The gut-brain and gut-macrophage contribution to gastrointestinal dysfunction with systemic inflammation.

The gastrointestinal tract is one of the main organs affected during systemic inflammation and disrupted gastrointestinal motility is a major clinical manifestation. Many studies have investigated the involvement of neuroimmune interactions in regulating colonic motility during localized colonic inflammation, i.e., colitis. However, little is known about how the enteric nervous system and intestinal macrophages contribute to dysregulated motility during systemic inflammation. Given that systemic inflammation commonly results from the innate immune response against bacterial infection, we mimicked bacterial infection by administering lipopolysaccharide (LPS) to rats and assessed colonic motility using ex vivo video imaging techniques. We utilized the Cx3cr1-Dtr rat model of transient depletion of macrophages to investigate the role of intestinal macrophages in regulating colonic motility during LPS infection. To investigate the role of inhibitory enteric neurotransmission on colonic motility following LPS, we applied the nitric oxide synthase inhibitor, Nω-nitro-L-arginine (NOLA). Our results confirmed an increase in colonic contraction frequency during LPS-induced systemic inflammation. However, neither the depletion of intestinal macrophages, nor the suppression of inhibitory enteric nervous system activity impacted colonic motility disruption during inflammation. This implies that the interplay between the enteric nervous system and intestinal macrophages is nuanced, and complex, and further investigation is needed to clarify their joint roles in colonic motility.

Incidental Gliosis in the Central Nervous System of Control Nonhuman Primates and Rats.

Gliosis, including microgliosis and astrocytosis, can be challenging to interpret in nonclinical studies. Incidences of glial foci in brains and spinal cords of control rats and nonhuman primates (NHPs) were reviewed in the historical control databases from two contract research organizations, including one specializing in neuropathology. In the brain, minimal to mild (grades 1-2) microgliosis was the most common diagnosis, especially in NHPs, although occasional moderate or marked microgliosis (grades 3 and 4) was encountered in both species. Microgliosis was more common in the cerebral cortex, cerebellum, and medulla oblongata in both species and was frequent in the white matter (brain), thalamus, and basal nuclei of NHPs. Gliosis ("not otherwise specified") of minimal severity was diagnosed in similar brain sub-sites for both species and was more common in NHPs compared with rats. Astrocytosis was most prominent in the cerebellum (molecular layer) of NHPs but was otherwise uncommon. In the spinal cord, microgliosis was most common in the lateral white matter tracts in rats and NHPs, and in the dorsal white matter tracts in NHPs. These data indicate that low-grade spontaneous glial responses occur with some frequency in control animals of two common nonclinical species.

Acrylates in artificial nails-Results of product analyses and glove penetration studies.

BACKGROUND: Artificial nail materials are mixtures that are prone to contain several sensitizing (meth)acrylates. It is not known whether the listing of (meth)acrylates is correct in these products' packages. Protective gloves suited for nail work are needed. OBJECTIVES: To analyse (meth)acrylates in gel nail and acrylic nail products chemically and to compare the results with the information in the product labels, and to study penetration of artificial nail materials through selected disposable gloves. METHODS: We analysed 31 gel nail products and 6 acrylic nail products for their (meth)acrylate content by gas chromatography-mass spectrometry (GC-MS). We tested the penetration of two nail products through three disposable gloves: nitrile rubber, neoprene rubber and polyvinyl chloride (PVC). RESULTS: Altogether 32/37 products contained (meth)acrylates. In all of them, there was discrepancy between the listed (meth)acrylates and those discovered in the analysis. The commonest (meth)acrylates were hydroxyethyl methacrylate (HEMA, 20/37 samples) and hydroxypropyl methacrylate (HPMA, 9/37 samples), but many of the product packages failed to declare them. Isobornyl acrylate (IBA) was discovered in nine gel nail products. The neoprene glove could withstand nail gel for 20 min and thin nitrile glove and PVC glove for 5 min. Acrylic nail liquid penetrated through disposable gloves quickly. CONCLUSIONS: Labelling of artificial nail products was notably incorrect on most products. Requirements for product labelling must be updated so that the risk of sensitization associated with artificial nail products is clearly indicated. Disposable gloves can probably be used short-term in gel nail work, whereas disposable gloves do not protect the user from acrylic nail liquids.

