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Diabetic retinopathy is characterized by multiple microcirculatory dysfunctions and angiogenesis resulting from hyperglycemia, oxidative stress, and inflammation. In this study, the retina and retinal pigmented epithelium of non-insulin-dependent diabetic Goto-Kakizaki (GK) rats were examined to detect microvascular alterations, gliosis, macrophage infiltration, lipid deposits, and fibrosis. Emphasis was given to the distribution of kinin B1 receptor (B1R) and vascular endothelial growth factor (VEGF), two major factors in inflammation and angiogenesis.30-week-old male GK rats and age-matched Wistar rats were used. The retinal vascular bed was examined using ADPase staining. The level of lipid accumulation was graded using triglyceride staining with Oil red O. Macrophage and retinal microglia activation, as well as other markers, were revealed by immunohistochemistry and studied with confocal laser scanning microscopy.Abundant lipid deposits were observed in the Bruch's membrane of GK rats. Immunohistochemistry and quantitative analysis showed significantly higher B1R, VEGF, Iba1 (microglia), CD11 (macrophages), fibronectin, and collagen I labeling in the diabetic retina. B1R immunolabeling was detected in the vascular layers of the GK retina. A strong VEGF staining within different retinal cell processes was detected and a pattern of GFAP staining suggested strong Müller cells/astrocytes reactivity. Microgliosis was apparent in the GK retina. A greater tortuosity of the retinal microvessels (an index of endothelial dysfunction) and their increased number were also observed in GK retinas.Data suggest retinal vascular bed alterations in spontaneous type 2 diabetic retinas at 30 weeks. Lipid and collagen accumulation in the retina and choroid, in addition to retinal upregulation of VEGF and B1R, microgliosis, and Müller cell reactivity, may contribute to vascular alterations and inflammatory processes.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1

Recent studies suggest that treatment with SGLT-2 inhibitors can reduce hepatic lipid storage and ameliorate non-alcoholic fatty liver disease (NAFLD) development beyond their glycemic benefits. However, the exact mechanism involved is still unclear. We investigated the hepatic metabolic effect of empagliflozin (10 mg/kg/day for eight weeks) on the development of NAFLD and its complications using HHTg rats as a non-obese prediabetic rat model. Empagliflozin treatment reduced neutral triacylglycerols and lipotoxic diacylglycerols in the liver and was accompanied by significant changes in relative mRNA expression of lipogenic enzymes (Scd-1, Fas) and transcription factors (Srebp1, Pparγ). In addition, alterations in the gene expression of cytochrome P450 proteins, particularly Cyp2e1 and Cyp4a, together with increased Nrf2, contributed to the improvement of hepatic lipid metabolism after empagliflozin administration. Decreased circulating levels of fetuin-A improved lipid metabolism and attenuated insulin resistance in the liver and in peripheral tissues. Our results highlight the beneficial effect of empagliflozin on hepatic lipid metabolism and lipid accumulation independent of obesity, with the mechanisms understood to involve decreased lipogenesis, alterations in cytochrome P450 proteins, and decreased fetuin-A. These changes help to alleviate NAFLD symptoms in the early phase of the disease and before the onset of diabetes.

Anxiety disorders are associated with a failure to sufficiently extinguish fear memories. The serotonergic system (5-hydroxytryptamine, 5-HT) with the 5-HT transporter (5-HTT, SERT) is strongly implicated in the regulation of anxiety and fear. In the present study, we examined the effects of SERT deficiency on fear extinction in a differential fear conditioning paradigm in male and female rats. Fear-related behavior displayed during acquisition, extinction, and recovery, was measured through quantification of immobility and alarm 22-kHz ultrasonic vocalizations (USV). Trait-like inter-individual differences in novelty-seeking, anxiety-related behavior, habituation learning, cognitive performance, and pain sensitivity were examined for their predictive value in forecasting fear extinction. Our results show that SERT deficiency strongly affected the emission of 22-kHz USV during differential fear conditioning. During acquisition, extinction, and recovery, SERT deficiency consistently led to a reduction in 22-kHz USV emission. While SERT deficiency did not affect immobility during acquisition, genotype differences started to emerge during extinction, and during recovery rats lacking SERT showed higher levels of immobility than wildtype littermate controls. Recovery was reflected in increased levels of immobility but not 22-kHz USV emission. Prominent sex differences were evident. Among several measures for trait-like inter-individual differences, anxiety-related behavior had the best predictive quality. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:22 issue:13 ispartof: location:Switzerland status: published

Rat glioma cell line C6 expressing human poliovirus receptor (PVR) and susceptible to polioviruses (C6-PVR-BFP) was used to produce a clone with knockout of IFNα/β (Ifnar1) receptor subunit 1 gene (Ifnar1). The sensitivity of C6-PVR-BFP cells to the vaccine strain of poliovirus type 3 (PV3) depended on the signaling pathways of the cell response to type 1 IFN. Using the model of subcutaneous tumor xenografts, we demonstrated oncolytic activity of PV3 against C6-PVR-BFP cells that depended on the expression of PVR and increased considerably upon disturbances in IFN response pathways.

Mechanisms underlying Purkinje cell (PC) death, which leads to many diseases in humans, are still poorly elucidated. Progressive PC degeneration occurs in shaker mutant rat due to an X-linked recessive mutation leading to gait ataxia and total-body tremors. Chemoablation of the inferior olive (IO) and olivocerebellar deafferentation temporally accelerated PC death from the natural 6-8 week time-course to 1-2 weeks in the shaker mutant rat. The present study posits that IO chemoablation leads to the accelerated and augmented upregulation of the executioner active caspase-3 that triggers apoptosis of at-risk PCs throughout the ordinary phenotypic manifestation of the shaker mutation.Immunofluorescence and double labeling for calbindin and active caspase-3 were used in vermal cerebellar sections from IO-chemoablated rats to demonstrate the effect of IO chemoablation on active caspase-3 expression in at-risk PCs.Active caspase-3 expression was enhanced in the anterior degeneration (ADC) and posterior degeneration (PDC) compartments to reach a peak in both degeneration compartments at 24 h following the injections for IO chemoablation.Consequently, it can be deduced that active caspase-3 expression in shaker mutant rats is modifiable suggesting the possibility of targeting it therapeutically in an attempt to rescue PCs from death. Abbreviation PC: Purkinje cell; IO: inferior olive; ADC: Anterior degeneration compartment; PDC: Posterior degeneration compartment; ISC: Intermediate survival compartment; FNSC: Flocculonodular survival compartment.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures., Charcot-Marie-Tooth disease 1 A (CMT1A) results from PMP22 gene duplication and is characterized by peripheral nerve myelination deficits. Here, the authors prevent the development of pathological features in a rat model of CMT1A through the local delivery of AAV2/9 expressing shRNAs against PMP22.

