Initiators of Classical and Lectin Complement Pathways Are Differently Engaged after Traumatic Brain Injury—Time-Dependent Changes in the Cortex, Striatum, Thalamus and Hippocampus in a Mouse Model

The complement system is involved in promoting secondary injury after traumatic brain injury (TBI), but the roles of the classical and lectin pathways leading to complement activation need to be clarified. To this end, we aimed to determine the ability of the brain to activate the synthesis of classical and lectin pathway initiators in response to TBI and to examine their expression in primary microglial cell cultures. We have modeled TBI in mice by controlled cortical impact (CCI), a clinically relevant experimental model. Using Real-time quantitative polymerase chain reaction (RT-qPCR) we analyzed the expression of initiators of classical the complement component 1q, 1r and 1s (C1q, C1r, and C1s) and lectin (mannose binding lectin A, mannose binding lectin C, collectin 11, ficolin A, and ficolin B) complement pathways and other cellular markers in four brain areas (cortex, striatum, thalamus and hippocampus) of mice exposed to CCI from 24 h and up to 5 weeks. In all murine ipsilateral brain structures assessed, we detected long-lasting, time- and area-dependent significant increases in the mRNA levels of all classical (C1q, C1s, C1r) and some lectin (collectin 11, ficolin A, ficolin B) initiator molecules after TBI. In parallel, we observed significantly enhanced expression of cellular markers for neutrophils (Cd177), T cells (Cd8), astrocytes (glial fibrillary acidic protein—GFAP), microglia/macrophages (allograft inflammatory factor 1—IBA-1), and microglia (transmembrane protein 119—TMEM119); moreover, we detected astrocytes (GFAP) and microglia/macrophages (IBA-1) protein level strong upregulation in all analyzed brain areas. Further, the results obtained in primary microglial cell cultures suggested that these cells may be largely responsible for the biosynthesis of classical pathway initiators. However, microglia are unlikely to be responsible for the production of the lectin pathway initiators. Immunofluorescence analysis confirmed that at the site of brain injury, the C1q is localized in microglia/macrophages and neurons but not in astroglial cells. In sum, the brain strongly reacts to TBI by activating the local synthesis of classical and lectin complement pathway activators. Thus, the brain responds to TBI with a strong, widespread and persistent upregulation of complement components, the targeting of which may provide protection in TBI.

Clozapine Prevents Poly (I:C) Induced Inflammation by Modulating NLRP3 Pathway in Microglial Cells

Schizophrenia is a complex psychiatric disorder that exhibits an interconnection between the immune system and the brain. Experimental and clinical studies have suggested the presence of neuroinflammation in schizophrenia. In the present study, the effect of antipsychotic drugs, including clozapine, risperidone, and haloperidol (10, 20 and 20 μM, respectively), on the production of IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IL-18, INF-γ, and TNF-α was investigated in the unstimulated and polyriboinosinic-polyribocytidilic acid [poly (I:C)]-stimulated primary microglial cell cultures. In the unstimulated cultures, clozapine, risperidone, and haloperidol did not influence the cytokine levels. Nevertheless, in cell cultures under strong inflammatory activation by poly (I:C), clozapine reduced the levels of IL-1α, IL-1β, IL-2, and IL-17. Risperidone and haloperidol both reduced the levels of IL-1α, IL-1β, IL-2, and IL-17, and increased the levels of IL-6, IL-10, INF-γ, and TNF-α. Based on the results that were obtained with the antipsychotic drugs and observing that clozapine presented with a more significant anti-inflammatory effect, clozapine was selected for the subsequent experiments. We compared the profile of cytokine suppression obtained with the use of NLRP3 inflammasome inhibitor, CRID3 to that obtained with clozapine, to test our hypothesis that clozapine inhibits the NLRP3 inflammasome. Clozapine and CRID3 both reduced the IL-1α, IL-1β, IL-2, and IL-17 levels. Clozapine reduced the level of poly (I:C)-activated NLRP3 expression by 57%, which was higher than the reduction thay was seen with CRID3 treatment (45%). These results suggest that clozapine might exhibit anti-inflammatory effects by inhibiting NLRP3 inflammasome and this activity is not typical with the use of other antipsychotic drugs under the conditions of strong microglial activation.

Biphalin, a Dimeric Enkephalin, Alleviates LPS-Induced Activation in Rat Primary Microglial Cultures in Opioid Receptor-Dependent and Receptor-Independent Manners

Neuropathic pain is relatively less responsive to opioids than other types of pain, which is possibly due to a disrupted opioid system partially caused by the profound microglial cell activation that underlines neuroinflammation. We demonstrated that intrathecally injected biphalin, a dimeric enkephalin analog, diminished symptoms of neuropathy in a preclinical model of neuropathic pain in rats (CCI, chronic constriction injury of the sciatic nerve) at day 12 postinjury. Using primary microglial cell cultures, we revealed that biphalin did not influence cell viability but diminished NO production and expression of Iba1 in LPS-stimulated cells. Biphalin also diminished MOP receptor level, as well as pronociceptive mediators (iNOS, IL-1β, and IL-18) in an opioid receptor-dependent manner, and it was correlated with diminished p-NF-κB, p-IκB, p-p38MAPK, and TRIF levels. Biphalin reduced IL-6, IL-10, TNFα, p-STAT3, and p-ERK1/2 and upregulated SOCS3, TLR4, and MyD88; however, this effect was not reversed by naloxone pretreatment. Our study provides evidence that biphalin diminishes neuropathy symptoms, which might be partially related to reduced pronociceptive mediators released by activated microglia. Biphalin may be a putative drug for future pain therapy, especially for the treatment of neuropathic pain, when the lower analgesic effects of morphine are correlated with profound microglial cell activation.

