Background & Aims

Diabetic peripheral neuropathy (DPN) affects approximately 50 % of diabetic patients leading to a loss of small fiber function indicated by increased warmth detection and heat pain thresholds (HPT) in quantitative sensory testing [1]. TRPV1 is a transmembrane cation channel, which is responsible for the detection of noxius heat in humans as well as other species [2], and TRPV1-positive fibers are involved in the pathophysiology of sensory loss in DPN. We developed novel methods to investigate nocifensive locomotive responses to heat as well as spontaneous locomotive behavior in a thermal gradient in larval zebrafish. First, we assessed whether heat nociception in larval zebrafish depends on TRPV1 in vivo. Next, we investigated TRPV1-dependent nociception in both glucose-driven and methylglyoxal-driven models of diabetes mellitus to determine the heat pain threshold as a sensory marker of DPN.

Methods

We injected zebrafish embryos with trpv1, pdx1, glo1 or control morpholino oligonucleotides to knock down TRPV1, or disrupt pancreatic development (pdx1) or methylglyoxal scavenging by glyoxalase 1 (glo1). Alternatively, we immersed larvae in glucose or mannitol (10, 20, 40 mM), or methylglyoxal (50, 200, 500 uM) from 24-96 hours post fertilization. Pdx1 morphants and glucose incubated larvae were also treated for hyperglycemia with 10 uM PK11195. To determine a stimulus response function, we randomly applied heat stimuli of different intensities (150 ms, 18-88 mW) to freely moving larvae with a near-infrared diode laser (Schäfter+Kirchhoff, Germany). The intensity eliciting nocifensive responses in 50 % of cases was considered the HPT. For an unbiased assessment of temperature preference we recorded larvae in a thermal gradient ranging from 18-36°C or 10-45°C for 30 minutes and tracked their behavior with DeepLabCut [3]. Extra sum-of-squares F test was conducted in GraphPad Prism 9.

Results

TRPV1 knockdown (HPT [mean + SD] 62.5 + 6.5 mW) results in increased HPT compared to controls (28.6 + 4.4 mW, p<0.0001), and in the thermal gradient larvae died when entering 41°C with a median survival of 5.58 mins for TRPV1 morphants and 60 mins for controls. This shows heat nociception in larval zebrafish depends on TRPV1 in vivo. Both pdx1 morphants (50.1 + 4.9 mW) and glucose incubated larvae (10 mM: 42.3 + 4.7 mW) show increased HPT (p<0.0001) compared to control morphants (33.3 + 3.6 mW), wildtype (31.2 + 3.5 mW) or mannitol incubated animals (10 mM: 33.5 + 3.6 mW). The glucose-dependent effects were prevented by PK11195, suggesting a downstream mediator of sensory loss. A similar phenotype was observed in glo1 knockdowns (45.5 + 4.9 mW) and methylglyoxal incubated larvae (50 uM: 47.3 + 4.9 mW, p<0.0001). Both pdx1 (37.0 %) and glo1 morphants (16.8 %) spent a smaller portion of time at 27°C than controls (69.5 %) and more time at higher temperatures in the thermal gradient.

Conclusions

We show that nocifensive responses to short laser heat stimuli can be quantitatively analyzed in larval zebrafish. The vital sense of heat nociception depends on the zebrafish TRPV1 and its genetic knockdown results in distinct thermal hypoalgesia. The low survival of TRPV1-deficient larvae in thermal gradients ranging up to 45°C underscores its evolutionary importance quo ad vitam. Further, we were able to confirm the thermal hypoalgesia observed in patients with DPN in all tested zebrafish models of diabetes, which is in contrast to thermal hyperalgesia observed in a majority of rodent models. The results also indicate the reactive dicarbonyl methylglyoxal (MG) as an important downstream mediator in the development of thermal hypoalgesia in DPN. This is in contrast to MG-induced thermal hyperalgesia in rodent models of diabetes, and may be due to zebrafish lacking Nav1.8, which was implicated in MG-driven hyperalgesia by a facilitation of nociceptive neuron firing [4].

References

[1] Themistocleous, A.C., Ramirez, J.D., Shillo, P.R., Lees, J.G., Selvarajah et al. (2016). The Pain in Neuropathy Study (PiNS): a cross-sectional observational study determining the somatosensory phenotype of painful and painless diabetic neuropathy. Pain 157, 1132-1145.
[2] Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., and Julius, D. (1997). The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816-824.
[3] Mathis, A., Mamidanna, P., Cury, K.M., Abe, T., Murthy, V.N. et al. (2018). DeepLabCut: markerless pose estimation of user-defined body parts with deep learning. Nat Neurosci 21, 1281-1289.
[4] Bierhaus, A., Fleming, T., Stoyanov, S., Leffler, A., Babes, A. et al. (2012). Methylglyoxal modification of Nav1.8 facilitates nociceptive neuron firing and causes hyperalgesia in diabetic neuropathy. Nat Med 18, 926-933.

Presenting Author

Jonathan R. Husk

Poster Authors

Jonathan Husk

Medical Faculty Mannheim, Heidelberg University, Germany

Lead Author

E-mail

Katrin Bennewitz

Department of Vascular Biology, Medical Faculty Mannheim, Heidelberg University, Germany

Lead Author

E-mail

Uta Binzen

Ph.D.

Department of Cardiovascular Physiology, Medical Faculty Mannheim, Heidelberg University, Germany

Lead Author

E-mail

Jens Kroll

Ph.D.

Department of Vascular Biology, Medical Faculty Mannheim, Heidelberg University, Germany

Lead Author

E-mail

Rolf-Detlef Treede

Heidelberg University

Lead Author

E-mail

J. Simon Wiegert

Ph.D.

Department of Neurophysiology, Medical Faculty Mannheim, Heidelberg University, Germany

Lead Author

E-mail

Wolfgang Greffrath

Heidelberg University

Lead Author

E-mail

Topics

  • Novel Experimental/Analytic Approaches/Tools