TY - THES A1 - Ben-Kraiem, Adel T1 - Temporal characterization of the blood nerve barrier and specialized pro resolving mediators as therapeutic targets in neuropathy T1 - Zeitliche Charakterisierung der Funktion der Blut-Nerven-Schranke und der entzündungsauflösenden Lipide als Targets bei Neuropathie N2 - Abstract Neuropathic pain affects 6.9 to 10% of the general population, arises from lesion or disease of the somatosensory nervous system and is still challenging to treat. Indeed, current treatments efficacy are relatively low and present strong side effects. To that extent, identifying new targets and developing new treatment strategies constitute a priority. The blood nerve barrier consists of the endoneurial micro-blood vessels and the perineurium sealed by tight junctions constituted of tight junction proteins such claudin-5 and claudin-1. As the functional blood nerve barrier allows nerve tissue protection from external elements and maintains homeostasis, a destabilization or a disruption leads to infiltration of immunocytes promoting neuroinflammation and increased inflammatory mediators that can sensitize nociceptors and enhance pain. Thus resealing the blood nerve barrier in case of neuropathic pain could be a possible treatment strategy. Specialised proresolving mediators such lipoxin A4 and resolvin D1 are small lipids that bind to receptors such the formylpeptide recptor 2 (FPR2) and resolve inflammation. Specially resolvin D1 as anti-inflammatory and analgesic properties. Thus using resolvin D1 or eventually other specialized proresolving mediators in neuropathic pain could reseal the blood nerve barrier and resolve neuropathic pain. The present work aimed to characterize the blood nerve barrier in a preclinical model of diabetic polyneuropathy and nerve injury (chronic constriction injury) and to identify specialized proresolving mediators that seal the blood nerve barrier and thereby alleviate neuropathic pain. In diabetic polyneuropathy, the blood nerve barrier is permeable only to small molecules, which is due to the loss of claudin-1 in the perineurium and a reduced number of blood vessel- associated macrophages. Interestingly, blood nerve barrier permeability did not occur until four to eight weeks after diabetes induction, whereas mechanical hyperalgesia was measurable as early as two weeks. This suggests a pain-maintaining rather than a pain-triggering role of the blood nerve barrier. In case of chronic constriction injury, a resolution process of both mechanical and thermal hyperalgesia occurs between three to six weeks after injury. Here, the blood nerve barrier is permeable to both small and large molecules from the beginning. The pain recovery process occurs primarily in parallel with the sealing of the endoneurial barrier to large molecules such as fibrinogen from the plasma and its degradation. Perineurium is still permeable nine weeks after injury. Metabolomic analyses show that especially precursors of Resolvin D1 as well as its receptor FPR2, are upregulated at the beginning of pain resolution. Application of resolvin D1 loaded nanoparticles or agonists of FPR2 at the injury site before the onset of pain resolution accelerates the process and fibrinogen is no longer detectable in the endoneurium. Depending on the nerve damage, the blood nerve barrier is affected to varying degrees. Direct mechanical trauma and the accompanying inflammation lead to a more pronounced and long-lasting permeability - independent hyperalgesia. Possibly permeability, at least for small molecules, is important for prolonged reparative processes. In the nerve, permeability of capillaries in particular depends not only on tight junctions but also on other cells: in addition to macrophages, pericytes could also have a sealing effect. Endoneurial fibrinogen triggers pain; the exact mechanism remains to be investigated. Resolvin-containing nanoparticles were particularly effective and could be used locally as they contain endogenous substances in non- toxic particles. N2 - Neuropathische Schmerzen betreffen 6,9 bis 10 % der Allgemeinbevölkerung. Sie entstehen durch eine Verletzung oder Erkrankung des somatosensorischen Nervensystems und deren Behandlung ist eine Herausforderung. Die derzeitigen Behandlungen sind relativ unwirksam und haben starke Nebenwirkungen. Insofern sind die Identifizierung neuer Angriffspunkte und die Entwicklung neuer Behandlungsstrategien besonders wichtig. Die Blut-Nerven-Schranke besteht aus den endoneuralen Kapillaren und dem Perineurium, die beide durch Tight Junctions versiegelt werden. Letztere werden aus Tight Junction-Proteinen wie Claudin-5 und -1 gebildet. Da die Blut-Nerven-Schranke das Nervengewebe vor äußeren Einflüssen schützt und die Homöostase aufrechterhält, führt eine Öffnung zur Diffusion von potential algetischen Mediatoren und Infiltration von Immunzellen. Dieser Prozess fördert Neuroinflammation