A simplified protocol for the automated production of 2-[18 F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine ([18 F]nifene) on an IBA Synthera® module.

The α4ß2 nicotinic acetylcholine receptor (nAChR) ligand 2-[18 F]fluoro-3-[2-((S)-3-pyrrolinyl)methoxy]pyridine ([18 F]nifene) has been synthesized in 10% decay-corrected radiochemical yield using the IBA Synthera® platform (IBA Cyclotron Solutions, Louvain-la-Neuve, Belgium) with an integrated fluidic processor (IFP). Boc-nitronifene served as the precursor, and 20% trifluoroacetic acid (TFA) was used to deprotect the Boc-group after radiolabeling. By omitting the solvent extraction step after radiolabeling, the process was simplified to a single step with no manual intervention. [18 F]Nifene was obtained in decay-corrected radiochemical yields of 10 ± 2% (n = 20) and radiochemical purity >99%. Typical specific radioactivities of 2700-4865 mCi/µmole (100-180 GBq/µmol) were measured at the end of synthesis; total synthesis times were about 1 h 40 min.

Chronic Variable Stress and Cafeteria Diet Combination Exacerbate Microglia and c-fos Activation but Not Experimental Anxiety or Depression in a Menopause Model.

The menopause transition is a vulnerable period for developing both psychiatric and metabolic disorders, and both can be enhanced by stressful events worsening their effects. The present study aimed to evaluate whether a cafeteria diet (CAF) combined with chronic variable stress (CVS) exacerbates anxious- or depressive-like behavior and neuronal activation, cell proliferation and survival, and microglia activation in middle-aged ovariectomized (OVX) rats. In addition, body weight, lipid profile, insulin resistance, and corticosterone as an index of metabolic changes or hypothalamus-pituitary-adrenal (HPA) axis activation, and the serum pro-inflammatory cytokines IL-6, IL-ß, and TNFα were measured. A CAF diet increased body weight, lipid profile, and insulin resistance. CVS increased corticosterone and reduced HDL. A CAF produced anxiety-like behaviors, whereas CVS induced depressive-like behaviors. CVS increased serum TNFα independently of diet. A CAF and CVS separately enhanced the percentage of Iba-positive cells in the hippocampus; the combination of factors further increased Iba-positive cells in the ventral hippocampus. A CAF and CVS increased the c-fos-positive cells in the hippocampus; the combination of factors increased the number of positive cells expressing c-fos in the ventral hippocampus even more. The combination of a CAF and CVS generates a slight neuroinflammation process and neuronal activation in a hippocampal region-specific manner and differentially affects the behavior.

High-fat diet, but not duration of lactation, increases mammary gland lymphatic vessel function and subsequent growth of inflammatory breast cancer cells.

