Whole-brain imaging for comparing the supraspinal effects of mPD5 to morphine in a mouse model of neuropathic pain

Hjernebilleddannelse til sammenligning af de supraspinale effekter af mPD5 og morfin i en musemodel for neuropatisk smerte.

Knudsen, Laura Reinholdt

4. term

Medicine with Industrial Specialisation, Master

2024

2024-05-30

46

Neuropatisk smerte rammer omkring 20 % af den voksne befolkning på verdensplan og er vanskelig at behandle. De nuværende muligheder har begrænset effekt, og blandt patienter, der får ordineret opioider, misbruger 21–29 % dem, mens 8–10 % udvikler afhængighed. På trods af den lave effekt bliver opioider fortsat brugt, hvilket understreger behovet for bedre behandlinger. En del af forklaringen på neuropatisk smerte er maladaptive ændringer i synapserne i baghornet i rygmarven. Protein interacting with C kinase 1 (PICK1) er et stillasprotein, som bidrager til disse ændringer. Myr-NPEG4-(HWLKV)2, kaldet mPD5, er en bivalent peptidhæmmer, der blokerer PICK1 med det formål at lindre kroniske smerter. Formålet med dette studie var at undersøge, om en spared nerve injury (SNI) musmodel medfører supraspinale (dvs. i hjernen) ændringer i c-Fos sammenlignet med naive mus, og at sammenligne de supraspinale effekter af mPD5 med morfin i udvalgte hjerneregioner, der bearbejder smerteinformation. Vi anvendte vævsklaring og immunofarvning for c-Fos som markør for nervecelleaktivitet. Denne metode muliggør helhjerneafbildning og tredimensionel visualisering af dybe hjernestrukturer med light-sheet mikroskopi. Musehjerner (n=74) blev afbildet, billederne blev sammensat, justeret til en museatlas og analyseret med en tilpasset Python-pipeline. Forskelle mellem behandlinger og fosfatbufferet saltvand (PBS) blev vurderet med ikke-parametriske statistiske test (Kruskal–Wallis for helhjerne og regionsvise Mann–Whitney U-test) og visualiseret med vulkanplot. Der var ingen signifikant forskel i det samlede antal c-Fos-positive celler i hele hjernen mellem ubehandlede naive og SNI-mus, men enkelte regionale forskelle blev observeret. Derimod sås signifikante forskelle mellem morfin og mPD5 samt mellem morfin og PBS i både naive og SNI-mus. mPD5 udløste aktivitet i hjerneregioner, der behandler smerte, men gav samlet set langt mindre hjerneaktivering end morfin. Studiet viser, at mPD5 påvirker smerterelaterede hjerneregioner, men i mindre omfang end morfin. Det peger på, at mPD5 muligvis ikke har de samme afhængighedsrelaterede egenskaber som morfin. Samlet set fremstår mPD5 som en lovende lægemiddelkandidat til yderligere prækliniske undersøgelser før eventuelle kliniske forsøg mod neuropatisk smerte.

Neuropathic pain affects about 20% of adults worldwide and is hard to treat. Current options often work poorly, and among patients prescribed opioids, 21–29% misuse them and 8–10% develop addiction. Despite limited benefit, opioids are still widely used, highlighting the need for better treatments. One cause of neuropathic pain involves maladaptive changes in synapses in the dorsal horn of the spinal cord. Protein interacting with C kinase 1 (PICK1) is a scaffold protein that contributes to these changes. Myr-NPEG4-(HWLKV)2, known as mPD5, is a bivalent peptide inhibitor that blocks PICK1 to relieve chronic pain. This study asked whether the spared nerve injury (SNI) mouse model produces supraspinal (i.e., brain) changes in c-Fos, a marker of neuronal activity, compared with naive mice, and compared the supraspinal effects of mPD5 versus morphine in selected brain regions that process pain signals. We used tissue clearing and immunolabeling for c-Fos to enable whole-brain, three-dimensional imaging with light-sheet microscopy. Mouse brains (n=74) were imaged, stitched, registered to a mouse atlas, and analyzed with a customized Python pipeline. Differences between treatments and phosphate-buffered saline (PBS) were assessed with non-parametric tests (Kruskal–Wallis for whole brain and region-wise Mann–Whitney U tests) and visualized with volcano plots. There was no significant difference in total whole-brain c-Fos–positive cell counts between untreated naive and SNI mice, although a few regional differences were observed. In contrast, significant differences were found between morphine and mPD5, and between morphine and PBS, in both naive and SNI mice. mPD5 induced activity in brain regions involved in pain processing but caused much less overall brain activation than morphine. These findings show that mPD5 engages pain-related brain regions while producing less widespread brain activation than morphine. This pattern suggests mPD5 may not share morphine’s addictive properties. Overall, mPD5 appears to be a promising drug candidate for further preclinical testing before potential clinical trials for neuropathic pain.

[This summary has been rewritten with the help of AI based on the project's original abstract]

brain ; mice ; tissue clearing ; c-Fos

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