und sensibilisiert Nozizeptoren und verstärkt letztendlich die Schmerzen. Daher könnte die Abdichtung der Blut-Nerven-Schranke bei neuropathischen Schmerzen eine mögliche Behandlung darstellen. „Specialised proresolving mediators“, wie Lipoxin A4 und Resolvin D1, sind kleine Lipide, die an Rezeptoren wie der Formlypeptid 2 Rezeptor (FPR2) binden und Entzündungsprozesse auflösen. Insbesondere Resolvin D1 hat nicht nur entzündungshemmende und schmerzlindernde Eigenschaften, sondern wirkt auch barriereabdichtend in Lungenepithelzellen. Der Einsatz von Resolvin D1 oder möglicherweise anderer „specialised proresolving mediators“ bei neuropathischen Schmerzen könnte also die Blut-Nerven-Schranke wieder abdichten und neuropathische Schmerzen lindern. In der vorliegenden Arbeit sollte die Blut-Nerven-Schranke in einem präklinischen Modell der diabetischen Polyneuropathie und einer Nervenverletzung („chronic constriction injury“) charakterisiert und „specialised proresolving mediators“ identifiziert werden, die die Blut-Nerven-Schranke abdichten und damit neuropathische Schmerzen lindern. Bei der diabetischen Polyneuropathie ist die Blut-Nerven-Schranke nur für kleine Moleküle durchlässig, was auf den Verlust von Claudin-1 im Perineurium und eine geringere Anzahl von gefäßassoziierten Makrophagen zurückzuführen ist. Interessanterweise tritt die Durchlässigkeit der Blut-Nerven-Schranke erst vier bis acht Wochen nach der Diabetesinduktion auf, während die mechanische Hyperalgesie bereits nach zwei Wochen messbar ist. Dies spricht eher für eine schmerzunterhaltende als für eine schmerzauslösende Rolle der Blut-Nerven-Schranke. Bei chronic constriction injury bilden sich die mechanische und thermische Hyperalgesie im Zeitraum von drei bis sechs Wochen nach der Verletzung zurück. Hier ist der Blut-Nerven-Schranke von Anfang an sowohl für kleine als auch für große Moleküle durchlässig. Der Schmerzrückbildungsprozess findet vor allem parallel zur Abdichtung der endoneurialen Barriere für große Moleküle wie Fibrinogen aus dem Plasma und dessen Abbau statt. Das Perineurium ist neun Wochen nach der Verletzung noch durchlässig. Metabolomanalysen zeigen, dass insbesondere Vorläufer von Resolvin D1 sowie die entsprechenden Rezeptoren wie FPR2, zu Beginn der Schmerzrückbildung hochreguliert sind. Applikation von Resolvin D1 Nanopartikeln bzw. Agonisten des FPR2 an der Verletzungsstelle vor dem Beginn der Schmerzauflösung beschleunigt sich den Prozess und Fibrinogen ist nicht mehr endoneurial nachweisbar. Zusammenfassend ist je nach Art der Nervenschädigung die Blut-Nervenschranke unterschiedlich stark betroffen. Ein direktes mechanisches Trauma und die begleitende Inflammation führen im Gegensatz zur metabolisch-toxischen Schädigung zu einer ausgeprägteren und langanhaltenden Durchlässigkeit. Gerade bei einer Nervenläsion sind verschiedene Bereiche (Ort der Nervendurchtrennung und distale Regenerationsbereiche) unterschiedlich betroffen von der Schädigung und der Reparatur. Insofern ist Hyperalgesie nur teilweise davon beeinflusst. Möglicherweise ist die Durchlässigkeit zumindest für kleine Moleküle für längerdauernde Reparaturvorgänge wichtig. Die Permeabilität der Kapillaren ist im Nerven nicht nur von den Tight Junctions sondern auch von anderen Zellen abhängig: neben gefäßassoziierten Makrophagen könnten auch Perizyten abdichtend wirken. Endoneuriales Fibrinogen löst Schmerzen aus, der genaue Mechanismus muss noch untersucht werden. Resolvinhaltige Nanopartikeln waren besonders effektiv und könnten lokal gut eingesetzt werden, da sie körpereigene Substanzen in nichttoxischen Partikeln enthalten. KW - Neuropathic pain KW - neuropaticher Schmerz KW - Specialized pro resolving mediators KW - Diabetic painful neuropathy KW - Traumatic neuropathy KW - Blood nerve barrier Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-320797 ER - TY - JOUR A1 - Ben-Kraiem, Adel A1 - Sauer, Reine-Solange A1 - Norwig, Carla A1 - Popp, Maria A1 - Bettenhausen, Anna-Lena A1 - Atalla, Mariam Sobhy A1 - Brack, Alexander A1 - Blum, Robert A1 - Doppler, Kathrin A1 - Rittner, Heike Lydia T1 - Selective blood-nerve barrier leakiness with claudin-1 and vessel-associated macrophage loss in diabetic polyneuropathy JF - Journal of Molecular Medicine N2 - Diabetic polyneuropathy (DPN) is the most common complication in diabetes and can be painful in up to 26% of all diabetic patients. Peripheral nerves are shielded by the blood-nerve barrier (BNB) consisting of the perineurium and endoneurial vessels. So far, there are conflicting results regarding the role and function of the BNB in the pathophysiology of DPN. In this study, we analyzed the spatiotemporal tight junction protein profile, barrier permeability, and vessel-associated macrophages in Wistar rats with streptozotocin-induced DPN. In these rats, mechanical hypersensitivity developed after 2 weeks and loss of motor function after 8 