Women with pulmonary arterial hypertension (PAH) exhibit better right ventricular (RV) function and survival than men; however, the underlying mechanisms are unknown. We hypothesized that 17β-estradiol (E2), through estrogen receptor α (ER-α), attenuates PAH-induced RV failure (RVF) by upregulating the procontractile and prosurvival peptide apelin via a BMPR2-dependent mechanism. We found that ER-α and apelin expression were decreased in RV homogenates from patients with RVF and from rats with maladaptive (but not adaptive) RV remodeling. RV cardiomyocyte apelin abundance increased in vivo or in vitro after treatment with E2 or ER-α agonist. Studies employing ER-α–null or ER-β–null mice, ER-α loss-of-function mutant rats, or siRNA demonstrated that ER-α is necessary for E2 to upregulate RV apelin. E2 and ER-α increased BMPR2 in pulmonary hypertension RVs and in isolated RV cardiomyocytes, associated with ER-α binding to the Bmpr2 promoter. BMPR2 is required for E2-mediated increases in apelin abundance, and both BMPR2 and apelin are necessary for E2 to exert RV-protective effects. E2 or ER-α agonist rescued monocrotaline pulmonary hypertension and restored RV apelin and BMPR2. We identified what we believe to be a novel cardioprotective E2/ER-α/BMPR2/apelin axis in the RV. Harnessing this axis may lead to novel RV-targeted therapies for PAH patients of either sex.

The angiotensin receptor/neprilysin inhibitor Sacubitril/Valsartan (Sac/Val) has been shown to be beneficial in patients suffering from heart failure with reduced ejection fraction (HFrEF). However, the impact of Sac/Val in patients presenting with heart failure with preserved ejection fraction (HFpEF) is not yet clearly resolved. The present study aimed to reveal the influence of the drug on the functionality of the myocardium, the skeletal muscle, and the vasculature in a rat model of HFpEF. Female obese ZSF-1 rats received Sac/Val as a daily oral gavage for 12 weeks. Left ventricle (LV) function was assessed every four weeks using echocardiography. Prior to organ removal, invasive hemodynamic measurements were performed in both ventricles. Vascular function of the carotid artery and skeletal muscle function were monitored. Sac/Val treatment reduced E/é ratios, left ventricular end diastolic pressure (LVEDP) and myocardial stiffness as well as myocardial fibrosis and heart weight compared to the obese control group. Sac/Val slightly improved endothelial function in the carotid artery but had no impact on skeletal muscle function. Our results demonstrate striking effects of Sac/Val on the myocardial structure and function in a rat model of HFpEF. While vasodilation was slightly improved, functionality of the skeletal muscle remained unaffected.

Mutations in the Crumbs homologue 1 (CRB1) gene cause inherited retinal dystrophies, such as early-onset retinitis pigmentosa and Leber congenital amaurosis. A Brown Norway rat strain was reported with a spontaneous insertion-deletion (indel) mutation in exon 6 of Crb1. It has been reported that these Crb1 mutant rats show vascular abnormalities associated with retinal telangiectasia and possess an early-onset retinal degenerative phenotype with outer limiting membrane breaks and focal loss of retinal lamination at 2 months of age. Here, we further characterized the morphological phenotype of new-born and adult Crb1 mutant rats in comparison with age-matched Brown Norway rats without a mutation in Crb1. A significantly decreased retinal function and visual acuity was observed in Crb1 mutant rats at 1 and 3 months of age, respectively. Moreover, in control rats, the subcellular localization of canonical CRB1 was observed at the subapical region in Müller glial cells while CRB2 was observed at the subapical region in both photoreceptors and Müller glial cells by immuno-electron microscopy. CRB1 localization was lost in the Crb1 mutant rats, whereas CRB2 was still observed. In addition, we determined the tropism of subretinal or intravitreally administered AAV5-, AAV9- or AAV6-variant ShH10Y445F vectors in new-born control and Crb1 mutant rat retinas. We showed that subretinal injection of AAV5 and AAV9 at postnatal days 5 (P5) or 8 (P8) predominantly infected the retinal pigment epithelium (RPE) and photoreceptor cells; while intravitreal injection of ShH10Y445F at P5 or P8 resulted in efficient infection of mainly Müller glial cells. Using knowledge of the subcellular localization of CRB1 and the ability of ShH10Y445F to infect Müller glial cells, canonical hCRB1 and hCRB2 AAV-mediated gene therapy were explored in new-born Crb1 mutant rats. Enhanced retinal function after gene therapy delivery in the Crb1 rat was not observed. No timely rescue of the retinal phenotype was observed using retinal function and visual acuity, suggesting the need for earlier onset of expression of recombinant hCRB proteins in Müller glial cells to rescue the severe retinal phenotype in Crb1 mutant rats.

The laboratory rat is widely used as a model for human diseases. Many of these diseases involve monocytes and tissue macrophages in different states of activation. Whilst methods forin vitrodifferentiation of mouse macrophages from embryonic stem cells (ESC) and bone marrow (BM) are well established, these are lacking for the rat. The gene expression profiles of rat macrophages have also not been characterised to the same extent as mouse. We have established the methodology for production of rat ESC-derived macrophages and compared their gene expression profiles to macrophages obtained from the lung and peritoneal cavity and those differentiated from BM and blood monocytes. We determined the gene signature of Kupffer cells in the liver using rats deficient in macrophage colony stimulating factor receptor (CSF1R). We also examined the response of BM-derived macrophages to lipopolysaccharide (LPS). The results indicate that many, but not all, tissue-specific adaptations observed in mice are conserved in the rat. Importantly, we show that unlike mice, rat macrophages express the CSF1R ligand, colony stimulating factor 1 (CSF1).

Deuterium Magnetic Resonance Spectroscopy (DMRS) is a non-invasive technique that allows the detection of deuterated compounds in vivo. DMRS has a large potential to analyze uptake, perfusion, washout or metabolism, since deuterium is a stable isotope and therefore does not decay during biologic processing of a deuterium labelled substance. Moreover, DMRS allows the distinction between different deuterated substances. In this work, we performed DMRS of deuterated 3-O-Methylglucose (OMG). OMG is a non-metabolizable glucose analog which is transported similar to D-glucose. DMRS of OMG was performed in phantom and in vivo measurements using a preclinical 7 Tesla MRI system. The chemical shift (3.51 ± 0.1 ppm) and relaxation times were determined. OMG was injected intravenously and spectra were acquired over a period of one hour to monitor the time evolution of the deuterium signal in tumor-bearing rats. The increase and washout of OMG could be observed. Three different exponential functions were compared in terms of how well they describe the OMG washout. A mono-exponential model with offset seems to describe the observed time course best with a time constant of 1910 ± 770 s and an offset of 2.5 ± 1.2 mmol/l (mean ± std, N = 3). Chemical shift imaging could be performed with a voxel size of 7.1 mm x 7.1 mm x 7.9 mm. The feasibility of DMRS with deuterium labelled OMG could be demonstrated. These data might serve as basis for future studies that aim to characterize glucose transport using DMRS.

