Neurosteroid [3α,5α]3-hydroxypregnan-20-one inhibition of chemokine monocyte chemoattractant protein-1 in alcohol-preferring rat brain neurons, microglia, and astroglia.

BACKGROUND: Neuroimmune dysfunction in alcohol use disorder (AUD) is associated with activation of myeloid differentiation primary response 88 (MyD88)-dependent Toll-like receptors (TLR) resulting in overexpression of the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). MCP-1 overexpression in the brain is linked to anxiety, higher alcohol intake, neuronal death, and activation of microglia observed in AUD. The neurosteroid [3α,5α][3-hydroxypregnan-20-one (3α,5α-THP) has been reported as an inhibitor of MyD88-dependent TLR activation and MCP-1 overexpression in mouse and human macrophages and the brain of alcohol-preferring (P) rats. METHODS: We investigated how 3α,5α-THP regulates MCP-1 expression at the cellular level in P rat nucleus accumbens (NAc) and central amygdala (CeA). We focused on neurons, microglia, and astrocytes, examining the individual voxel density of MCP-1, neuronal marker NeuN, microglial marker IBA1, astrocytic marker GFAP, and their shared voxel density, defined as intersection. Ethanol-naïve male and female P rats were perfused 1 h after IP injections of 15 mg/kg of 3α,5α-THP, or vehicle. The NAc and CeA were imaged using confocal microscopy following double-immunofluorescence staining for MCP-1 with NeuN, IBA1, and GFAP, respectively. RESULTS: MCP-1 intersected with NeuN predominantly and IBA1/GFAP negligibly. 3α,5α-THP reduced MCP-1 expression in NeuN-labeled cells by 38.27 ± 28.09% in male and 56.11 ± 21.46% in female NAc, also 37.99 ± 19.53% in male and 54.96 ± 30.58% in female CeA. In females, 3α,5α-THP reduced the MCP-1 within IBA1 and GFAP-labeled voxels in the NAc and CeA. Conversely, in males, 3α,5α-THP did not significantly alter the MCP-1 within IBA1 in NAc or with GFAP in the CeA. Furthermore, 3α,5α-THP decreased levels of IBA1 in both regions and sexes with no impact on GFAP or NeuN levels. Secondary analysis performed on data normalized to % control values indicated that no significant sex differences were present. CONCLUSIONS: These data suggest that 3α,5α-THP inhibits neuronal MCP-1 expression and decreases the proliferation of microglia in P rats. These results increase our understanding of potential mechanisms for 3α,5α-THP modulation of ethanol consumption.

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.

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.

Immuno-surveillance and protection of the human cochlea.

Background: Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Materials and methods: Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Results: Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. Discussion: The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

Expression of ChAT, Iba-1, and nNOS in the Central Nervous System following Facial Nerve Injury.

Facial nerve injury can cause significant functional impairment, impacting both the peripheral and central nervous systems. The present study evaluated changes in facial motor function, numbers of cholinergic neurons and microglia, and nNOS levels in the facial nucleus of the central nervous system (CNS) following peripheral facial nerve injury. Facial nerve function, as determined by eyeblink and whisker-movement reflexes, was evaluated at baseline and 1, 2, 3, 4, 8, and 12 weeks after inducing facial nerve injury through compression or axotomy. The expression of choline acetyltransferase (ChAT), ionized calcium-binding adaptor molecule 1 (Iba-1), and neuronal nitric oxide synthase (nNOS) in the facial nucleus of the CNS was analyzed 2, 4, and 12 weeks after peripheral facial nerve injury. Compression-induced facial nerve injury was found to lead to temporary facial motor impairment, whereas axotomy resulted in persistent impairment. Moreover, both compression and axotomy reduced ChAT expression and increased Iba-1 and nNOS expression in the facial nucleus, indicating upregulation of an inflammatory response and neurodegeneration. These results indicate that, compared with compression-induced injury, axotomy-induced facial nerve injury results in greater facial motor dysfunction and more persistent microglial and nitric oxide activation in the facial nucleus of the CNS.

Congenital localized cutaneous Langerhans cell histiocytosis in a Holstein calf.

