Dark Agouti Rats Research Articles

Resveratrol nanoparticles reduce retinal ganglion cell loss in glaucoma

AbstractPurposeResveratrol is a natural polyphenol found in red wine and dark chocolate known for its antioxidant, anti‐inflammatory and anti‐apoptotic properties. All these properties can be used to treat neurodegenerative diseases such as glaucoma. To date, resveratrol low solubility in water and bioavailability limits its clinical use. The goal of this study was to develop a novel nanoparticle formulation of resveratrol and to assess it in an ocular hypertension (OHT) rat model of glaucoma.MethodsResveratrol nanoparticles (RNs) were formulated using a thin film rehydration technique. OHT rat model was induced with the injection of 1.85 M normal saline solution in two episcleral veins of the left eye of 10 Dark Agouti rats. The right eye was used as an internal control. RNs (n = 5) or vehicle (n = 5) were given topically as an eye drop daily for 3 weeks from induction. After 3 weeks, rats were imaged with the Detection of Apoptosing Retinal Cell (DARC) technology. DARC count was defined as the number of apoptosing retinal cells counted by a masked investigator. Next, all rats were sacrificed, and their retinas immunostained with Brn3a antibody. Retinal Ganglion Cell (RGC) density ratio was defined as the ratio between the left treated and the right untreated eye (control).ResultsRNs were formulated with an encapsulation efficiency >70% and a stability >90 days. They were well‐tolerated without any sign of ocular irritation and neuroprotective in the OHT glaucoma model as evidenced by the reduction of RGC apoptosis compared to the vehicle (1.02 ± 0.05 vs 0.70 ± 0.11 RGC density ratio, p < 0.05). RNs did not reduce the Intraocular Pressure (IOP) suggesting that their effect was independent from IOP modulation. However, RNs were not able to reduce significantly DARC count compared to the vehicle (493 ± 83 vs 612 ± 84 DARC count, p > 0.05).ConclusionsThese promising results show that topical administration of RNs is neuroprotective in an OHT glaucoma rat model by reducing significantly RGC apoptosis. DARC count reduction is not significant owing to the low number of animals used. Therefore, bigger studies are needed to confirm these encouraging results.

Cytochrome P450 2D (CYP2D) enzyme dysfunction associated with aging and serotonin deficiency in the brain and liver of female Dark Agouti rats

Among the enzymes that support brain metabolism, cytochrome P450 (CYP) enzymes occupy an important place. These enzymes catalyze the biotransformation pathways of neuroactive endogenous substrates (neurosteroids, neurotransmitters) and are necessary for the detoxification processes. The aim of the present study was to assess changes in the CYP2D activity and protein level during the aging process and as a result of serotonin deficiency in the female brain. The CYP2D activity was measured in brain and liver microsomes of Dark Agouti wild type (WT) female rats (mature 15-week-old and senescent 18-month-old rats) and in tryptophan hydroxylase 2 (TPH2)-deficient senescent female rats. The CYP2D activity in mature WT Dark Agouti females was independent of the changing phases of the estrous cycle. In senescent WT females rats, the CYP2D activity and protein level were decreased in the cerebral cortex, hippocampus, cerebellum and liver, but increased in the brain stem. In the other examined structures (frontal cortex, hypothalamus, thalamus, striatum), the enzyme activity did not change. In aging TPH2-deficient females, the CYP2D activity and protein levels were decreased in the frontal cortex, hypothalamus and brain stem (activity only), remaining unchanged in other brain structures and liver, relative to senescent WT females. In summary, the aging process and TPH2 deficit affect the CYP2D activity and protein level in female rats, which may have a negative impact on the compensatory capacity of CYP2D in the synthesis of serotonin and dopamine in cerebral structures involved in cognitive and emotional functions. In the liver, the CYP2D-catalyzed drug metabolism may be diminished in elderly females. The results in female rats are compared with those obtained previously in males. It is concluded that aging and serotonin deficiency exert sex-dependent effects on brain CYP2D, which seem to be less favorable in females concerning CYP2D-mediated neurotransmitter synthesis, but beneficial regarding slower neurosteroid metabolism.