Inflammatory breast cancer (IBC) presents as rapid-onset swelling and breast skin changes caused by tumor emboli in the breast and breast skin lymphatics. IBC has been linked with obesity and duration of breastfeeding, but how these factors affect IBC tumor progression is not clear. We modeled the simultaneous effects of diet and weaning in mice on in vivo lymphatic function; on IBC tumor growth; and on aspects of the mammary gland microenvironment before and after IBC (SUM149) xenograft inoculation. We hypothesized that weaning status and diet would have synergistic effects on lymphatic function and the breast microenvironment to enhance IBC tumor growth. Changes in lymphatic structure and function were characterized with in vivo near-infrared fluorescence (NIRF) imaging. Mice were fed either a high-fat diet (HFD; 60 kcal%) or a normal/low-fat diet (LFD; 10 kcal%), bred twice, and subjected to either normal-duration nursing (NW) or forced weaning (FW). SUM149 IBC tumors were implanted at 14 months; images were obtained before and after implantation. Multiparous mice fed HFD showed increased pre-tumor lymphatic pulsing in both the FW and NW groups relative to mice fed LFD. HFD promoted tumor growth independent of weaning time (P = 0.04). Pre-tumor lymphatic pulsing was associated with tumor volume at 8 weeks (P = 0.02) and was significantly correlated with expression of the lymphatic tracking ligand CCL21 (P = 0.05, Table 1). HFD significantly increased the numbers of monocyte-derived IBA1+, CD163+, and CD11c+ cells (P < 0.0001, P < 0.0001, P = 0.0005) in the contralateral, non-tumor-bearing mammary gland. Numbers of lymphangiogenic podoplanin+/IBA1+ macrophages were increased in the ducts of HFD and FW mice (all P < 0.003). HFD in nulliparous mice had a similar increase in lymphatic pulsing at 14 weeks (P = 0.006), indicating that this functional change was independent of parity. We conclude that HFD induced increases in mammary gland lymphatic function, assessed as pulsing rate before tumor initiation, and correlated with inflammation in the mammary gland and increased SUM149 tumor growth. The relationship between diet, lymphatic pulsing, and tumor growth warrants further investigation.

Effects of Whole-Body Vibration and Manually Assisted Locomotor Therapy on Neurotrophin-3 Expression and Microglia/Macrophage Mobilization Following Thoracic Spinal Cord Injury in Rats.

Microglial cells play an important role in neuroinflammation and secondary damages after spinal cord injury (SCI). Progressive microglia/macrophage inflammation along the entire spinal axis follows SCI, and various factors may determine the microglial activation profile. Neurotrophin-3 (NT-3) is known to control the survival of neurons, the function of synapses, and the release of neurotransmitters, while also stimulating axon plasticity and growth. We examined the effects of whole-body vibration (WBV) and forms of assisted locomotor therapy, such as passive flexion-extension (PFE) therapy, at the neuronal level after SCI, with a focus on changes in NT-3 expression and on microglia/macrophage reaction, as they play a major role in the reconstitution of CNS integrity after injury and they may critically account for the observed structural and functional benefits of physical therapy. More specifically, the WBV therapy resulted in the best overall functional recovery when initiated at day 14, while inducing a decrease in Iba1 and the highest increase in NT-3. Therefore, the WBV therapy at the 14th day appeared to be superior to the PFE therapy in terms of recovery. Functional deficits and subsequent rehabilitation depend heavily upon the inflammatory processes occurring caudally to the injury site; thus, we propose that increased expression of NT-3, especially in the dorsal horn, could potentially be the mediator of this favorable outcome.

Clinical, radiological, biochemical and molecular characterization of a new case with multiple mitochondrial dysfunction syndrome due to IBA57: Lysine and tryptophan metabolites as potential biomarkers.

Iron­sulfur clusters (FeS) are one of the most primitive and ubiquitous cofactors used by various enzymes in multiple pathways. Biosynthesis of FeS is a complex multi-step process that is tightly regulated and requires multiple machineries. IBA57, along with ISCA1 and ISCA2, play a role in maturation of [4Fe-4S] clusters which are required for multiple mitochondrial enzymes including mitochondrial Complex I, Complex II, lipoic acid synthase, and aconitase. Pathogenic variants in IBA57 have been associated with multiple mitochondrial dysfunctions syndrome 3 (MMDS3) characterized by infantile to early childhood-onset psychomotor regression, optic atrophy and nonspecific dysmorphism. Here we report a female proband who had prenatal involvement including IUGR and microcephaly and developed subacute psychomotor regression at the age of 5 weeks in the setting of preceding viral infection. Brain imaging revealed cortical malformation with polymicrogyria and abnormal signal alteration in brainstem and spinal cord. Biochemical analysis revealed increased plasma glycine and hyperexcretion of multiple organic acids in urine, raising the concern for lipoic acid biosynthesis defects and mitochondrial FeS assembly defects. Molecular analysis subsequently detected compound heterozygous variants in IBA57, confirming the diagnosis of MMDS3. Although the number of MMDS3 patients are limited, certain degree of genotype-phenotype correlation has been observed. Unusual brain imaging in the proband highlights the need to include mitochondrial disorders as differential diagnoses of structural brain abnormalities. Lastly, in addition to previously known biomarkers including high blood lactate and plasma glycine levels, the increase of 2-hydroxyadipic and 2-ketoadipic acids in urine organic acid analysis, in the appropriate clinical context, should prompt an evaluation for the lipoic acid biosynthesis defects and mitochondrial FeS assembly defects.