Distinct solitary dermal nodules, either covered by an alopecic, or sometimes ulcerated, epidermis, were noticed on the head of a stillborn Holstein calf. The head was submitted for autopsy, and the nodules were found to consist of homogeneous, diffuse pale-yellow, soft-tissue masses with distinct margins that elevated the epidermis above the adjacent skin. Histologically, the dermal nodules were well-delineated on the deep margin approaching the cutaneous muscle and consisted of perivascular neoplastic infiltrates of round cells that in some places coalesced into sheets that extended into the dermis and subcutis. Neoplastic cells separated adnexa and collagen. Immunohistochemistry revealed intense tumor cell expression of vimentin, Iba1, E-cadherin, and CD204; expression of CD18 was faint. The masses were diagnosed as Langerhans cell histiocytosis. Congenital cutaneous Langerhans cell histiocytosis has not been reported previously in cattle, to our knowledge, and should be included in the differential diagnosis of congenital nodular skin lesions.

Short Working Memory Impairment Associated with Hippocampal Microglia Activation in Chronic Hepatic Encephalopathy.

Hepatic encephalopathy (HE) is a major neuropsychological condition that occursas a result of impaired liver function. It is frequently observed in patients with advanced liver disease or cirrhosis. Memory impairment is among the symptoms of HE; the pathophysiologic mechanism for this enervating condition remains unclear. However, it is possible that neuroinflammation may be involved, as recent studies have emphasized such phenomena. Therefore, the aim of the present study is to assess short working memory (SWM) and examine the involvement of microglia in a chronic model of HE. The study was carried out with male Wistar rats that were induced by repeated thioacetamide (TAA) administration (100 mg/kg i.p injection for 10 days). SWM function was assessed through Y-maze, T-Maze, and novel object recognition (NOR) tests, together with an immunofluorescence study of microglia activation within the hippocampal areas. Our data showed impaired SWM in TAA-treated rats that was associated with microglial activation in the three hippocampal regions, and which contributed to cognitive impairment.

A role for immunohistochemical stains in perinatal brain autopsies.

Identification of central nervous system injury is a critical part of perinatal autopsies; however, injury is not always easily identifiable due to autolysis and immaturity of the developing brain. Here, the role of immunohistochemical stains in the identification of perinatal brain injury was investigated. Blinded semiquantitative scoring of injury was performed on sections of frontal lobe from 76 cases (51 liveborn and 25 stillborn) using H&E, GFAP, Iba-1, and ß-APP stains. Digital image analysis was used to quantify GFAP and Iba-1 staining. Commonly observed pathologies included diffuse white matter gliosis (DWMG) and white matter necrosis (WMN). DWMG scores were very similar on H&E and GFAP stains for liveborn subjects. For stillborn subjects, DWMG scores were significantly higher on GFAP stain than H&E. ß-APP was needed for identification of WMN in 71.4% of stillborn subjects compared to 15.4% of liveborn subjects. Diffuse staining for Iba-1 within cortex and white matter was positively correlated with subject age. Staining quantification on digital image analysis was highly correlated to semiquantitative scoring. Overall, GFAP and ß-APP stains were most helpful in identifying white matter injury not seen on H&E in stillborn subjects. Immunostains may therefore be warranted as an integral part of stillborn brain autopsies.

Proteomic study of left ventricle and cortex in rats after myocardial infarction.

Myocardial infarction (MI) induces neuroinflammation indirectly, chronic neuroinflammation may cause neurodegenerative diseases. Changes in the proteomics of heart and brain tissue after MI may shed new light on the mechanisms involved in neuroinflammation. This study explored brain and heart protein changes after MI with a data-independent acquisition (DIA) mode proteomics approach. Permanent ligation of the left anterior descending coronary artery (LAD) was performed in the heart of rats, and the immunofluorescence of microglia in the brain cortex was performed at 1d, 3d, 5d, and 7d after MI to detect the neuroinflammation. Then proteomics was accomplished to obtain the vital proteins in the heart and brain post-MI. The results show that the number of microglia was significantly increased in the Model-1d group, the Model-3d group, the Model-5d group, and the Model-7d group compared to the Sham group. Various proteins were obtained through DIA proteomics. Linking to key targets of brain disease, 14 proteins were obtained in the brain cortex. Among them, elongation of very long chain fatty acids protein 5 (ELOVL5) and ATP-binding cassette subfamily G member 4 (ABCG4) were verified through western blotting (WB). The results of WB were consistent with the proteomics results. Therefore, these proteins may be related to the pathogenesis of neuroinflammation after MI.