Gender-Related Differences in BMP Expression and Adult Hippocampal Neurogenesis within Joint-Hippocampal Axis in a Rat Model of Rheumatoid Arthritis.

BMPs regulate synovial quiescence and adult neurogenesis in the hippocampus in non-stress conditions. However, changes in BMP expression that are induced by inflammation during rheumatoid arthritis (RA) have not yet been reported. Here, we show that signalling with synovial BMPs (BMP-4 and -7) mediates the effect of systemic inflammation on adult neurogenesis in the hippocampus during pristane-induced arthritis (PIA) in Dark Agouti (DA) rats, an animal model of RA. Moreover, we show gender differences in BMP expressions and their antagonists (Noggin and Gremlin) during PIA and their correlations with the clinical course and IL-17A and TNF-α levels in serum. Our results indicate gender differences in the clinical course, where male rats showed earlier onset and earlier recovery but a worse clinical course in the first two phases of the disease (onset and peak), which correlates with the initial increase of serum IL-17A level. The clinical course of the female rats worsened in remission. Their prolonged symptoms could be a reflection of an increased TNF-α level in serum during remission. Synovial inflammation was greater in females in PIA-remission with greater synovial BMP and antagonist expressions. More significant correlations between serum cytokines (IL-17A and TNF-α), and synovial BMPs and their antagonists were found in females than in males. On the other hand, males showed an increase in hippocampal BMP-4 expression during the acute phase, but both genders showed a decrease in antagonist expressions during PIA in general. Both genders showed a decrease in the number of Ki-67+ and SOX-2+ and DCX+ cells and in the ratio of DCX+ to Ki67+ cells in the dentate gyrus during PIA. However, in PIA remission, females showed a faster increase in the number of Ki67+, SOX-2+, and DCX+ cells and a faster increase in the DCX/Ki67 ratio than males. Both genders showed an increase of hippocampal BMP-7 expression during remission, although males constantly showed greater BMP-7 expression at all time points. Our data show that gender differences exist in the BMP expressions in the periphery–hippocampus axis and in the IL-17A and TNF-α levels in serum, which could imply differences in the mechanisms for the onset and progression of the disease, the clinical course severity, and adult neurogenesis with subsequent neurological complications between genders.

Non-invasive visual evoked potentials under sevoflurane versus ketamine-xylazine in rats

BackgroundVisual Evoked Potential (VEP) quantifies electrical signals produced in visual cortex in response to visual stimuli. VEP elicited by light flashes is a useful biomarker to evaluate visual function in preclinical models and it can be recorded in awake or anaesthetised state. Different types of anaesthesia influence VEP properties, such as latency, which measures the propagation speed along nerve fibers, and amplitude that quantifies the power of electrical signal. AimThe goal of this work is to compare VEPs elicited in Dark Agouti rats under two types of anaesthesia: volatile sevoflurane or injectable ketamine-xylazine. MethodsVEP latency, amplitude, signal-to-noise ratio and recording duration were measured in Dark Agouti rats randomly assigned to two groups, the first subjected to volatile sevoflurane and the second to injectable ketamine-xylazine. Taking advantage of non-invasive flash-VEP recording through epidermal cup electrodes, three time points of VEP recordings were assessed in two weeks intervals. ResultsVEP recorded under ketamine-xylazine showed longer latency and higher amplitude compared with sevoflurane, with analogous repeatability over time. However, sevoflurane tended to suppress electrical signals from visual cortex, resulting in a lower signal-to-noise ratio. Moreover, VEP procedure duration lasted longer in rats anaesthetised with sevoflurane than ketamine-xylazine. ConclusionsIn Dark Agouti rats, the use of different anaesthesia can influence VEP components in terms of latency and amplitude. Notably, sevoflurane and ketamine-xylazine revealed satisfying repeatability over time, which is critical to perform reliable follow-up studies. Ketamine-xylazine allowed to obtain more clearly discernible VEP components and less background noise, together with a quicker recording procedure and a consequently improved animal safety and welfare.