Dynamic changes in microglia in the mouse hippocampus during administration and withdrawal of the CSF1R inhibitor PLX3397.

Pexidartinib (PLX3397), a colony-stimulating factor-1 receptor (CSF1R) inhibitor, is currently in phase 1-3 clinical trials as a treatment for a variety of tumours. CSF1R signalling regulates the development, survival and maintenance of microglia, the resident brain innate immune cells. In this study, we examined the effects of PLX3397 in the drinking water of mice on microglia in the hippocampus using ionized calcium-binding adapter molecule 1 (Iba1, a microglial marker) immunocytochemistry. A high concentration of PLX3397 (1 mg/mL) significantly decreased the density of Iba1-immunoreactive cells after 7 days of exposure, but a low concentration of PLX3397 (0.5 mg/mL) did not. In addition, both low and high concentrations of PLX3397 significantly increased the intersection number, total length and maximum length of microglial processes in male mice. PLX3397 administered for 21 days eliminated microglia with 78% efficiency in males and 84% efficiency in females. Significant increases in microglial processes were found after both seven and 21 days of PLX3397 exposure in males, whereas decreases in microglial processes were observed after both 14 and 21 days of exposure in females. After PLX3397 withdrawal following its administration for 14 days in males, the soma size quickly returned to normal levels within a week. However, the microglial density, intersection number and total length of microglial processes after 3 days of recovery stabilized to untreated levels. In summary, these findings provide detailed insight into the dynamic changes in microglial number and morphology in the hippocampus in a dose- and time-dependent manner after PLX3397 treatment and withdrawal.

Retinal pathological features and proteome signatures of Alzheimer's disease.

Alzheimer's disease (AD) pathologies were discovered in the accessible neurosensory retina. However, their exact nature and topographical distribution, particularly in the early stages of functional impairment, and how they relate to disease progression in the brain remain largely unknown. To better understand the pathological features of AD in the retina, we conducted an extensive histopathological and biochemical investigation of postmortem retina and brain tissues from 86 human donors. Quantitative examination of superior and inferior temporal retinas from mild cognitive impairment (MCI) and AD patients compared to those with normal cognition (NC) revealed significant increases in amyloid ß-protein (Aß42) forms and novel intraneuronal Aß oligomers (AßOi), which were closely associated with exacerbated retinal macrogliosis, microgliosis, and tissue atrophy. These pathologies were unevenly distributed across retinal layers and geometrical areas, with the inner layers and peripheral subregions exhibiting most pronounced accumulations in the MCI and AD versus NC retinas. While microgliosis was increased in the retina of these patients, the proportion of microglial cells engaging in Aß uptake was reduced. Female AD patients exhibited higher levels of retinal microgliosis than males. Notably, retinal Aß42, S100 calcium-binding protein B+ macrogliosis, and atrophy correlated with severity of brain Aß pathology, tauopathy, and atrophy, and most retinal pathologies reflected Braak staging. All retinal biomarkers correlated with the cognitive scores, with retinal Aß42, far-peripheral AßOi and microgliosis displaying the strongest correlations. Proteomic analysis of AD retinas revealed activation of specific inflammatory and neurodegenerative processes and inhibition of oxidative phosphorylation/mitochondrial, and photoreceptor-related pathways. This study identifies and maps retinopathy in MCI and AD patients, demonstrating the quantitative relationship with brain pathology and cognition, and may lead to reliable retinal biomarkers for noninvasive retinal screening and monitoring of AD.