α-Linolenic Acid Induces Microglial Activation and Extracellular Tau Internalization.

Neuroinflammation is the brain condition that occurs due to the hyper-activation of brain's immune cells and microglia, over the stimulation of extracellular aggregated proteins such as amyloid plaques and by extracellular Tau as well. The phenotypic changes of microglia from inflammatory to anti-inflammatory can be triggered by many factors, which also includes dietary fatty acids. The classes of omega-3 fatty acids are the majorly responsible in maintaining the anti-inflammatory phenotype of microglia. The enhanced phagocytic ability of microglia might induce the clearance of extracellular aggregated proteins, such as amyloid beta and Tau. In this study, we emphasized on the effect of α-linolenic acid (ALA) on the activation of microglia and internalization of the extracellular Tau seed in microglia.

The role of glial cells in mental illness: a systematic review on astroglia and microglia as potential players in schizophrenia and its cognitive and emotional aspects.

Schizophrenia is a complex and severe mental disorder that affects approximately 1% of the global population. It is characterized by a wide range of symptoms, including delusions, hallucinations, disorganized speech and behavior, and cognitive impairment. Recent research has suggested that the immune system dysregulation may play a significant role in the pathogenesis of schizophrenia, and glial cells, such as astroglia and microglia known to be involved in neuroinflammation and immune regulation, have emerged as potential players in this process. The aim of this systematic review is to summarize the glial hallmarks of schizophrenia, choosing as cellular candidate the astroglia and microglia, and focusing also on disease-associated psychological (cognitive and emotional) changes. We conducted a systematic review following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We searched PubMed, Scopus, and Web of Science for articles that investigated the differences in astroglia and microglia in patients with schizophrenia, published in the last 5 years. The present systematic review indicates that changes in the density, morphology, and functioning of astroglia and microglia may be involved in the development of schizophrenia. The glial alterations may contribute to the pathogenesis of schizophrenia by dysregulating neurotransmission and immune responses, worsening cognitive capabilities. The complex interplay of astroglial and microglial activation, genetic/epigenetic variations, and cognitive assessments underscores the intricate relationship between biological mechanisms, symptomatology, and cognitive functioning in schizophrenia.

Multisession anodal epidural direct current stimulation of the auditory cortex delays the progression of presbycusis in the Wistar rat.

Presbycusis or age-related hearing loss (ARHL) is one of the most prevalent chronic health problems facing aging populations. Along the auditory pathway, the stations involved in transmission and processing, function as a system of interconnected feedback loops. Regulating hierarchically auditory processing, auditory cortex (AC) neuromodulation can, accordingly, activate both peripheral and central plasticity after hearing loss. However, previous ARHL-prevention interventions have mainly focused on preserving the structural and functional integrity of the inner ear, overlooking the central auditory system. In this study, using an animal model of spontaneous ARHL, we aim at assessing the effects of multisession epidural direct current stimulation of the AC through stereotaxic implantation of a 1-mm silver ball anode in Wistar rats. Consisting of 7 sessions (0.1 mA/10 min), on alternate days, in awake animals, our stimulation protocol was applied at the onset of hearing loss (threshold shift detection at 16 months). Click- and pure-tone auditory brainstem responses (ABRs) were analyzed in two animal groups, namely electrically stimulated (ES) and non-stimulated (NES) sham controls, comparing recordings at 18 months of age. At 18 months, NES animals showed significantly increased threshold shifts, decreased wave amplitudes, and increased wave latencies after click and tonal ABRs, reflecting a significant, spontaneous ARHL evolution. Conversely, in ES animals, no significant differences were detected in any of these parameters when comparing 16 and 18 months ABRs, indicating a delay in ARHL progression. Electrode placement in the auditory cortex was accurate, and the stimulation did not cause significant damage, as shown by the limited presence of superficial reactive microglial cells after IBA1 immunostaining. In conclusion, multisession DC stimulation of the AC has a protective effect on auditory function, delaying the progression of presbycusis.