The Ihara epileptic rat (IER) is a mutant model with limbic-like seizures whose pathology and causative gene remain elusive. In this report, via linkage analysis, we identified Down syndrome cell adhesion molecule-like 1(Dscaml1) as the responsible gene for IER. A single base mutation in Dscaml1 causes abnormal splicing, leading to lack of DSCAML1. IERs have enhanced seizure susceptibility and accelerated kindling establishment. Furthermore, GABAergic neurons are severely reduced in the entorhinal cortex (ECx) of these animals. Voltage-sensitive dye imaging that directly presents the excitation status of brain slices revealed abnormally persistent excitability in IER ECx. This suggests that reduced GABAergic neurons may cause weak sustained entorhinal cortex activations, leading to natural kindling via the perforant path that could cause dentate gyrus hypertrophy and epileptogenesis. Furthermore, we identified a single nucleotide substitution in a human epilepsy that would result in one amino acid change in DSCAML1 (A2105T mutation). The mutant DSCAML1A2105T protein is not presented on the cell surface, losing its homophilic cell adhesion ability. We generated knock-in mice (Dscaml1A2105T) carrying the corresponding mutation and observed reduced GABAergic neurons in the ECx as well as spike-and-wave electrocorticogram. We conclude that DSCAML1 is required for GABAergic neuron placement in the ECx and suppression of seizure susceptibility in rodents. Our findings suggest that mutations in DSCAML1 may affect seizure susceptibility in humans.

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

Although women are more susceptible to pulmonary arterial hypertension (PAH) than men, their right ventricular (RV) function is better preserved. Estrogen receptor-α (ERα) has been identified as a likely mediator for estrogen protection in the RV. However, the role of ERα in preserving RV function and remodeling during pressure overload remains poorly understood. We hypothesized that loss of functional ERα removes female protection from adverse remodeling and is permissive for the development of a maladapted RV phenotype. Male and female rats with a loss-of-function mutation in ERα (ERαMut) and wild-type (WT) littermates underwent RV pressure overload by pulmonary artery banding (PAB). At 10 wk post-PAB, WT and ERαMut demonstrated RV hypertrophy. Analysis of RV pressure waveforms demonstrated RV-pulmonary vascular uncoupling and diastolic dysfunction in female, but not male, ERαMut PAB rats. Similarly, female, but not male, ERαMut exhibited increased RV fibrosis, comprised primarily of thick collagen fibers. There was an increased protein expression ratio of TIMP metallopeptidase inhibitor 1 (Timp1) to matrix metalloproteinase 9 (Mmp9) in female ERαMut compared with WT PAB rats, suggesting less collagen degradation. RNA-sequencing in female WT and ERαMut RV revealed kallikrein-related peptidase 10 (Klk10) and Jun Proto-Oncogene (Jun) as possible mediators of female RV protection during PAB. In summary, ERα in females is protective against RV-pulmonary vascular uncoupling, diastolic dysfunction, and fibrosis in response to pressure overload. ERα appears to be dispensable for RV adaptation in males. ERα may be a mediator of superior RV adaptation in female patients with PAH. NEW & NOTEWORTHY Using a novel loss-of-function mutation in estrogen receptor-α (ERα), we demonstrate that female, but not male, ERα mutant rats display right ventricular (RV)-vascular uncoupling, diastolic dysfunction, and fibrosis following pressure overload, indicating a sex-dependent role of ERα in protecting against adverse RV remodeling. TIMP metallopeptidase inhibitor 1 (Timp1), matrix metalloproteinase 9 (Mmp9), kallikrein-related peptidase 10 (Klk10), and Jun Proto-Oncogene (Jun) were identified as potential mediators in ERα-regulated pathways in RV pressure overload. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/role-of-estrogen-receptor-alpha-in-right-ventricular-remodeling/.

We recently demonstrated that aspartoacylase (Aspa) and hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1) genes were causative of essential tremor (ET) in rats. This finding was obtained using Aspaem34Kyo/Hcn1A354V double-mutant rats, but they were bred on a heterogeneous genetic background of two strains, F344 and WTC. Here, we developed an Aspaem34Kyo/Hcn1em1Kyo double-knockout rat strain with a homogenous F344 genetic background and studied the ability of glutamate receptor antagonists to suppress ET. The F344-Aspa/Hcn1 double-knockout rats exhibited spontaneous, intense body tremor equivalent to that in the double-mutant rats. N-acetyl-aspartate (NAA), a substrate of ASPA, showed accumulation in all brain regions and in the spinal cord. However, N-acetyl-aspartyl-glutamate (NAAG), which is derived from NAA and interacts with glutamatergic receptors, was decreased in the medulla oblongata of the double-knockout rats. The tremor was suppressed by 3-[(R)-2-carboxypiperazin-4-yl]-prop-2-enyl-1-phosphonic acid, an N-methyl-D-aspartate (NMDA) receptor antagonist, in F344-Aspa/Hcn1 double-knockout rats. The non-NMDA glutamate receptor antagonist NBQX weakly inhibited the tremor, while the metabotropic glutamate receptor antagonist LY341495 showed no effect. In addition, both NR2B subunit-specific (Ro 25-6981) and NR2C/NR2D subunit-specific (cis-piperidine dicarboxylic acid) NMDA receptor antagonists suppressed the tremor. These data indicated that the pathogenesis of tremor in Aspa/Hcn1 double-knockout rats involved ionotropic glutamate receptors, particularly NMDA receptors.

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a neurodegenerative disease due to mutations in TUBB4A. Patients suffer from extrapyramidal movements, spasticity, ataxia, and cognitive deficits. Magnetic resonance imaging features are hypomyelination and atrophy of the striatum and cerebellum. A correlation between the mutations and their cellular, tissue and organic effects is largely missing. The effects of these mutations on sensory functions have not been described so far. We have previously reported a rat carrying a TUBB4A (A302T) mutation and sharing most of the clinical and radiological signs with H-ABC patients. Here, for the first time, we did a comparative study of the hearing function in an H-ABC patient and in this mutant model. By analyzing hearing function, we found that there are no significant differences in the auditory brainstem response (ABR) thresholds between mutant rats and WT controls. Nevertheless, ABRs show longer latencies in central waves (II-IV) that in some cases disappear when compared to WT. The patient also shows abnormal AEPs presenting only Waves I and II. Distortion product of otoacoustic emissions and immunohistochemistry in the rat show that the peripheral hearing function and morphology of the organ of Corti are normal. We conclude that the tubulin mutation severely impairs the central hearing pathway most probably by progressive central white matter degeneration. Hearing function might be affected in a significant fraction of patients with H-ABC; therefore, screening for auditory function should be done on patients with tubulinopathies to evaluate hearing support therapies.