Periapical lesions in two inbred strains of rats differing in immunological reactivity.

To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. DA rats developed significantly larger (p<.05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p<.05). In DA rats, periapical lesions had a significantly higher (p<.05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p<.05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1β, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p<.05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p<.05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p<.05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p<.05) in the supernatant in the periapical lesions of DA rats. After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.

Neuroimmunological complications arising from chemotherapy-induced gut toxicity and opioid exposure in female dark agouti rats.

Cancer patients may experience symptom clusters, including chemotherapy-induced (CI) gut toxicity (CIGT) and cognitive impairment. Analgesic selection for pain associated with CIGT is difficult as opioids induce glial reactivity and unwanted side effects. This study quantified central glial reactivity and proinflammatory effects in rats with CIGT using three mechanistically different analgesics. Regional adaptations were indicative of immune-to-brain signaling routes. Utilizing a 5-fluorouracil-induced GT (5IGT) rat model and analgesic intervention (carprofen (CAR), buprenorphine (BUP), and tramadol (TRAM)), spinal and brain neuroimmune modulation was examined via microglial, astrocyte, and proinflammatory (cluster of differentiation molecule 11b; CD11b, glial fibrillary associated protein; GFAP, and interleukin-1 beta; IL1β) reactivity marker expression changes by western blot analysis. 5IGT significantly increased thoracic GFAP (p<0.05) and IL-1β (p<0.0001) expression, CAR and BUP ameliorated these effects. BUP and TRAM with 5-FU synergistically increased hippocampal GFAP expression. CAR administered with 5IGT significantly elevated hippocampal and thoracic CD11b expression levels (p<0.05). The neuroimmune responses observed in this study suggest activation of peripheral-to-central immune signaling pathways. We speculate that the opioid-induced hippocampal changes inferred a humorally mediated mechanism, whereas thoracic neuroimmune modifications indicated activation of an indirect neural route. Although TRAM ameliorated 5IGT-intestinal inflammation, this opioid presents complications relating to bodyweight and regional glial dysregulation (neuroinflammation) and may not be optimal in the management of pain associated with 5IGT. The chemotherapy-induced gut-derived neuroimmune consequences observed suggest a potential mechanistic contribution to central components of the cancer symptom cluster experience, while the opioid-related glial changes have implications for optimal pain management in this setting warranting further investigation.

Lactobacillus rhamnosus Affects Rat Peritoneal Cavity Cell Response to Stimulation with Gut Microbiota: Focus on the Host Innate Immunity

Gut microbiota contribute to shaping the immune repertoire of the host, whereas probiotics may exert beneficial effects by modulating immune responses. Having in mind the differences in both the composition of gut microbiota and the immune response between rats of Albino Oxford (AO) and Dark Agouti (DA) rat strains, we investigated if intraperitoneal (i.p.) injection of live Lactobacillus rhamnosus (LB) may influence peritoneal cavity cell response to in vitro treatments with selected microbiota in the rat strain-dependent manner. Peritoneal cavity cells from AO and DA rats were lavaged two (d2) and seven days (d7) following i.p. injection with LB and tested for NO, urea, and H2O2 release basally, or upon in vitro stimulation with autologous E.coli and Enterococcus spp. Whereas the single i.p. injection of LB nearly depleted resident macrophages and increased the proportion of small inflammatory macrophages and monocytes on d2 in both rat strains, greater proportion of MHCIIhiCD163- and CCR7+ cells and increased NO/diminished H2O2 release in DA compared with AO rats suggest a more intense inflammatory priming by LB in this rat strain. Even though E.coli- and/or Enterococcus spp.-induced rise in H2O2 release in vitro was abrogated by LB in cells from both rat strains, LB prevented microbiota-induced increase in NO/urea ratio only in cells from AO and augmented it in cells from DA rats. Thus, the immunomodulatory properties may not be constant for particular probiotic bacteria, but shaped by innate immunity of the host.