Nano-encapsulated anandamide reduces inflammatory cytokines in vitro and lesion severity in a murine model of cutaneous lupus erythematosus.

Cutaneous lupus erythematosus (CLE) is a heterogeneous autoimmune skin disease which occurs independently and in conjunction with systemic lupus erythematosus. Drug development for CLE is severely lacking. Anandamide (AEA) is a primary endocannabinoid which exhibits immunomodulatory effects through mixed cannabinoid receptor agonism. We evaluated AEA as topical treatment for CLE and assessed benefits of nanoparticle encapsulation (AEA-NP) on cutaneous drug penetration, delivery and biological activity. Compared to untreated controls, AEA-NP decreased IL-6 and MCP-1 in UVB-stimulated keratinocytes (p < 0.05) in vitro. In BALB/c mice, AEA-NP displayed improved cutaneous penetration, extended release and persistence of AEA in the follicular unit extending to the base after 24 h. Utilizing the MRL-lpr lupus murine model, twice weekly treatment of lesions with topical AEA-NP for 10 weeks led to decreased clinical and histologic lesion scores compared to unencapsulated AEA and untreated controls (p < 0.05). Prophylactic application of AEA-NP to commonly involved areas on MRL-lpr mice similarly resulted in decreased clinical and histologic scores when compared to controls (p < 0.05), and reduced C3 and IBA-1 in lesional tissue (p < 0.05). The demonstrated clinical and immunomodulatory effects of treatment with AEA support its potential as therapy for CLE. This work also suggests that encapsulation of AEA improves penetration and treatment efficacy. Future studies will be conducted to assess full therapeutic potential.

Lutetium177-Labeled DOTA-Ibandronate: A Novel Radiopharmaceutical for Targeted Treatment of Bone Metastases.

Bone metastases of malignant tumors significantly threaten the patient survival and quality of life. We designed and synthesized a novel bisphosphonate radiopharmaceutical [68Ga- or 177Lu-labeled DOTA-Ibandronate(68Ga/177Lu-DOTA-IBA)] for targeted diagnosis and treatment of bone metastases. This study explored the basic biological characteristics of 177Lu-DOTA-IBA, guiding clinical translation and providing evidence for future clinical applications. The control variable method was used to optimize the optimal labeling conditions. The in vitro properties, biological distribution, and toxicity of 177Lu-DOTA-IBA were studied. Normal mice and tumor-bearing mice were imaged using micro SPECT/CT. With Ethics Committee approval, five volunteers were recruited for a preliminary clinical translation study. 177Lu-DOTA-IBA has a radiochemical purity of more than 98%, with good biological properties and safety. Blood clearance is fast and soft tissue uptake is low. Tracers are excreted mainly through the urinary system, targeting and continuously concentrating in the bones. Three patients experienced significant pain relief within 3 days after 177Lu-DOTA-IBA treatment (740-1110 MBq), lasting more than 2 months, with no toxic side effects. 177Lu-DOTA-IBA is easy to prepare and exhibits good pharmacokinetic characteristics. Low-dose 177Lu-DOTA-IBA is effective, well tolerated, and was associated with no significant adverse reactions. It is a promising radiopharmaceutical for the targeted treatment of bone metastases, controlling the progress of bone metastasis and improving survival and quality of life of patients with advanced bone metastasis.

Toxicologic Pathology Forum Opinion: Interpretation of Gliosis in the Brain and Spinal Cord Observed During Nonclinical Safety Studies.