Many animal models that are currently used in appetite and obesity research share at least some main features of human obesity and its comorbidities. Hence, even though no animal model replicates all aspects of "common" human obesity, animal models are imperative in studying the control of energy balance and reasons for its imbalance that may eventually lead to overt obesity. The most frequently used animal models are small rodents that may be based on mutations or manipulations of individual or several genes and on the exposure to obesogenic diets or other manipulations that predispose the animals to gaining or maintaining excessive weight. Characteristics include hyperphagia or changes in energy metabolism and at least in some models the frequent comorbidities of obesity, like hyperglycemia, insulin resistance, or diabetes-like syndromes. Some of the most frequently used animal models of obesity research involve animals with monogenic mutations of the leptin pathway which in fact are useful to study specific mechanistic aspects of eating controls, but typically do not recapitulate "common" obesity in the human population. Hence, this review will mention advantages and disadvantages of respective animal models in order to build a basis for the most appropriate use in biomedical research.

Human induced pluripotent stem cells (hiPSC)-derived retinal pigment epithelium (RPE) cells are therapeutic cells that have been shown to be promising in the rescue of lost photoreceptors. In this study, we generated hiPSC from human epidermal keratinocytes and subsequently differentiated them into RPE cells to investigate their ability to influence the retinal functions of the Royal College of Surgeon (RCS) rats. Keratinocytes were reprogrammed to hiPSC using a non-integrating Sendai reprogramming system. Established hiPSCs were differentiated into RPE cells, and complete characterization was performed. Next, the suspension of hiPSC-RPE cells was transplanted into the subretinal space of 3-week-old RCS rats (n = 12). Posttransplantation evaluations were performed using optical coherence tomography (OCT), electroretinography, and immunohistochemical analysis. The hiPSC colonies were identical to embryonic stem-like cells that revealed the expression of pluripotency markers and retention of the normal genome. These cells exhibited the ability to differentiate into an amalgam of germ layers and produce RPE cells. The differentiated RPE cells exhibited an identical pigmented morphology that expressed RPE-specific markers, such as CRALBP, BESTROPHIN, RPE65, and MERTK. At 8 weeks of longitudinal culture, the RPE cells exhibited maximum pigmentation with in vitro phagocytotic activity. Furthermore, transplantation data showed improved retinal function till week 12 post-transplantation and a significantly higher number of rod/cone ratios in transplanted eyes compared to non-surgery control eyes. hiPSC-derived RPE cells exhibited naïve RPE cell properties and functionality that provided trophic support and the transient rescue of photoreceptor cells.

The interstitial nucleus of the posterior limb of the anterior commissure (IPAC) is exclusively innervated by tyrosine hydroxylase-immunoreactive (TH-IR) fibers as observed in the other nuclei of the rat forebrain such as the striatum and nucleus accumbens. Distinguishing TH-IR afferents to the IPAC from those projecting to neighboring nuclei has been difficult. However, we previously showed that the TH-IR fibers projecting to the IPAC were invulnerable to neurodegeneration in zitter mutant rats, whereas almost all TH-IR afferents fibers to the dorsolateral striatum were lost, indicating that these two groups of TH-IR afferents have distinct neurochemical properties. Here, to explore this observation further, we injected Fluorogold (FG) retrograde tracers to identify neurons projecting to the IPAC or dorsal striatum. We also determined the distribution of attractin mRNA and protein, causative factors for the pathological phenotypes of zitter mutant rats, within the normal rat midbrain. In rats injected with FG into the dorsal striatum, we detected many FG-positive neurons in the ventral aspect of the substantia nigra pars compacta (SNC). In contrast, many FG-positive neurons were observed in the dorsal aspect of the SNC of rats injected with FG into the IPAC. Immunohistochemistry and in situ hybridization studies of intact animals revealed that both attractin mRNA and protein were expressed at higher levels in the ventral aspect of the SNC, whereas both attractin mRNA and protein were expressed at lower levels in the dorsal aspect of the SNC. Taken together, these results indicate that TH-IR afferents to the IPAC have distinct neurochemical properties from those to the striatum and may account for the differential vulnerability to neurodegeneration observed in zitter mutant rats.

Background Clinical trials of supra-lateral medial forebrain bundle (MFB) Deep Brain Stimulation (DBS) in treatment resistant major depressive patients have shown rapid and long-term benefits. Objective/hypothesis The study used Flinders Sensitive Line (FSL) rats with previously identified depressive-like phenotype to assess the range of behavior modification achieved by MFB DBS. Methods Male FSL and wild-type Sprague-Dawley rats as Controls were tested on mood/anxiety/exploration, cognitive and motor behaviors. The animals were implanted with bipolar stimulation electrodes in the MFB, and recovery was followed by 10 days of bilateral, chronic and continuous stimulation. Results Weight dynamics was assessed continuously and indicated similar growth rates although the FSL rats weighed approximately 20–25% less. MFB DBS had no impact on ultrasound calls emitted and the FSL rats continued to vocalize significantly less in the positive affect frequency compared to controls. Similarly, stimulation did not influence the FSL's exploration level (Elevated Plus Maze), nor locomotion (Open Field), although it reduced their freezing behavior (Open Field). Importantly, MFB DBS improved cognitive performance (Double-H) compared to Controls by reducing the time required and the number of errors committed to complete a spatial task. Conclusion MFB DBS in the FSL animals selectively affected certain types of behaviors. Exploration and vocalization remained unaltered, but cognitive performance such as speed and precision of memory recall improved compared to unstimulated and stimulated controls. Future studies should focus on the mechanisms of action of MFB DBS, and in particular on the role of dopamine in the stimulation-dependent phenotype changes.

