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

Abstract

Indledning: Neuropatisk smerte, en global sundhedsmæssig bekymring, der påvirker 20% af den voksne befolkning, har vist sig at være vanskelig at behandle. De nuværende behandlingsmuligheder har vist ringe effekt og resultater. Derudover misbruger 21-29% af patienter, der får ordineret opioider disse, hvoraf 8-10% udvikler en opioidafhængighed. På trods af den lave effektivitet af opioider fortsætter neuropatiske smertepatienter med at få ordineret disse medikamenter. Dette understreger behovet for bedre smertehåndteringsmuligheder for neuropatiske smertepatienter. En af årsagerne til neuropatisk smerte er maladaptive ændringer i synapserne i dorsalhornet i medulla spinalis. PICK1 er et scaffold-protein involveret i de maladaptive ændringer, der observeres i neuropatiske smertetilstande. mPD5 er en bivalent peptidinhibitor af PICK1. Dens funktion er at blokere virkningerne af PICK1 og mediere smertelindring ved blandt andet neuropatiske smertetilstande. Formål: Formålet med denne undersøgelse er at vurdere, om SNI-modellen hos mus resulterer i supraspinale c-Fos-ændringer sammenlignet med naive mus. Desuden er formålet med denne undersøgelse at sammenligne de supraspinale effekter af mPD5 med morfin i en SNI-model med fokus på udvalgte hjerneområder, der behandler nociceptive informationer. Metode: Vævs-clearing og farvning med c-Fos som en proxy for neuronal aktivitet var den valgte metode til denne undersøgelse, da det letter helhjerneafbildning og muliggør 3D-visualisering af dybe strukturer i hjernen. Efter at mussehjernerne (n=74) blev afbildet med et lightsheet-mikroskop, blev billederne sammenstykket, justeret til et museatlas og analyseret ved hjælp af et tilpasset Python-script. For at undersøge forskellene i hele hjernens c-Fos positive celler mellem behandlings- og PBS-grupperne, blev en non-parametrisk Kruskal-Wallis test udført. Dernæst blev en regionvis sammenligningsanalyse (Mann-Whitney U test) udført og præsenteret ved hjælp af volcanoplots for at undersøge forskellene i hjerneregionernes c-Fos positive celler mellem behandlings- og PBS-grupperne. Resultater: Mann-Whitney U-testen viste ingen signifikant forskel i hele hjernens c-Fos positive celleantal mellem de ubehandlede naive og SNI-mus, men på hjerne regionsniveau blev der observeret nogle få forskelle. Kruskal-Wallis-testen viste en signifikant (p<0,05) forskel mellem morfin og mPD5 for både naive og SNI-mus (SNI p=0,0014 and naive p=0,023) samt en signifikant (p<0,05) forskel mellem morfin og PBS i både naive og SNI-mus (SNI p=0,007 and naive p=0,0002). Volcano plotsene viste, at mPD5 inducerede aktivitet i hjerneregioner, der processerer nociceptive informationer. Konklusion: Denne undersøgelse demonstrerede de aktivitetsinducerende effekter af mPD5 i hjerneregioner, der processerer nociceptive informationer. Desuden viste den, at mPD5 har langt mindre aktivitetsinducerende effekt på hjernen sammenlignet med morfin og ser ikke ud til at præsentere de samme afhængighedsskabende egenskaber som morfin. Dette tyder på, at mPD5 repræsenterer en lovende lægemiddelkandidat til videre præklinisk testning, før den går videre til kliniske forsøg og behandling af neuropatisk smerte.

Introduction: Neuropathic pain, a global health concern affecting 20% of the adult population, has proven difficult to treat. The current treatment options have demonstrated poor efficacy and outcomes. Additionally, 21-29% of patients prescribed opioids misuse them, with 8-10% of patients developing an opioid addiction. Despite the low efficacy of opioids, neuropathic pain patients continue to be prescribed these medications. This underscores the need for better pain management options for neuropathic pain patients. One of the etiologies of neuropathic pain is maladaptive changes in the synapses of the dorsal horn in the medulla spinalis. Protein interacting with C kinase 1 (PICK1) is a scaffold protein involved in the maladaptive changes observed in neuropathic pain conditions. Myr-NPEG4-(HWLKV)2, commonly referred to as mPD5, is a bivalent peptide inhibitor of the scaffold protein PICK1. Its function is to block the effects of PICK1 and mediate pain relief in chronic pain conditions. Aim: The aim of this study is to assess whether the spared nerve injury (SNI) mouse model, results in supraspinal c-Fos changes compared to naive mice. Moreover, the aim of this study is to compare the supra spinal effects of mPD5 to morphine in an SNI model, with focus on selected brain areas processing nociceptive information. Method: Tissue clearing and immunolabeling with c-Fos as a proxy for neuronal activity was the chosen method for this study, as it facilitates whole-brain imaging and enables three-dimensional visualization of deep structures in the brain. After mice brains (n=74) were imaged with a light sheet microscope, the images were stitched, aligned to a mouse atlas, and analyzed using a customized Python script. To investigate the differences in whole-brain c-Fos positive cells between the treatments and phosphate buffered saline (PBS) groups, a non-parametric Kruskal-Wallis test was performed. Next, a region-wise comparison analysis (Mann-Whitney U test) was conducted and presented using volcano plots, to examine the differences in brain regions’ c-Fos positive cells between the treatments and PBS groups. Results: The Mann-Whitney U test showed no significant difference in whole-brain total c-Fos positive cell counts between the non-treated naive and SNI mice, but on a region wise level a few differences were observed. The Kruskal-Wallis test showed a significant (p<0.05) difference between morphine and mPD5 for both naive and SNI mice (SNI p=0.0014 and naive p=0.023) as well as a significant (p<0.05) difference between morphine and PBS in both naive and SNI mice (SNI p=0.007 and naive p=0.0002). The volcano plots showed that mPD5 induced activity in brain regions processing nociceptive information. Conclusion: This study demonstrated the activity-inducing effects of mPD5 in brain areas processing nociceptive information. Moreover, it showed that mPD5 has far less activity-inducing effect on the brain compared to morphine and does not appear to present the same addictive properties as morphine. This advocates that mPD5 represents a promising drug candidate for further preclinical testing before proceeding to clinical trials and treatment of neuropathic pain.

A master thesis from Aalborg University

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.]