Gliosis, defined as a nonneoplastic reaction (hypertrophy and/or proliferation) of astrocytes and/or microglial cells, is a frequent finding in the central nervous system (CNS [brain and/or spinal cord]) in nonclinical safety studies. Gliosis in rodents and nonrodents occurs at low incidence as a spontaneous finding and is induced by various test articles (e.g., biomolecules, cell and gene therapies, small molecules) delivered centrally (i.e., by injection or infusion into cerebrospinal fluid or neural tissue) or systemically. Several CNS gliosis patterns occur in nonclinical species. First, gliosis may accompany degeneration and/or necrosis of cells (mainly neurons) or neural parenchyma (neuron processes and myelin). Second, gliosis often follows inflammation (i.e., leukocyte accumulation causing parenchymal damage) or neoplasm formation. Third, gliosis may appear as variably sized, randomly scattered foci of reactive glial cells in the absence of visible parenchymal damage or inflammation. In interpreting test article-related CNS gliosis, adversity is indicated by parenchymal injury (e.g., degeneration, necrosis, or inflammation) and not the mere existence of a glial reaction. In the absence of clear structural damage to the parenchyma, gliosis as a standalone CNS finding should be interpreted as a nonadverse reaction to regional alterations in microenvironmental conditions rather than as evidence of a glial reaction associated with neurotoxicity.

The many faces of mouse histiocytic sarcoma in C57BL/6J mice.

Histiocytic sarcoma is a tumor of the hematopoietic system considered to be derived from macrophages. Although rare in humans, it occurs frequently in mice. Histiocytic sarcoma can be a difficult tumor to diagnose due to its diverse cellular morphologies, growth patterns, and organ distributions. The varying morphology of histiocytic sarcomas makes it easy to confuse them with other types of neoplasia, including hepatic hemangiosarcoma, uterine schwannoma, leiomyosarcoma, uterine stromal cell tumor, intramedullary osteosarcoma, and myeloid leukemia. As such, immunohistochemistry (IHC) is often needed to differentiate histiocytic sarcomas from other common tumors in mice that they can morphologically mimic. The goal of this article is to present a broader perspective of the diverse cellular morphologies, growth patterns, organ distributions, and IHC labeling of histiocytic sarcomas encountered by the authors. This article describes these features in a set of 62 mouse histiocytic sarcomas, including the IHC characterization of the tumors using a panel of markers for the macrophage antigens F4/80, IBA1, MAC2, CD163, CD68, and lysozyme, and describes differentiating features of histiocytic sarcomas from other morphologically similar tumors. The genetic changes underlying the pathogenesis of histiocytic sarcoma in humans are beginning to be elucidated, but this is difficult due to its rarity. The higher prevalence of this tumor in mice provides opportunities to investigate mechanisms of its development and to test potential treatments.

Partial blood replacement ameliorates middle cerebral artery occlusion generated neurological aberrations by intervening TLR4 and NLRP3 cascades in rats.

Acute ischemic stroke is a catastrophic medical condition that causes severe disability and mortality if the sufferer escapes treatment within a stipulated timeframe. While timely intervention with clot-bursting agents like tissue-plasminogen activators abrogates some post-stroke neurologic deficits, no neuroprotective therapy is yet promisingly addresses the post-recanalization neuroinflammation in post-stroke survivors. Herein, we investigated the effect of partial blood replacement therapy (BRT), obtained from healthy and treadmill-trained donor rats, on neurological deficits, and peripheral and central inflammatory cascades using the ischemia-reperfusion animal paradigm. The cerebral ischemia-reperfusion was induced in rats by occlusion of the middle cerebral artery (MCAO) for 90 min, followed by reperfusion. Rats underwent MCAO surgery displayed remarkable sensorimotor and motor deficits in rotarod, foot fault, adhesive removal, and paw whisker tests till 5 days post-surgery. These behavior abnormalities were ameliorated in the BRT-recipient MCAO rats. BRT also reduced the infarct volume and neuronal death in the ipsilateral hemisphere revealed by TTC and cresyl violet staining compared to the MCAO group. Rats received BRT infusion exhibited the reduced expression of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule-1 (Iba-1), and MyD88 on day 5 post-MCAO in immunohistochemistry and immunofluorescent assays. Moreover, elevated levels of toll-like receptor 4 (TLR4) and mRNA expression of IL-1ß, TNF-α, matrix metalloproteinase-9 and NLRP3, and decreased levels of zonula occludens-1 in MCAO rats, were reversed following BRT. These findings suggest that the partial BRT may rescind MCAO-induced neurological dysfunctions and cerebral injury by intervening in the TLR4 and NLRP3 pathways in rats.