Rats affected by the MENX syndrome spontaneously develop multiple neuroendocrine tumors (NETs) including adrenal, pituitary and thyroid gland neoplasms. MENX was initially reported to be inherited as a recessive trait and affected rats were found to be homozygous for the predisposingCdkn1bmutation encoding p27. We here report that heterozygous MENX-mutant rats (p27+/mut) develop the same spectrum of NETs seen in the homozygous (p27mut/mut) animals but with slower progression. Consequently, p27+/mut rats have a significantly shorter lifespan compared with their wild-type (p27+/+) littermates. In the tumors of p27+/mut rats, the wild-typeCdkn1ballele is neither lost nor silenced, implying that p27 is haploinsufficient for tumor suppression in this model. Transcriptome profiling of rat adrenal (pheochromocytoma) and pituitary tumors having different p27 dosages revealed a tissue-specific, dose-dependent effect of p27 on gene expression. In p27+/mut rats, thyroid neoplasms progress to invasive and metastatic medullary thyroid carcinomas (MTCs) accompanied by increased calcitonin levels, as in humans. Comparison of expression signatures of late-stage vs early-stage MTCs from p27+/mut rats identified genes potentially involved in tumor aggressiveness. The expression of a subset of these genes was evaluated in human MTCs and found to be associated with aggressive RET-M918T-positive tumors. Altogether, p27 haploinsufficiency in MENX rats uncovered a novel, representative model of invasive and metastatic MTC exploitable for translational studies of this often aggressive and incurable cancer.

In this study, we analyze the neuropathological and biochemical alterations involved in the pathogenesis of a neurodegenerative/movement disorder during different developmental stages in juvenile rats with a mutant Myosin5a (Myo5a). In mutant rats, a spontaneous autosomal recessive mutation characterized by the absence of Myo5a protein expression in the brain is associated with a syndrome of locomotor dysfunction, altered coat color, and neuroendocrine abnormalities. Myo5a encodes a myosin motor protein required for transport and proper distribution of subcellular organelles in somatodendritic processes in neurons. Here we report marked hyperphosphorylation of alpha-synuclein and tau, as well as region-specific buildup of the autotoxic dopamine metabolite, 3,4-dihydroxyphenyl-acetaldehyde (DOPAL), related to decreased aldehyde dehydrogenases activity and neurodegeneration in mutant rats. Alpha-synuclein accumulation in mitochondria of dopaminergic neurons is associated with impaired enzymatic respiratory complex I and IV activity. The behavioral and biochemical lesions progress after 15 days postnatal, and by 30–40 days the animals must be euthanized because of neurological impairment. Based on the obtained results, we propose a pleiotropic pathogenesis that links the Myo5a gene mutation to deficient neuronal development and progressive neurodegeneration. This potential model of a neurodevelopmental disorder with neurodegeneration and motor deficits may provide further insight into molecular motors and their associated proteins responsible for altered neurogenesis and neuronal disease pathogenesis.

The taiep rat undergoes hypomyelination and progressive demyelination caused by an abnormal microtubule accumulation in oligodendrocytes, which elicits neuroinflammation and motor behavior dysfunction. Based on taurine antioxidant and proliferative actions, this work explored whether its sustained administration from the embryonic age to adulthood could prevent neuroinflammation, stimulate cell proliferation, promote myelination, and relieve motor impairment. Taurine (50 mg/L of drinking water = 50 ppm) was given to taiep pregnant rats on gestational day 15 and afterward to the male offspring until eight months of age. We measured the levels of nitric oxide (NO), malondialdehyde + 4-hydroxyalkenals (MDA + 4-HDA), CXCL1, CXCR2 receptor, growth factors (BNDF and FGF2), cell proliferation, and myelin content over time. Integral motor behavior was also evaluated. Our results showed that taurine administration significantly decreased NO and MDA + 4-HDA levels, increased cell proliferation, and promoted myelination in an age- and brain region-dependent fashion compared with untreated taiep rats. Taurine effect on chemokines and growth factors was also variable. Taurine improved vestibular reflexes and limb muscular strength in perinatal rats and fine movements and immobility episodes in adult rats. These results show that chronic taurine administration partially alleviates the taiep neuropathology.

Central orexinergic system contributes to the regulation of cardiovascular function. Orexinergic neurons receiving projections of nerve fibers from multiple structures of brain which involved in control and regulation of cardiovascular function locate in hypothalamus, and their axon terminals widely project to various central structures where orexins receptors are expressed. Here, we summarize the present knowledge that describes the influence of central orexinergic system on cardiovascular activity, the relevance of dysfunction in central orexinergic system with hypertension and psychological stress induced cardiovascular reactivity which are serious risk factors for cardiovascular disease and cardiovascular death. We propose that central orexinergic system may be potentially important targets for the prevention of cardiovascular disease and cardiovascular death, and different orexinergic system involved neuronal circuits may be involved in distinct cardiovascular functions. Acupuncture having bidirectional regulatory ability and a much lower incidence of side effects can prevent disease. We review the improvement of acupuncture on hypertension and psychological stress induced cardiovascular reactivity. We think that acupuncture intervenes hypertension and psychological stress induced cardiovascular reactivity to prevent cardiovascular disease and cardiovascular death. We also summarize relation between acupuncture and central orexinergic system. We propose a hypothesis that acupuncture improve hypertension and psychological stress induced cardiovascular reactivity through regulating central orexinergic system. The knowledge is beneficial for the development of potential therapeutic targets and methods to prevent cardiovascular disease and cardiovascular death.

Aloe vera (L.) Burm. f. extract has been medicinally used for over 5000 years in different cultures for its curative and therapeutic properties ranging from dermatitis to diabetes. It has been demonstrated to alleviate diabetes through its protective effects on pancreatic islets and by improving insulin secretion.To investigate the simultaneous effect of ethanolic A. vera gel extract on diabetes and obesogenic milieu in Streptozotocin-induced WNIN/GR-Ob mutant obese rats.A total of 30 rats were grouped equally into WNIN/GR-Ob control (received water as a vehicle), WNIN/GR-Ob Diabetic rats (Streptozotocin-35 mg/kg bw), WNIN/GR-Ob Diabetic rats + Sitagliptin (10 mg/kg bw), WNIN/GR-Ob Diabetic rats + A. vera (300 mg/kg bw) and GR-Ob control + A. vera (300 mg/kg bw). After 4 weeks of treatment, fasting blood glucose, serum insulin, Homeostatic Model Assessment - Insulin Resistance and β-cell function, glucose-stimulated insulin secretion, Dipeptidyl peptidase-IV activity, and lipid profiles were studied. In addition, ultrastructural analysis of isolated islets and dual-energy X-ray absorptiometry analysis for body composition were also carried out.The A. vera treated group showed a significant reduction (p 0.05) in triglyceride, Very low-density lipoprotein levels, Triglyceride to High-density lipoprotein ratio as well as fasting blood glucose levels and DPP-IV activity with a concomitant increase in the serum insulin levels. The increase in IR was observed in both WNIN/GR-Ob control and diabetic rats with a significant decrease in β-cell function in the diabetic rats as per Homeostatic Model Assessment values. Oral administration of A. vera was effective in both reducing Homeostatic Model Assessment-Insulin Resistance and increasing Homeostatic Model Assessment-β values. Also, the treated group demonstrated preservation of islets and a significant increase (p 0.05) in the diameter of β-cell as evident through Scanning electron microscope analysis. The increase in lean body mass was manifested in the treated group with a reduction in Fat percent in comparison with other groups.The beneficial effects of A. vera in WNIN/GR-Ob strain may be attributed to its ability to lower lipid profile thus improve insulin sensitivity and/or modulating β-cell function. Thus, it has great therapeutic potential as an herbal remedy for the treatment of diabetes and associated adverse effects such as obesity. The exact mechanism underlying the observation needs to be investigated further to explore the anti-obesity and anti-diabetic properties of A. vera and advocate its potential application as alternative medicine.

