 1, 14 and 21 (black arrows in Fig. 9). On day 21, the belly of the mice was shaved by wet shaving, further tape stripped and 100 µg OVA allergen was applied epicutaneously by the patch test method for one week period.
Each mouse had a total of three one week allergen exposures at the same site on the skin with a two week intervals in between without any allergen.
To better understand the intrinsic role of TNF in skin inflammation, different mediators and specific antibodies were applied intradermal (i.d) to the mouse skin one day before the patch, half a day before the patch renewal and in the middle of the patch-free week (blue arrows in Fig. 9). The dose and timing schedule of the antibody application was based on data from the literature i.e. anti-TSLP 20 µg/mice i.d.133, anti-c-Kit 40 µg/mice i.d134.
On day 71, mice were anesthetised by isoflurane and sacrificed by cervical dislocation. Blood was collected for further analysis of immunoglobulin and cytokines levels in the serum. Photographs of the patch area were taken for the
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assessment of the symptom score. 5 mm2 skin biopsies from lesional skin were taken for immunohistochemistry in O.C.T compound and frozen slowly into liquid nitrogen or in formalin for paraffin embedding. Rest of the skin was frozen into liquid nitrogen for mRNA isolation. All frozen samples were stored at -80 °C until further analysis.
2.2.1.5 Assessment of AD symptoms
AD severity was evaluated by using a skin score which nearly resemble to a score which is widely used in clinical practice. The SCORAD (scoring of atopic dermatitis) considers different clinical features to determine the severity of AD in humans135. In our model such typical features used to evaluate the severity were papulation, erythema, excoriation/crusting, dryness and extension of the lesions. Each parameter was evaluated independently in a blinded manner by six individuals in a randomized order. Severity for each parameter was rated as following: 0, no symptom; 1, mild symptoms; 2, intermediate symptoms; and 3, severe symptoms.
The score from all the six individuals for each of these factors were then summed up together and the total skin score was taken as AD severity with maximal skin score considered as 15 and minimal 5.
Functional skin barrier assessment
AD severity was further evaluated at a functional level by measuring TEWL in the skin. This method measures the barrier dysfunction which is developing in eczematous skin. During the measurement, the probe was placed on the belly of the mice to measure TEWL. The vapor gradient density was measured indirectly by two pairs of sensors i.e. sensors of temperature and relative humidity inside the hollow cylinder of defined volume and analyzed by a microprocessor. The measurement of TEWL is based on diffusion principle in an open chamber TEWL machine136.
Blood samples
Blood samples were taken on days 0 and 35 from the vena facialis with a micro lancet by punction. On day 71, complete blood was withdrawn from retro orbital
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venous sinus located behind the eyes. The blood was collected into special serum separator tubes and centrifuged at 14,000 rpm for 10 min. Serum was further stored at -80 °C until further analysis.