Proteomic analysis identifies HSP90AA1, PTK2B, and ANXA2 in the human entorhinal cortex in Alzheimer's disease: Potential role in synaptic homeostasis and Aß pathology through microglial and astroglial cells.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder worldwide, is clinically characterized by cognitive deficits. Neuropathologically, AD brains accumulate deposits of amyloid-ß (Aß) and tau proteins. Furthermore, these misfolded proteins can propagate from cell to cell in a prion-like manner and induce native proteins to become pathological. The entorhinal cortex (EC) is among the earliest areas affected by tau accumulation along with volume reduction and neurodegeneration. Neuron-glia interactions have recently come into focus; however, the role of microglia and astroglia in the pathogenesis of AD remains unclear. Proteomic approaches allow the determination of changes in the proteome to better understand the pathology underlying AD. Bioinformatic analysis of proteomic data was performed to compare ECs from AD and non-AD human brain tissue. To validate the proteomic results, western blot, immunofluorescence, and confocal studies were carried out. The findings revealed that the most disturbed signaling pathway was synaptogenesis. Because of their involvement in synapse function, relationship with Aß and tau proteins and interactions in the pathway analysis, three proteins were selected for in-depth study: HSP90AA1, PTK2B, and ANXA2. All these proteins showed colocalization with neurons and/or astroglia and microglia and with pathological Aß and tau proteins. In particular, ANXA2, which is overexpressed in AD, colocalized with amoeboid microglial cells and Aß plaques surrounded by astrocytes. Taken together, the evidence suggests that unbalanced expression of HSP90AA1, PTK2B, and ANXA2 may play a significant role in synaptic homeostasis and Aß pathology through microglial and astroglial cells in the human EC in AD.

Memory impairment induced by aluminum nanoparticles is associated with hippocampal IL-1 and IBA-1 upregulation in mice.

OBJECTIVES: Increasing evidence indicates a link between aluminum (Al) intake and Alzheimer's disease (AD). The main entry of Al into the human body is through oral route, and in the digestive tract, under the influence of the pH change, Al can be transformed into Al nanoparticles (Al-NP). However, studies related to the effect of Al-NP on the brain are limited and need further investigation. Neuro-inflammation is considered as one of the principal features of AD. Microglial activation and expression of the inflammatory cytokine IL-1ß (interleukin-1ß) in the brain have been used as hallmarks of brain inflammation. Therefore, in the present study, the hippocampal levels of ionized calcium-binding adaptor molecule 1 (IBA-1), as the marker of microglia activation, and IL-1ß were assessed. METHODS: Adult male NMRI mice were treated with Al-NP (5 or 10 mg/kg) for 5 days. A novel object recognition (NOR) test was used to assess memory. Following cognitive assessments, the hippocampal tissues were isolated to analyze the levels of IL-1ß and IBA-1 as well as beta actin proteins using western blot technique. RESULTS: Al-NP in both doses of 5 and 10 mg/kg impaired NOR memory in mice. In addition, Al-NP increased IL-1ß and IBA-1 in the hippocampus. DISCUSSION: These findings indicate that the memory impairing effect of Al-NP coincides with hippocampal inflammation. According to the proposed relationship between AD and Al toxicity, this study can increase the knowledge about the toxic effects of Al-NP and highlight the need to limit the use of this nanoparticle.

Genetic and Immunohistochemistry Tools to Visualize Rat Macrophages In Situ.

Macrophages contribute to many aspects of development and homeostasis, innate and acquired immunity, immunopathology, and tissue repair. Every tissue contains an abundant resident macrophage population. Inflammatory stimuli promote the recruitment of monocytes from the blood and their adaptation promotes the removal of the stimulus and subsequent restoration of normal tissue architecture. Dysregulation of this response leads to chronic inflammation and tissue injury. In many tissues, their differentiation and survival are dependent on the colony stimulating factor 1 receptor (CSF1R) signalling axis, which is highly conserved across all vertebrates. Complete loss of either CSF1R or its cognate ligands, colony stimulating factor 1 (CSF1), and interleukin 34 (IL-34), results in the loss of many tissue-resident macrophage populations. This provides a useful paradigm to study macrophages.There are many tools used to visualize tissue-resident macrophages and their precursors, monocytes, in mice and humans. Particularly in mice there are genetic tools available to delete, enhance and manipulate monocytes and macrophages and their gene products to gain insight into phenotype and function. The laboratory rat has many advantages as an experimental model for the understanding of human disease, but the analytical resources are currently more limited than in mice. Here, we describe available genetic models, antibodies, and immunohistochemistry (IHC) methods that may be used to visualize tissue-resident macrophages in rats.

