GoKawiilAnimals
Male Uchl1-eGFP and Cd68-eGFP mice (aged 8 weeks) on the C57BL/6J background or wild-type C57BL/6J mice were fed either a chow diet or a high-fat diet (60% fat, D12492i from Research Diets) for 16–18 weeks ad libitum. Mice were maintained on a 12 h–12 h light–dark cycle. The set points in the animal room were adjusted to 20–24 °C temperature and 45–65% humidity. Body composition was determined using an EchoMRI-100H system (EchoMRI). For insulin-tolerance tests, mice were fasted for 6 h and intraperitoneally (i.p.) injected with 0.75 U kg−1 insulin. Blood glucose was measured from the tail vein at the indicated timepoints using glucose test stripes. Mice were euthanized after deep anaesthesia with a mix of ketamine and xylazine, followed by intracardiac perfusion with heparinized PBS (10 U ml−1 heparin) and by a perfusion with 4% paraformaldehyde (PFA). Mice were post-fixed overnight in 4% PFA and subsequently washed five times with PBS shaking (300 rpm) at room temperature for 1 h for each wash step. Animal experimentation was performed in accordance with the European Union directives and the German animal welfare act (Tierschutzgesetz). They have been approved by the state ethics committee and the government of Upper Bavaria (ROB-55.2-2532.Vet_02-21-133, ROB-55.2-2532.Vet_02-16-117, ROB-55.2-2532.Vet_02-17-49, ROB-55.2-2532.Vet_02-19-166).
Human participants
Trigeminal ganglion samples were dissected post-mortem from body donors at the Institute of Anatomy, University of Leipzig, Germany and fixed in 4% Histofix. Body donors gave their informed and written consent to explore the cadavers for research and educational purposes (ethical approval number 129/21-ck, Medizinische Fakultät Ethik-Kommission). The participants were divided into lean (BMI < 25) or obese (BMI > 30). Data on age and sex can be found in Supplementary Table 11. We dissected three regions of interest from each trigeminal ganglion per individual for proteomic profiling.
Whisker stimulation test
The whisker test paradigm was adapted from the methods described previously40,41,42,43 and the Neuroscore test44. To avoid introducing confounding variables, mice were kept in their original cages. A cotton swab with a wooden end was used to administer the test. Initially, the cotton swab was presented in front of the mouse’s head and allowed to touch it. This was followed by four consecutive strokes, first to the whiskers on the right side and then on the left side of the face. The response to the cotton swab stimulation was evaluated using a modified whisker score test. A normal behavioural response to the stimulation, such as turning the head towards or away from the cotton swab or initiating grooming, was assigned a score of one. A lack of response to the stimulation was assigned a score of zero. Both sides of the face were stimulated four times, and the scores were recorded by a blinded evaluator. The maximum whisker score was 8, in which mice would have responded to all stimuli. The total score was then averaged for both sides. High scores (3–4) indicated normal responses to the stimulation, while low scores (0–2) suggested a lack of reaction, consistent with sensory deficits.
vDISCO nanobody labelling and clearing
vDISCO was performed as previously described2,45 in combination with active pumping GFP-Nanobooster labelling (Atto647N-conjugated anti-GFP nanobooster Chromotek, gba647n-100) for 6 days and passive labelling for 3 days. This approach amplifies the endogenous eGFP signal in reporter mice and shifts it into the far-red spectrum, substantially improving signal-to-noise ratios throughout the tissue. Mice underwent DISCO clearing46 using a tetrahydrofuran (THF)/H 2 O series (50% THF, 70% THF twice, 90% THF, 100% THF) for 24 h per step followed by an incubation in dichloromethane for 6 h. Tissues were incubated in benzyl alcohol/benzyl benzoate (BABB, 1:2 (v/v)) until tissue transparency was reached (>48 h).
WildDISCO antibody labelling and clearing
WildDISCO antibody labelling was performed as previously described in combination with anti-UCHL1 (14730-1-AP1, Proteintech, 26 µl per 200 ml immunostaining buffer) and anti-CGRP (ab36001, Abcam, 26 µl per 200 ml immunostaining buffer)28. Mice underwent DISCO clearing as described above.
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