Titin, a giant sarcomeric protein, is largely responsible for the diastolic properties of the heart. It has two major isoforms, N2B and N2BA due to pre-mRNA splicing regulated mainly by a splicing factor RNA binding motif 20 (RBM20). Mis-splicing of titin pre-mRNA in response to external stimuli may lead to altered ratio of N2B to N2BA, and thus, impaired cardiac contractile function. However, little is known about titin alternative splicing in response to external stimuli. Here, we reported the detailed mechanisms of titin alternative splicing in response to insulin. Insulin treatment in cultured neonatal rat cardiomyocytes (NRCMs) activated the PI3K-Akt-mTOR Kinase axis, leading to increased N2B expression in the presence of RBM20, but not in NRCMs in the absence of RBM20. By inhibiting this kinase axis with inhibitors, decreased N2B isoform was observed in NRCMs and also in diabetic rat model treated with streptozotocin, but not in NRCMs and diabetic rats in the absence of RBM20. In addition to the alteration of titin isoform ratios in response to insulin, we found that RBM20 expression was increased in NRCMs with insulin treatment, suggesting that RBM20 levels were also regulated by insulin-induced kinase axis. Further, knockdown of p70S6K1 with siRNA reduced both RBM20 and N2B levels, while knockdown of 4E-BP1 elevated expression levels of RBM20 and N2B. These findings reveal a major signal transduction pathway for insulin-induced titin alternative splicing, and place RBM20 in a central position in the pathway, which is consistent with the reputed role of RBM20 in titin alternative splicing. Findings from this study shed light on gene therapeutic strategies at the molecular level by correction of pre-mRNA mis-splicing.

The SHR and SLA16 inbred strains present behavioral differences in anxiety/emotionality that could be under the influence of dopaminergic neurotransmission. In order to investigate the role of D2 receptors in modulating such differences, an agonist (quinpirole) and an antagonist (haloperidol) of this receptor were administered, either via systemic injection (IP), or microinjected into the ventral area of the hippocampus (vHIP). Quinpirole and haloperidol IP decreased locomotor activity, only in SLA16 rats in the open-field (OF), and in both strains in the elevated plus-maze (EPM). Quinpirole also increased the preference for the aversive areas of the EPM. Quinpirole vHIP decreased locomotor activity in both strains. Haloperidol vHIP did not elicit behavioural changes and no differences in the levels of D2 receptors and of dopamine transporter in the hippocampus were found. Results indicate that systemic activation/blocking of D2 receptors caused a strain-dependent hypolocomotion, whereas activation of D2 receptors in the vHIP, but not D2 receptor antagonism, regardless of dose, decreased general locomotor activity in the two strains. Therefore, we suggest that genomic differences in the chromosome 4 can influence the locomotor activity regulated by the D2 dopaminergic receptor, especially in the vHIP.

Contains fulltext : 175064.pdf (Publisher’s version ) (Closed access) Alcohol use disorder (AUD) is characterized by excessive alcohol use and persistent alcohol seeking despite knowledge of its negative consequences. Importantly, AUD typically develops after chronic excessive alcohol use in a subgroup of individuals who drink alcohol, suggesting that AUD results from an interaction between individual vulnerability and prolonged alcohol exposure. The present study assessed the contribution of prolonged exposure to alcohol and individual levels of alcohol intake to the development of loss of control over alcohol seeking in a conditioned suppression model. To investigate the impact of prolonged alcohol exposure, conditioned suppression of alcohol seeking was assessed after 2 and 4 months of intermittent alcohol access (IAA) in a subgroup of rats drinking moderate amounts of alcohol. We observed that suppression of alcohol seeking was reduced after 4 months compared with 2 months of IAA. The influence of individual levels of alcohol intake on loss of control over alcohol seeking was subsequently determined by assessing conditioned suppression in subgroups of low and high alcohol drinking rats. Unlike the low alcohol drinking rats, the high alcohol drinking rats showed aversion-resistant alcohol seeking after 2 months of IAA, although both groups showed comparable levels of conditioned freezing. These findings show that the development of loss of control over alcohol seeking, a key characteristic of AUD in humans, is dependent on both the extent of alcohol exposure and the individual's propensity to consume alcohol.

Purpose: Our previous study demonstrated that photoelectric dye-coupled polyethylene film (Okayama University-type retinal prosthesis), which was implanted in subretinal space of the eyes of Royal College of Surgeons (RCS) rats, prevented retinal neurons from apoptotic death. In this study, we aimed to examine whether photoelectric dye itself would protect retinal neurons from apoptosis in RCS rats. Methods: RCS rats received intravitreous injection of different concentrations of the dye in the left eye and housed under a 12-h light–dark cycle. Saline injection in the right eye served as control. In addition, RCS rats with dye injection were kept in 24-h daily dark condition. Sections were processed for terminal deoxynucleotidyl transferase-mediated fluorescein-conjugated-dUTP nick-end-labeling (TUNEL) assay and immunohistochemical staining of glial fibrillary acidic protein (GFAP) and protein kinase Cα (PKCα). Results: The number of TUNEL-positive cells significantly decreased in the retina of dye-injected eyes compared with those in saline-injected eyes (P = 0.0001, 2-factor analysis of variance [ANOVA]), under 12-h light–dark cycle. Significant decrease of TUNEL-positive cells was noted in the retina of rats with dye injection compared with those with saline injection, kept under 24-h dark condition (P = 0.0001, 2-factor ANOVA). Immunoreactive area for GFAP decreased significantly in the retina of dye-injected eyes compared with that in controls (P = 0.0001, 2-factor ANOVA), whereas immunoreactive area for PKCα increased significantly in the retina of dye-injected eyes compared with that in controls (P = 0.01, 2-factor ANOVA). Conclusions: Photoelectric dye inhibits apoptotic death of photoreceptor cells in RCS rats and downregulates GFAP expression in retinal Müller cells. Photoelectric dye may be a candidate agent for neuroprotection in retinitis pigmentosa and other retinal diseases.