Effects of trans-resveratrol on type 1 diabetes-induced up-regulation of apoptosis and mitogen-activated protein kinase signaling in retinal pigment epithelium of Dark Agouti rats

Microvascular changes and retinal degeneration precede diabetic retinopathy. Oxidative stress alters several intracellular signaling pathways, which form the basis of diabetic retinopathy. Many antioxidants have been investigated as possible preventive and therapeutic remedies for diabetic retinopathy. The current study investigated the modulatory effects of trans-resveratrol on streptozotocin-induced type 1 diabetes mediated changes in the transcription and levels of apoptosis-related proteins and mitogen-activated protein kinases (MAPKs) in the retinal pigment epithelium (RPE) of adult male dark Agouti rats. In control rats, 5 mg/kg/d trans-resveratrol administration for 30 days increased gene expressions of tumor suppressor protein 53, Bcl2-associated X protein, B-cell lymphoma-2 (Bcl2), Caspase-3 (CASP3), CASP8 and CASP9, p38αMAPK, c-Jun N-terminal kinase-1 (JNK1), and extracellular signal-regulated kinase-1 (ERK1). On the other hand, diabetes decreased gene expressions of CASP3, CASP8, p38αMAPK, JNK, and ERK1. Trans-resveratrol reversed the inhibited gene expressions of CASP8, p38αMAPK, JNK, and ERK1 to normal control levels in diabetic rats. Trans-resveratrol normalized diabetes-induced upregulation of CASP3 and -9, cytochrome-c, Bcl-2, and ERK1 proteins. In conclusion, Trans-resveratrol-induced alterations in gene expressions do not seem to affect RPE functions as they do not reflect as altered protein functions. Trans-resveratrol imparts its protective effects by normalizing apoptosis-related proteins and ERK1 but does not affect JNK proteins. Trans-resveratrol causes cytostasis in RPE of normal rats by upregulating Bcl2 protein and apoptotic proteins.

Trichinella spiralis excretory-secretory products downregulate MMP-9 in Dark Agouti rats affected by experimental autoimmune encephalomyelitis

Matrix metalloproteinases (MMPs), are implicated in the pathogenesis of multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). Our aim was to investigate whether amelioration of EAE in Dark Agouti (DA) rats, induced by Trichinella spiralis muscle larvae excretory-secretory products (ES L1), could be related to the level and activity of gelatinases, MMP-9 and MMP-2. Serum levels of MMP-9, MMP-2, NGAL/MMP-9, TIMP-1, and cytokines, evaluated by gel-zymography or ELISA, as well as gelatinases and TIMP-1 expression in the spinal cord (SC), were determined in: i) EAE induced, ii) ES L1-treated EAE induced animals. Milder clinical signs in ES L1-treated EAE induced DA rats were accompanied with lower serum levels of MMP-9 and NGAL/MMP-9 complex. However, the correlation between the severity of EAE and the level of serum MMP-9 was found only in the peak of the disease, with MMP-9/TIMP-1 ratio higher in EAE animals without ES L1 treatment. Lower expression of MMP-9 in SC of ES L1-treated, EAE induced rats, correlated with the reduced number of SC infiltrating cells. In SC infiltrates, in the effector and the recovery phase, production of anti-inflammatory cytokines IL-4 and IL-10 was higher in animals treated with ES L1 prior to EAE induction, compared to untreated EAE animals. Reduced expression of MMP-9 in SC tissue, which correlated with the reduced number of infiltrating cells, might be ascribed to regulatory mechanisms, among which is IL-10.

Physiological response of the retinal pigmented epithelium to 3-ns pulse laser application, in vitro and in vivo.