Caffeine Administration Mitigates Chronic Stress-Induced Behavioral Deficits, Neurochemical Alterations, and Glial Disruptions in Rats.

Prolonged exposure to stress has detrimental effects on health, and the consumption of caffeine, mostly contained in energy drinks, has become a widely adopted stress coping strategy. Currently, there is limited information regarding the effects of caffeine intake on chronic stress exposure. Thus, this study investigated the effects of caffeine administration on chronic stress-induced behavioral deficits, neurochemical alterations, and glial disruptions in experimental rats. Thirty male Wistar rats were randomly assigned to five groups (n = 6): non-stress control, stress control, and caffeine groups of doses 12.5, 25, and 50 mg/kg. The stress control and caffeine groups were subjected to an unpredictable chronic mild stress (UCMS) protocol daily for 14 days. The rats were evaluated for phenotypic and neurobehavioral assessments. Thereafter, the rat brains were processed for biochemical and immunohistochemical assays. Caffeine administration was found to ameliorate behavioral dysfunctions in rats exposed to UCMS. The UCMS-induced changes in brain levels of monoamines, cholinesterases, and some oxidative stress biomarkers were reversed by caffeine. Caffeine administration also produced mild protective effects against UCMS-induced changes in GFAP and Iba-1 expression in stress-specific brain regions. These results showed that low and moderate doses of caffeine reversed most of the stress-induced changes, suggesting its ameliorative potential against chronic stress-induced alterations.

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.

Carob Extract (Ceratonia siliqua L.): Effects on Dyslipidemia and Obesity in a High-Fat Diet-Fed Rat Model.

Dyslipidemia and obesity are recognized as two of the major global health issues and main risk factors for coronary heart disease and cerebrovascular disease. In recent years, carob has shown certain antioxidant and anti-dyslipidemic potential. In this study, Wistar rats were fed with a standard and cholesterol-enriched diet and treated orally with carob extract and simvastatin for four weeks. After sacrifice, blood samples were collected for biochemical analysis, and liver tissue was taken for histological and immunohistochemical assessment. Weight gain was significantly higher in groups fed with cholesterol-fortified granules; total cholesterol was found to be significantly lower in the hypercholesterolemic groups treated with simvastatin and simvastatin/carob combined regimens compared with hypercholesterolemic animals treated with saline (p < 0.05). The same was true for low-density lipoprotein cholesterol and the LDL/HDL ratio (p < 0.05). Adiponectin was remarkably higher in animals treated with simvastatin compared to all other groups (p < 0.05). Leptin was significantly lower in groups treated with carob and simvastatin compared to the hypercholesterolemic group treated with saline (p < 0.05). Carob/simvastatin co-administration reduced hepatocyte damage and improved liver morphology. A study confirmed the anti-dyslipidemic, anti-obesity, and hepatoprotective potential of carob pulp alone or in combination with simvastatin in the treatment of high-fat diet-fed rats.

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.

Developmental profile of microglia distribution in nuclei of the superior olivary complex.

In the brain, microglia are involved in immune responses and synaptic maturation. During early development, these cells invade the brain, proliferate, and morphologically mature to achieve coverage of the surrounding tissue with their fine processes. Their developmental proliferation overlaps with the postnatal development of neuronal circuits. Within the superior olivary complex (SOC), an auditory brainstem structure, microglia, and their early postnatal development have been documented. A quantification over the full developmental profile of the arrangement and morphological changes in single microglia cells is missing. Here, we used immunofluorescence labeling to quantify their distribution, morphological changes, and coverage during early and late postnatal development in the SOC of Mongolian gerbils. Microglia distributed rather homogenously within each nucleus with a bias to the nucleus borders at postnatal day (P) 5 and more centrally in the nucleus in mature stages. We found a nucleus-specific transient increase in microglia cell number and density reaching its peak at P17 with a subsequent decline to P55 values. Length and branching of microglia protrusions increased especially after P12. The stronger ramification together with the increase in cell density allows coverage of the surrounding tissue from P5 to mature stages, despite the large developmental increase in nucleus size. The transient increase in density during synaptic refinement in SOC nuclei suggests that microglia are important during the pruning period, compensating for developmental increase in tissue volume, and that in mature stages their main function appears tissue surveillance.