Methylmercury (CH 3 Hg + ) is an environmental toxicant that may lead to significant pathologies in exposed individuals. The current study assessed the disposition and toxicological effects of 2.5 or 7.5 mg kg −1 CH 3 Hg + , conjugated to cysteine (Cys; Cys- S -CH 3 Hg) and administered orally to pregnant and non-pregnant Wistar and TR − rats. Rats were euthanized on gestational day 20 and the content of mercury in each fetus, amniotic sac, and placenta was determined. The brain, liver, and kidneys were removed from each fetus for estimation of mercury content. From the dams, a sample of blood, kidneys, liver, and brain were removed at the time of euthanasia. The findings from this study indicate that pregnancy leads to significant changes in the handling of mercuric ions, particularly in the liver. Furthermore, there are significant differences in the handling of non-nephrotoxic and nephrotoxic doses of Cys- S -CH 3 Hg by maternal and fetal organs.

Several studies have suggested a role of BDNF in the development of schizophrenia. For example, post-mortem studies have shown significantly reduced levels of BDNF protein expression in the brain of schizophrenia patients. We investigated the relationship between reduced levels of BDNF in the brain and the regulation of prepulse inhibition (PPI), a behavioral endophenotype of schizophrenia. We used BDNF heterozygous mutant rats which display a 50% decrease of mature BDNF protein levels. Previously, we observed normal baseline PPI and responses to the dopamine D1/D2 receptor agonist, apomorphine, in these rats. Here, we focused on the effects of the NMDA receptor antagonist, MK-801, its interaction with mGluR2/3 and mGluR5 receptors, and the PPI response to serotonergic drugs. MK-801 administration caused a dose-dependent reduction of PPI and increase of startle amplitudes. Baseline PPI and the effect of 0.02-0.1mg/kg of MK-801 were not significantly altered in male or female BDNF heterozygous rats, although the MK-801-induced increase in startle levels was reduced. Co-treatment with the mGluR2/3 agonist, LY379,268, or the mGluR5 antagonist, MPEP, did not alter the effect of MK-801 on PPI in controls or BDNF mutant rats. Treatment with the serotonin-1A receptor agonist, 8-OH-DPAT, the serotonin-2A receptor agonist, DOI, or the serotonin releaser, fenfluramine, induced differential effects on PPI and startle but these effects were not different between the genotypes. These results show that a significant decrease of BDNF protein expression does not lead to reduced PPI at baseline or changes in the regulation of PPI via NMDA receptors or serotonergic mechanisms. These findings in a genetic rat model of BDNF deficiency do not support a role for similar reductions of BDNF levels in schizophrenia in the disruption of PPI, widely reported as an endophenotype of the illness. The potential implications of these results for our understanding of changes in PPI and BDNF expression in schizophrenia are discussed.

Objectives Cardiac surgery with cardiopulmonary bypass (CPB) provokes ischaemia and reperfusion injury (IRI). Superoxide is a main mediator of IRI and is detoxified by superoxide dismutases (SODs). Extracellular SOD (SOD3) is the prevailing isoform in the cardiovascular system. Its mutation is associated with elevated risk for ischaemic heart disease as epidemiological and experimental studies suggest. We investigated the influence of SOD3 on IRI in the context of CPB and hypothesized a protective role for this enzyme. Methods Mutant rats with loss of SOD3 function induced by amino acid shift, SOD3-E124D, (SOD3 mutant; n = 9) were examined in a model of CPB with deep hypothermic circulatory arrest provoking global IRI and compared with SOD3 competent controls (n = 8) as well as sham animals (n = 7). SOD3 plasma activity was photometrically measured with a diazo dye-forming reagent. Activation of cardioprotective rescue pathways (p44-42 MAPK and STAT3), cleavage of PARP-1, expression of SOD isoforms (SOD1, 2 and 3) and nitric oxide metabolism were analysed on the protein level by western blot. To evaluate whether SOD3 inactivity directly affects the myocardium, we isolated adult cardiac myocytes, which underwent hypoxia prior to protein analyses. Results Relative SOD3 plasma activity in SOD3 mutant rats was significantly decreased by at least 50% compared with that in SOD3 competent controls (prior to euthanasia P = 0.008). Effectively, physiological parameters [heart rate and mean arterial pressure (MAP)] indicated a trend toward impaired handling of ischaemia and reperfusion in SOD3 mutants: after reperfusion, mean heart rate was 46 bpm lower (P = 0.083) and MAP 8 mmHg lower (P = 0.288) than that in SOD competent controls. Decreased SOD3 activity led to reduced activation of cardioprotective rescue pathways in vivo and in vitro: relative activation of p44-42 MAPK (P = 0.074) and STAT3 (P = 0.027) was more than 30% decreased in heart and aortic tissue of SOD3 mutants (activity normalized to sham control as 1). After CPB, cleavage of PARP-1 was doubled in the control group (P = 0.017), but increased 3-fold in SOD3 mutants (P = 0.002). Furthermore, 3-nitrotyrosine as a measure of decreased nitric oxide bioavailability and other SOD isoforms (SOD1 and 2) were increased. Conclusions Collectively, SOD3 has a significant cardioprotective role in cases of IRI and directly affects the myocardium as hypothesized. Exploration of intervention strategies targeting SOD3 may provide therapeutic options against IRI and associated systemic inflammation.

In genetically predisposed WAG/Rij rats and healthy Wistar rats, we studied functioning of the paralemniscal region of the thalamo-cortical system. The responses of neurons of the somatosensory cortex to single electrical stimulation of the posterior nucleus of the thalamus were recorded in two- to three-monthold rats within the period when the epileptic activity was not developed. We revealed lower number of shortterm inhibitory responses in WAG/Rij rats as compared to Wistar rats. This may create preconditions for the spreading of spike-wave activity in the somatosensory cortex, which is an electrophysiological sign of absence epilepsy.

Although silver nanoparticles are considered as promising antibacterial agents because of their antibacterial activity, the acute cytotoxicity of Ag+ released from Ag nanoparticles restricts their potential practical applications. Herein, porous Ag@Au nanoplates, which could balance the Ag+ release and the toxicity of Ag naoparticles, were fabricated by stepwise seed-mediated growth and oxidation. Laser irradiation further boosted their antimicrobial activity, and significantly accelerated the curing rate of wound. Comparing with Ag nanoplates, the irradiated porous Ag@Au nanoplates showed the similar antibiotic ability against S. aureus strains and lower cytotoxicity in vitro. When the porous Ag@Au nanoplates were applied to treat S. aureus-infected wound, they had the best curing effect. Thus, these porous Ag@Au nanoplates could act as promising antibacterial agents for wound healing applications.