To treat healthy retinal pigmented epithelium (RPE) with the 3-ns retinal rejuvenation therapy (2RT) laser and to investigate the subsequent wound-healing response of these cells. Primary rat RPE cells were treated with the 2RT laser at a range of energy settings. Treated cells were fixed up to 7 days post-irradiation and assessed for expression of proteins associated with wound-healing. For in vivo treatments, eyes of Dark Agouti rats were exposed to laser and tissues collected up to 7 days post-irradiation. Isolated wholemount RPE preparations were examined for structural and protein expression changes. Cultured RPE cells were ablated by 2RT laser in an energy-dependent manner. In all cases, the RPE cell layer repopulated completely within 7 days. Replenishment of RPE cells was associated with expression of the heat shock protein, Hsp27, the intermediate filament proteins, vimentin and nestin, and the cell cycle-associated protein, cyclin D1. Cellular tight junctions were lost in lased regions but re-expressed when cell replenishment was complete. In vivo, 2RT treatment gave rise to both an energy-dependent localised denudation of the RPE and the subsequent repopulation of lesion sites. Cell replenishment was associated with the increased expression of cyclin D1, vimentin and the heat shock proteins Hsp27 and αB-crystallin. The 2RT laser was able to target the RPE both in vitro and in vivo, causing debridement of the cells and the consequent stimulation of a wound-healing response leading to layer reformation.

Loss of microRNA-147 function alleviates synovial inflammation through ZNF148 in rheumatoid and experimental arthritis.

MicroRNA-147 (miR-147) had been previously found induced in synoviocytes by inflammatory stimuli derived from Tcells in experimental arthritis. This study was designed to verify whether loss of its function might alleviate inflammatory events in joints of experimental and rheumatoid arthritis (RA). Dark Agouti (DA) rats were injected intradermally with pristane to induce arthritis, and rno-miR-147 antagomir was locally administrated into individual ankle compared with negative control or rno-miR-155-5p antagomir (potential positive control). Arthritis onset, macroscopic severity, and pathological changes were monitored. While in vitro, gain or loss function of hsa-miR-147b-3p/hsa-miR-155-5p and ZNF148 was achieved in human synovial fibroblast cell line SW982 and RA synovial fibroblasts (RASF). The expression of miRNAs and mRNAs was detected by using RT-quantitative PCR, and protein expression was detected by using Western blotting. Anti-miR-147 therapy could alleviate the severity, especially for the synovitis and joint destruction in experimental arthritis. Gain of hsa-miR-147b-3p/hsa-miR-155-5p function in TNF-α stimulated SW982 and RASF cells could upregulate, in contrast, loss of hsa-miR-147b-3p/hsa-miR-155-5p function could downregulate the gene expression of TNF-α, IL-6, MMP3, and MMP13. Hence, such alteration could participate in synovial inflammation and joint destruction. RNAi of ZNF148, a miR-147's target, increased gene expression of TNF-α, IL-6, MMP3, and MMP13 in SW982 and RASF cells. Also, mRNA sequencing data showed that hsa-miR-147b-3p mimic and ZNF148 siRNA commonly regulated the gene expression of CCL3 and DEPTOR as well as some arthritis and inflammation-related pathways. Taken together, miR-147b-3p contributes to synovial inflammation through repressing ZNF148 in RA and experimental arthritis.

Complete Freund's adjuvant-free experimental autoimmune encephalomyelitis in Dark Agouti rats is a valuable tool for multiple sclerosis studies

Experimental autoimmune encephalomyelitis (EAE) is classically induced with complete Freund's adjuvant (CFA). The immune response against CFA has a confounding influence on the translational capacity of EAE as a multiple sclerosis model. Here, we compare clinical, cellular and molecular properties between syngeneic spinal cord homogenate (SCH)- and SCH + CFA-immunized Dark Agouti rats. EAE signs were observed earlier and the cumulative clinical score was higher without CFA. Also, a higher number of immune cells infiltrates in the spinal cords was noticed at the peak of EAE without CFA. High spinal cord abundance of CD8+CD11bc+MHC class II+ cells was detected in SCH-immunized rats. Myelin basic protein -specific response can be elicited in the cells from the lymph nodes draining the site of SCH immunization. This CFA-free EAE is a reliable multiple sclerosis model.

Auditory brainstem responses in aging dark agouti rats.