The Arabidopsis NRT1/PTR FAMILY protein NPF7.3/NRT1.5 is an indole-3-butyric acid transporter involved in root gravitropism.

Active membrane transport of plant hormones and their related compounds is an essential process that determines the distribution of the compounds within plant tissues and, hence, regulates various physiological events. Here, we report that the Arabidopsis NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER FAMILY 7.3 (NPF7.3) protein functions as a transporter of indole-3-butyric acid (IBA), a precursor of the major endogenous auxin indole-3-acetic acid (IAA). When expressed in yeast, NPF7.3 mediated cellular IBA uptake. Loss-of-function npf7.3 mutants showed defective root gravitropism with reduced IBA levels and auxin responses. Nevertheless, the phenotype was restored by exogenous application of IAA but not by IBA treatment. NPF7.3 was expressed in pericycle cells and the root tip region including root cap cells of primary roots where the IBA-to-IAA conversion occurs. Our findings indicate that NPF7.3-mediated IBA uptake into specific cells is required for the generation of appropriate auxin gradients within root tissues.

Microglia in Cultured Porcine Retina: Qualitative Immunohistochemical Analyses of Reactive Microglia in the Outer Retina.

A late stage of several retinal disorders is retinal detachment, a complication that results in rapid photoreceptor degeneration and synaptic damages. Experimental retinal detachment in vivo is an invasive and complicated method performed on anesthetized animals. As retinal detachment may result in visual impairment and blindness, research is of fundamental importance for understanding degenerative processes. Both morphological and ethical issues make the porcine retina a favorable organotypic model for studies of the degenerative processes that follow retinal detachment. In the cultured retina, photoreceptor degeneration and synaptic injuries develop rapidly and correlate with resident microglial cells' transition into a reactive phenotype. In this immunohistochemical study, we have begun to analyze the transition of subsets of reactive microglia which are known to localize close to the outer plexiform layer (OPL) in degenerating in vivo and in vitro retina. Biomarkers for reactive microglia included P2Ry12, CD63 and CD68 and the general microglial markers were CD11b, Iba1 and isolectin B4 (IB4). The reactive microglia markers labeled microglia subpopulations, suggesting that protective or harmful reactive microglia may be present simultaneously in the injured retina. Our findings support the usage of porcine retina cultures for studies of photoreceptor injuries related to retinal detachment.

Effects of Therapeutic Hypothermia on Macrophages in Mouse Cochlea Explants.

Globally, over the next few decades, more than 2.5 billion people will suffer from hearing impairment, including profound hearing loss, and millions could potentially benefit from a cochlea implant. To date, several studies have focused on tissue trauma caused by cochlea implantation. The direct immune reaction in the inner ear after an implantation has not been well studied. Recently, therapeutic hypothermia has been found to positively influence the inflammatory reaction caused by electrode insertion trauma. The present study aimed to evaluate the hypothermic effect on the structure, numbers, function and reactivity of macrophages and microglial cells. Therefore, the distribution and activated forms of macrophages in the cochlea were evaluated in an electrode insertion trauma cochlea culture model in normothermic and mild hypothermic conditions. In 10-day-old mouse cochleae, artificial electrode insertion trauma was inflicted, and then they were cultured for 24 h at 37 °C and 32 °C. The influence of mild hypothermia on macrophages was evaluated using immunostaining of cryosections using antibodies against IBA1, F4/80, CD45 and CD163. A clear influence of mild hypothermia on the distribution of activated and non-activated forms of macrophages and monocytes in the inner ear was observed. Furthermore, these cells were located in the mesenchymal tissue in and around the cochlea, and the activated forms were found in and around the spiral ganglion tissue at 37 °C. Our findings suggest that mild hypothermic treatment has a beneficial effect on immune system activation after electrode insertion trauma.