Microglial Inhibition Influences XCL1/XCR1 Expression and Causes Analgesic Effects in a Mouse Model of Diabetic Neuropathy

Background Recent studies indicated the involvement of some chemokines in the development of diabetic neuropathy; however, participation of the chemokine-C-motif ligand (XCL) subfamily remains unknown. The goal of this study was to examine how microglial inhibition by minocycline hydrochloride (MC) influences chemokine-C-motif ligand 1 (XCL1)–chemokine-C-motif receptor 1 (XCR1)/G protein–coupled receptor 5 expression and the development of allodynia/hyperalgesia in streptozotocin-induced diabetic neuropathy. Methods The studies were performed on streptozotocin (200 mg/kg, intraperitoneally)-induced mouse diabetic neuropathic pain model and primary glial cell cultures. The MC (30 mg/kg, intraperitoneally) was injected two times daily until day 21. XCL1 and its neutralizing antibody were injected intrathecally, and behavior was evaluated with von Frey and cold plate tests. Quantitative analysis of protein expression of glial markers, XCL1, and/or XCR1 was performed by Western blot and visualized by immunofluorescence. Results MC treatment diminished allodynia (0.9 ± 0.1 g; n = 7 vs. 3.8 ± 0.7 g; n = 7) and hyperalgesia (6.5 ± 0.6 s; n = 7 vs. 16.5 ± 1 s; n = 7) in the streptozotocin-induced diabetes. Repeated MC administration prevented microglial activation and inhibited the up-regulation of the XCL1/XCR1 levels. XCL1 administration (10 to 500 ng/5 μl; n = 9) in naive mice enhanced nociceptive transmission, and injections of neutralizing XCL1 (4 to 8 μg/5 μl; n = 10) antibody into the mice with diabetic neuropathic pain diminished allodynia/hyperalgesia. Microglia activation evoked in primary microglial cell cultures resulted in enhanced XCL1 release and XCR1 expression. Additionally, double immunofluorescence indicated the widespread coexpression of XCR1-expressing cells with spinal neurons. Conclusions In diabetic neuropathy, declining levels of XCL1 evoked by microglia inhibition result in the cause of analgesia. The putative mechanism corroborating this finding can be related to lower spinal expression of XCR1 together with the lack of stimulation of these XCR1 receptors, which are localized on neurons.

Parthenolide Relieves Pain and Promotes M2 Microglia/Macrophage Polarization in Rat Model of Neuropathy

Neuropathic pain treatment remains a challenge because pathomechanism is not fully understood. It is believed that glial activation and increased spinal nociceptive factors are crucial for neuropathy. We investigated the effect of parthenolide (PTL) on the chronic constriction injury to the sciatic nerve (CCI)-induced neuropathy in rat. We analyzed spinal changes in glial markers and M1 and M2 polarization factors, as well as intracellular signaling pathways. PTL (5 µg;i.t.) was preemptively and then daily administered for 7 days after CCI. PTL attenuated the allodynia and hyperalgesia and increased the protein level of IBA1 (a microglial/macrophage marker) but did not change GFAP (an astrocyte marker) on day 7 after CCI. PTL reduced the protein level of M1 (IL-1β, IL-18, and iNOS) and enhanced M2 (IL-10, TIMP1) factors. In addition, it downregulated the phosphorylated form of NF-κB, p38MAPK, and ERK1/2 protein level and upregulated STAT3. In primary microglial cell culture we have shown that IL-1β, IL-18, iNOS, IL-6, IL-10, and TIMP1 are of microglial origin. Summing up, PTL directly or indirectly attenuates neuropathy symptoms and promotes M2 microglia/macrophages polarization. We suggest that neuropathic pain therapies should be shifted from blanketed microglia/macrophage suppression toward maintenance of the balance between neuroprotective and neurotoxic microglia/macrophage phenotypes.

Minocycline Enhances the Effectiveness of Nociceptin/Orphanin FQ during Neuropathic Pain

Nociceptin/orphanin FQ (N/OFQ) antinociception, which is mediated selectively by the N/OFQ peptide receptor (NOP), was demonstrated in pain models. In this study, we determine the role of activated microglia on the analgesic effects of N/OFQ in a rat model of neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve. Repeated 7-day administration of minocycline (30 mg/kg i.p.), a drug that affects microglial activation, significantly reduced pain in CCI-exposed rats and it potentiates the analgesic effects of administered N/OFQ (2.5–5 μg i.t.). Minocycline also downregulates the nerve injury-induced upregulation of NOP protein in the dorsal lumbar spinal cord. Ourin vitrostudy showed that minocycline reducedNOPmRNA, but not protein, level in rat primary microglial cell cultures. In [35S]GTPγS binding assays we have shown that minocycline increases the spinal N/OFQ-stimulated NOP signaling. We suggest that the modulation of the N/OFQ system by minocycline is due to the potentiation of its neuronal antinociceptive activity and weakening of the microglial cell activation. This effect is beneficial for pain relief, and these results suggest new targets for the development of drugs that are effective against neuropathic pain.