The present study examined auditory function across age in the dark agouti (DA) rat strain. Auditory brainstem responses (ABRs) were measured for frequencies 8, 16, and 32 kHz in male and female DA rats from 3 to 18 months of age. Hearing thresholds and absolute and interpeak latencies (IPLs) were analyzed. Male hearing thresholds remained stable for the first year of life and then significantly increased at 18 months across all frequencies; female hearing remained stable at all tested ages out to 18 months. At 12 months, male DA rats showed significantly longer absolute latencies by age (i.e., compared with 3-month-old males) and sex (compared with 12-month-old females), with no differences in IPLs. At 18 months, female DA rats showed significantly longer absolute latencies with age (compared with 3-month-old females) and sex (compared with 18-month-old males), particularly for the later waves. Female IPLs were also significantly longer with age and by sex for the later waves. This report supports the feasibility of using male DA rats in studies to investigate age-related hearing loss (ARHL; presbycusis).

CD161a-positive natural killer (NK) cells and α-smooth muscle actin-positive myofibroblasts were upregulated by extrarenal DPP4 in a rat model of acute renal rejection

AimsSystemic inhibition of dipeptidyl peptidase 4 (DPP4) showed a protective effect in several transplant models. Here we assessed the specific role of extrarenal DPP4 in renal transplant rejection. Methods: Kidneys from wildtype (wt) F344 rats were either transplanted in wt Dark Agouti or congenic rats not expressing DPP4. The remaining, not transplanted donor kidney served as healthy controls. To investigate early inflammatory events rats were sacrificed 3 days after transplantation and kidneys were evaluated for inflammatory cells, capillary rarefaction, proliferation, apoptosis and myofibroblasts by immunohistochemistry. ResultsCapillary ERG-1-positive endothelial cells were significantly more abundant in renal cortex when transplanted into DPP4 deficient compared to wt recipients. In contrast, TGF-ß and myofibroblasts were reduced by more than 25% in kidneys transplanted into DPP4 deficient compared to wt recipients. Numbers of CD161a-positive NK-cells were significantly lower in allografts in DPP4 deficient compared to wt recipients. Numbers of all other investigated immune cells were not affected by the lack of extrarenal DPP4. ConclusionIn early transplant rejection extrarenal DPP4 is involved in the recruitment of NK-cells and early fibrosis. Beneficial effects were less pronounced than reported for systemic DPP4 inhibition, indicating that renal DPP4 is an important player in transplantation-mediated injury.

Therapeutic potential of agmatine in the experimental autoimmune encephalomyelitis

Background/Aim. Experimental autoimmune encephalomyelitis (EAE) is a model of multiple sclerosis (MS), in which we investigated the neuroprotective effect of agmatine (AGM), known as a primary amine produced via the decarboxylation of L-arginine. Methods. Dark Agouti rats were divided into groups: control (C), Complete Freund`s Adjuvant (CFA), EAE rats decapitated on the 13th day post immunization (dpi) (EAE13) and on the 20th dpi (EAE20), EAE animals given three (EAE+AGM13) and 10 (EAE+AGM20) doses of AGM, and healthy animals ad-ministered three/10 doses of AGM (AGM). Thiobarbituric acid-reacting substances (TBARS), SH groups (SH), total glutathione (GSH), glutathione peroxidase activity (GPx), superoxide dismutase activities (tSOD, MnSOD, CuZ-nSOD) and nitrite/nitrate concentration (NO2+NO3) were assessed in plasma and brain structures [whole encephalitic mass (WEM) and brainstem (BS)]. Results. The obtained results showed that AGM treatment successfully attenuated severe clinical deficits in EAE. Applications of AGM in EAE rats induced normalized TBARS, SH, GSH, GPx and NO in WEM. In BS, AGM expressed less prominent effects, inducing normalized TBARS, GPx and NO, but no effect on SH and GSH. In both brain structures, tSOD activity lowered and normalized at the peak and in the remission phase of the disease, post-AGM treatment. The effect of AGM on the MnSOD in EAE was expressed in WEM/BS only in the remission phase as a reduced activity. Conclusion. Milder clinical form of developed EAE in rats indicates promising therapeutic effect of AGM in MS. The activated antioxidant system and suppressed oxidative/nitrosative stress development may denote a successful blockade of neu-roinflammation initiated by EAE immunization. The study implies the capability of AGM to attenuate oxidative/nitrosative damage at the peak of EAE by modulating antioxidative defense capacity during the time-course of the disease. Thus, AGM may be considered as an agent with a beneficial effect on neuroinflammation in EAE.