Switching Rat Resident Macrophages from M1 to M2 Phenotype by Iba1 Silencing Has Analgesic Effects in SNL-Induced Neuropathic Pain.

Resident macrophages from dorsal root ganglia are important for the development of traumatic-induced neuropathic pain. In the first 5-7 days after a traumatic sciatic nerve injury (i.e., spinal nerve ligation (SNL), spared nerve injury (SNI), sciatic nerve transection or sciatic nerve ligation and transection), Ionized binding adapter protein 1 (Iba1) (+) resident macrophages cluster around dorsal root ganglia neurons, possibly contributing to nerve injury-induced hypersensitivity. Since infiltrating macrophages gradually recruited to the lesion site peak at about 7 days, the first few days post-lesion offer a window of opportunity when the contribution of Iba1 (+) resident macrophages to neuropathic pain pathogenesis could be investigated. Iba1 is an actin cross-linking cytoskeleton protein, specifically located only in macrophages and microglia. In this study, we explored the contribution of rat Iba1 (+) macrophages in SNL-induced neuropathic pain by using intra-ganglionic injections of naked Iba1-siRNA, delivered at the time the lesion occurred. The results show that 5 days after Iba1 silencing, Iba1 (+) resident macrophages are switched from an M1 (pro-inflammatory) phenotype to an M2 (anti-inflammatory) phenotype, which was confirmed by a significant decrease of M1 markers (CD32 and CD86), a significant increase of M2 markers (CD163 and Arginase-1), a reduced secretion of pro-inflammatory cytokines (IL-6, TNF-α and IL-1ß) and an increased release of pro-regenerative factors (BDNF, NGF and NT-3) which initiated the regrowth of adult DRG neurites and reduced SNL-induced neuropathic pain. Our data show for the first time, that it is possible to induce macrophages towards an anti-inflammatory phenotype by interacting with their cytoskeleton.

Microglia and Astroglia-The Potential Role in Neuroinflammation Induced by Pre- and Neonatal Exposure to Lead (Pb).

Neuroinflammation is one of the postulated mechanisms for Pb neurotoxicity. However, the exact molecular mechanisms responsible for its pro-inflammatory effect are not fully elucidated. In this study, we examined the role of glial cells in neuroinflammation induced by Pb exposure. We investigated how microglia, a type of glial cell, responded to the changes caused by perinatal exposure to Pb by measuring the expression of Iba1 at the mRNA and protein levels. To assess the state of microglia, we analyzed the mRNA levels of specific markers associated with the cytotoxic M1 phenotype (Il1b, Il6, and Tnfa) and the cytoprotective M2 phenotype (Arg1, Chi3l1, Mrc1, Fcgr1a, Sphk1, and Tgfb1). Additionally, we measured the concentration of pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α). To assess the reactivity and functionality status of astrocytes, we analyzed the GFAP (mRNA expression and protein concentration) as well as glutamine synthase (GS) protein level and activity. Using an electron microscope, we assessed ultrastructural abnormalities in the examined brain structures (forebrain cortex, cerebellum, and hippocampus). In addition, we measured the mRNA levels of Cxcl1 and Cxcl2, and their receptor, Cxcr2. Our data showed that perinatal exposure to Pb at low doses affected both microglia and astrocyte cells' status (their mobilization, activation, function, and changes in gene expression profile) in a brain-structure-specific manner. The results suggest that both microglia and astrocytes represent a potential target for Pb neurotoxicity, thus being key mediators of neuroinflammation and further neuropathology evoked by Pb poisoning during perinatal brain development.