weeks, while the BNB and the blood-DRG barrier were leakier for small, but not for large molecules after 8 weeks only. The blood-spinal cord barrier remained sealed throughout the observation period. No gross changes in tight junction protein or cytokine expression were observed in all barriers to blood. However, expression of Cldn1 mRNA in perineurium was specifically downregulated in conjunction with weaker vessel-associated macrophage shielding of the BNB. Our results underline the role of specific tight junction proteins and BNB breakdown in DPN maintenance and differentiate DPN from traumatic nerve injury. Targeting claudins and sealing the BNB could stabilize pain and prevent further nerve damage. KW - macrophages KW - neuropathy KW - barrier KW - pain Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265237 VL - 99 IS - 9 ER - TY - JOUR A1 - Oehler, Beatrice A1 - Kloka, Jan A1 - Mohammadi, Milad A1 - Ben-Kraiem, Adel A1 - Rittner, Heike L. T1 - D-4F, an ApoA-I mimetic peptide ameliorating TRPA1-mediated nocifensive behaviour in a model of neurogenic inflammation JF - Molecular Pain N2 - Background High doses of capsaicin are recommended for the treatment of neuropathic pain. However, low doses evoke mechanical hypersensitivity. Activation of the capsaicin chemosensor transient receptor potential vanilloid 1 (TRPV1) induces neurogenic inflammation. In addition to the release of pro-inflammatory mediators, reactive oxygen species are produced. These highly reactive molecules generate oxidised phospholipids and 4-hydroxynonenal (4-HNE) which then directly activate TRP ankyrin 1 (TRPA1). The apolipoprotein A-I mimetic peptide D-4F neutralises oxidised phospholipids. Here, we asked whether D-4F ameliorates neurogenic hypersensitivity in rodents by targeting reactive oxygen species and 4-HNE in the capsaicin-evoked pain model. Results Co-application of D-4F ameliorated capsaicin-induced mechanical hypersensitivity and allodynia as well as persistent heat hypersensitivity measured by Randell–Selitto, von Frey and Hargreaves test, respectively. In addition, mechanical hypersensitivity was blocked after co-injection of D-4F with the reactive oxygen species analogue H2O2 or 4-HNE. In vitro studies on dorsal root ganglion neurons and stably transfected cell lines revealed a TRPA1-dependent inhibition of the calcium influx when agonists were pre-incubated with D-4F. The capsaicin-induced calcium influx in TRPV1-expressing cell lines and dorsal root ganglion neurons sustained in the presence of D-4F. Conclusions D-4F is a promising compound to ameliorate TRPA1-dependent hypersensitivity during neurogenic inflammation. KW - TRPA1 KW - capsaicin KW - reactive oxygen species KW - oxidised lipids KW - pain KW - targeting Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-236061 VL - 16 ER - TY - JOUR A1 - Lux, Thomas J. A1 - Hu, Xiawei A1 - Ben-Kraiem, Adel A1 - Blum, Robert A1 - Chen, Jeremy Tsung-Chieh A1 - Rittner, Heike L. T1 - Regional differences in tight junction protein expression in the blood−DRG barrier and their alterations after nerve traumatic injury in rats JF - International Journal of Molecular Sciences N2 - The nervous system is shielded by special barriers. Nerve injury results in blood–nerve barrier breakdown with downregulation of certain tight junction proteins accompanying the painful neuropathic phenotype. The dorsal root ganglion (DRG) consists of a neuron-rich region (NRR, somata of somatosensory and nociceptive neurons) and a fibre-rich region (FRR), and their putative epi-/perineurium (EPN). Here, we analysed blood–DRG barrier (BDB) properties in these physiologically distinct regions in Wistar rats after chronic constriction injury (CCI). Cldn5, Cldn12, and Tjp1 (rats) mRNA were downregulated 1 week after traumatic nerve injury. Claudin-1 immunoreactivity (IR) found in the EPN, claudin-19-IR in the FRR, and ZO-1-IR in FRR-EPN were unaltered after CCI. However, laser-assisted, vessel specific qPCR, and IR studies confirmed a significant loss of claudin-5 in the NRR. The NRR was three-times more permeable compared to the FRR for high and low molecular weight markers. NRR permeability was not further increased 1-week after CCI, but significantly more CD68\(^+\) macrophages had migrated into the NRR. In summary, NRR and FRR are different in naïve rats. Short-term traumatic nerve injury leaves the already highly permeable BDB in the NRR unaltered for small and large molecules. Claudin-5 is downregulated in the NRR. This could facilitate macrophage invasion, and thereby neuronal sensitisation and hyperalgesia. Targeting the stabilisation of claudin-5 in microvessels and the BDB barrier could be a future approach for neuropathic pain therapy. KW - tight junction KW - claudin-5 KW - neuropathic pain KW - nerve injury KW - dorsal root ganglion Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285029 SN - 1422-0067 VL - 21 IS - 1 ER -