Treatment with the calcineurin inhibitor and immunosuppressant cyclosporine A impairs sensorimotor gating in Dark Agouti rats

RationaleCalcineurin is a protein regulating cytokine expression in T lymphocytes and calcineurin inhibitors such as cyclosporine A (CsA) are widely used for immunosuppressive therapy. It also plays a functional role in distinct neuronal processes in the central nervous system. Disturbed information processing as seen in neuropsychiatric disorders is reflected by deficient sensorimotor gating, assessed as prepulse inhibition (PPI) of the acoustic startle response (ASR).ObjectivePatients who require treatment with immunosuppressive drugs frequently display neuropsychiatric alterations during treatment with calcineurin inhibitors. Importantly, knockout of calcineurin in the forebrain of mice is associated with cognitive impairments and symptoms of schizophrenia-like psychosis as seen after treatment with stimulants.MethodsThe present study investigated in rats effects of systemic acute and subchronic administration of CsA on sensorimotor gating. Following a single injection with effective doses of CsA, adult healthy male Dark Agouti rats were tested for PPI. For subchronic treatment, rats were injected daily with the same doses of CsA for 1 week before PPI was assessed. Since calcineurin works as a modulator of the dopamine pathway, activity of the enzyme tyrosine hydroxylase was measured in the prefrontal cortex and striatum after accomplishment of the study.ResultsAcute and subchronic treatment with the calcineurin inhibitor CsA disrupted PPI at a dose of 20 mg/kg. Concomitantly, following acute CsA treatment, tyrosine hydroxylase activity was reduced in the prefrontal cortex, which suggests that dopamine synthesis was downregulated, potentially reflecting a stimulatory impact of CsA on this neurotransmitter system.ConclusionsThe results support experimental and clinical evidence linking impaired calcineurin signaling in the central nervous system to the pathophysiology of neuropsychiatric symptoms. Moreover, these findings suggest that therapy with calcineurin inhibitors may be a risk factor for developing neurobehavioral alterations as observed after the abuse of psychomotor stimulant drugs.

Upregulated PKM2 in Macrophages Exacerbates Experimental Arthritis via STAT1 Signaling.

Recent studies indicate that glucose metabolism is altered in rheumatoid arthritis. We hypothesize that Pkm2, as a key regulatory enzyme of glycolysis pathway, triggers the activation of macrophages (Mφ), which results in proinflammatory cytokine production during the arthritis progress. In this study, Pkm2 was found to be overexpressed in ED1-positive Mφ in spleens and synovial tissues from arthritic rats via immunofluorescence, Western blotting, and quantitative RT-PCR. To reveal the role of Pkm2, Dark Agouti rats were treated with either Pkm2 enzyme inhibitor shikonin or the RNA interference plasmids of Pkm2 and negative control plasmids, respectively, via i.p. injection. Pkm2 intervention could alleviate the severity of pristane-induced arthritis in aspects of the macroscopic arthritis score, perimeter changes of midpaw, and the synovitis and destruction of the bone and cartilage as well as reduce the ED1 and p-Stat1-positive cell population in rat synovial tissues. Silencing Pkm2 by RNA interference in classical activated rat and mouse Mφ resulted in less Tnf-α, Il-1β production via Stat1 signaling. Collectively, Pkm2 is highly expressed in ED1-positive Mφ of spleens and synovial tissues from arthritic rats and promotes Mφ activation via Stat1 signaling. Pkm2 might be a promising selective metabolic target molecule for rheumatoid arthritis treatment.

Imaging of Therapeutic Effects of Anti-Vascular Endothelial Growth Factor Inhibitors by Optical Coherence Tomography Angiography in a Rat Model.

PurposeThe aim of the study was to investigate optical coherence tomography angiography (OCTA) as a high-resolution in vivo imaging modality for monitoring therapeutic response to different vascular endothelial growth factor inhibitors in the rat model of laser-induced choroidal neovascularization (CNV). Further, OCTA findings were compared with fluorescein angiography (FA) and fluorescence microscopy.MethodsLaser treatment at day (D)0 was followed by intravitreal injection of aflibercept, AF564, and NaCl in dark agouti rats. Imaging with OCTA and FA was performed at D2, D7, D14, and D21. OCTA was compared to FA as well as confocal imaged flat mounts and analysis included quantification of CNV area, pixel intensity, vessel density, and number of vessel junctions.ResultsWithin laser lesions, neovascularization were visible especially in deeper retinal layers on OCTA, but not on FA images. Using OCTA, mean CNV area (D21) at the level of the outer nuclear layer (ONL) was 0.017 mm² following aflibercept administration, 0.016 mm² following AF564 and 0.026 mm² following NaCl injection (P = 0.04 and P = 0.03). Similar differences between treatment groups were determined by FA and histology, although the overall CNV area was always larger on FA due to dye leakage (P ≤ 0.0001, all layers).ConclusionsCompared to FA, OCTA imaging allows for a more precise and quantitative analysis of new blood vessel formation and therapeutic response to vascular endothelial growth factor (VEGF)-inhibitors, whereas it does not permit assessment of leakage.Translational RelevanceThese findings suggest that OCTA may be particularly useful for the investigation of new treatment targets in the animal model.

Sex differences in Tfh cell help to B cells contribute to sexual dimorphism in severity of rat collagen-induced arthritis

The study examined germinal centre (GC) reaction in lymph nodes draining inflamed joints and adjacent tissues (dLNs) in male and female Dark Agouti rat collagen type II (CII)-induced arthritis (CIA) model of rheumatoid arthritis. Female rats exhibiting the greater susceptibility to CIA mounted stronger serum CII-specific IgG response than their male counterparts. This correlated with the higher frequency of GC B cells in female compared with male dLNs. Consistently, the frequency of activated/proliferating Ki-67+ cells among dLN B cells was higher in females than in males. This correlated with the shift in dLN T follicular regulatory (Tfr)/T follicular helper (Tfh) cell ratio towards Tfh cells in females, and greater densities of CD40L and CD40 on their dLN T and B cells, respectively. The higher Tfh cell frequency in females was consistent with the greater dLN expression of mRNA for IL-21/27, the key cytokines involved in Tfh cell generation and their help to B cells. Additionally, in CII-stimulated female rat dLN cell cultures IFN-γ/IL-4 production ratio was shifted towards IFN-γ. Consistently, the serum IgG2a(b)/IgG1 CII-specific antibody ratio was shifted towards an IgG2a(b) response in females. Thus, targeting T-/B-cell interactions should be considered in putative further sex-based translational pharmacology research.

Dermatotoxicity of oral cadmium is strain-dependent and related to differences in skin stress response and inflammatory/immune activity

Adverse effects of non-occupational exposure to cadmium (Cd) are increasingly acknowledged. Since our previous study has showed that orally acquired Cd affects skin, the contribution of genetic background to dermatotoxicity of oral cadmium was examined in two rat strains, Albino Oxford (AO) and Dark Agouti (DA), which differed in response to chemicals. While similar accumulation of Cd in the skin of both strains was noted, the skin response to the metal differed. DA rat individuals mounted antioxidant enzyme defense in the skin already at lower Cd dose, in contrast to AO rats which reacted to higher metal dose solely (and less pronounced), implying higher susceptibility of DA strain to Cd dermatotoxicity. Epidermal cells from both strains developed stress response, but higher intensity of antioxidant response in AO rats implied this strain`s better ability to defend against Cd insult. Cd induced epidermal cells’ proinflammatory cytokine response only in DA rats. Increased IL-10 seems responsible for the lack of response in AO rats. Differences in the pattern of skin/epidermal cell responsiveness to cadmium give a new insight into repercussion of genetic variability to dermatotoxicity of orally acquired cadmium, bearing relevance for variations in the link between dietary cadmium and inflammation-based skin pathologies.