Background Rodents are born with morphological immature lungs and an intact surfactant system. CD26/DPP4 is a multifactorial transmembrane integral type II protein, which is involved in physiological and pathophysiological processes and is already expressed during development. CD26/DPP4, called CD26 in the following, is able to enhance or dampen differently triggered inflammation. LPS exposure often used to simulate perinatal infection delays lung development. Objective A perinatal LPS rat model was used to test the hypothesis that CD26 deficiency modulates LPS-induced retardation in morphological lung development. Methods New born Fischer CD26 positive (CD26+) and deficient (CD26−) rats were exposed to LPS on postnatal day (day post partum, dpp) 3 and 5. Morphological parameters of lung development were determined stereologically. Lung development was analysed in 7, 10 14 and 21day old rats. Results Compared to controls LPS application resulted (1) in a mild inflammation independent of the strain, (2) in significantly lower total surface and volume of alveolar septa combined with significantly higher total volume of airspaces and alveolar size on dpp 7 in both substrains. However, compared to controls in LPS treated CD26− rats significant lower values of total septal surface and volume combined with higher values of total parenchymal airspaces and alveolar size were found until the end of classical alveolarization (dpp14). In LPS treated CD26+ rat pups the retardation was abolished already on dpp 10. Conclusion In absence of CD26, LPS enhances the delay of morphological lung development. Morphological recovery was slower after the end of LPS exposure in CD26 deficient lungs.

Sirtuin 2 (SIRT2) is a deacetylase that belongs to class III family of histone deacetylases (HDACs). Although it is the most abundantly expressed member of HDAC-III in human bone tissues, it is unclear whether SIRT2 plays a role in bone metabolism. In this study, the role of SIRT2 in bone metabolism, and the underlying mechanism were investigated. In in vivo experiments, micro-CT analysis revealed that there were no differences in bone microstructures between SIRT2-KO and WT rats at 12 weeks of age. However, in 36-week-old rats, increased Tb. BMD, bone volume fraction (BV/TV) and trabecular number (Tb. N) of distal femurs were observed in SIRT2-KO rats, when compared with those of WT rats. Moreover, reduced serum β-CTX was identified in the 36-week old rats. In in vitro studies, inhibition of SIRT2 with its specific inhibitor, AGK2, suppressed the differentiation of bone marrow-derived mononuclear cells (BMMs) into osteoclasts via reduction of the expressions of c-Fos and NFATc1. These results suggest that SIRT2 plays a role in age-related bone loss, probably by regulating osteoclastogenesis.

MicroRNAs (miRNAs) have been shown to play critical roles in the pathogenesis and progression of degenerative retinal diseases like age-related macular degeneration (AMD). In this study, we first demonstrated that miR-24 plays an important role in maintaining retinal structure and visual function of rats by targeting chitinase-3-like protein 1 (CHI3L1). In the retinal pigment epithelial (RPE) cells of Royal College of Surgeons (RCS) rats, an animal model of genetic retinal degeneration (RD), miR-24 was found lower and CHI3L1 level was higher in comparison with those in Sprague-Dawley (SD) rats. Other changes in the eyes of RCS rats include activated AKT/mTOR and ERK pathways and abnormal autophagy in the RPE cells. Such roles of miR-24 and CHI3L1 were further confirmed in RCS rats by subretinal injection of agomiR-24, which decreased CHI3L1 level and preserved retinal structure and function. Upstream, NF-κB was identified as the regulator of miR-24 in the RPE cells of these rats. On the other hand, in SD rats, intraocular treatment of antagomiR-24 induced pathological changes similar to those in RCS rats. The results revealed the protective roles for miR-24 to RPE cells and a mechanism for RD in RCS rats was proposed: extracellular stress stimuli first activate the NF-κB signaling pathway, which lowers miR-24 expression so that CHI3L1 increased. CHI3L1 sequentially results in aberrant autophagy and RPE dysfunction by activating AKT/mTOR and ERK pathways. Taken together, although the possibility, that the therapeutic effects in RCS rats are caused by other transcriptional changes regulated by miR-24, cannot be excluded, these findings indicate that miR-24 protects rat retina by targeting CHI3L1. Thus, miR-24 and CHI3L1 might be the targets for developing more effective therapy for degenerative retinal diseases like AMD.

Preserving of vision is the main goal in vision research. The presented research evaluates the preservation of visual function in Royal College of Surgeon (RCS) rats using a depth perception test. Rats were placed on a stage with one side containing an illusory steep drop (“cliff”) and another side with a minimal drop (“table”). Latency of stage dismounting and the percentage of rats that set their first foot on the “cliff” side were determined. Nondystrophic Long–Evans (LE) rats were tested as control. Electroretinogram and histology analysis were used to determine retinal function and structure. Four-week-old RCS rats presented a significantly shorter mean latency to dismount the stage compared with 6-week-old rats (mean ± standard error, 13.7 ± 1.68 vs. 20.85 ± 6.5 s, P = 0.018). Longer latencies were recorded as rats aged, reaching 45.72 s in 15-week-old rats (P < 0.00001 compared with 4-week-old rats). All rats at the age of 4 weeks placed their first foot on the table side. By contrast, at the age of 8 weeks, 28.6% rats dismounted on the cliff side and at the age of 10 and 15 weeks, rats randomly dismounted the stage to either table or cliff side. LE rats dismounted the stage faster than 4-week-old RCS rats, but the difference was not statistically significant (7 ± 1.58 s, P = 0.057) and all LE rats dismounted on the table side. The latency to dismount the stage in RCS rats correlated with maximal electroretinogram b-wave under dark and light adaptation (Spearman’s rho test = −0.603 and −0.534, respectively, all P < 0.0001), outer nuclear layer thickness (Spearman’s rho test = −0.764, P = 0.002), and number of S- and M-cones (Spearman’s rho test = −0.763 [P = 0.002], and −0.733 [P = 0.004], respectively). The cliff avoidance test is an objective, quick, and readily available method for the determination of RCS rats’ visual function.

Most research in diabetes mellitus (DM) has been conducted in animals, and their replacement is currently a chimera. As compared to when they started to be used by modern science in the 17th century, a very high number of animal models of diabetes is now available, and they provide new insights into almost every aspect of diabetes. Approaches combining human, in vitro, and animal studies are probably the best strategy to improve our understanding of the underlying mechanisms of diabetes, and the choice of the best model to achieve such objective is crucial. Traditionally classified based on pathogenesis as spontaneous or induced models, each has its own advantages and disadvantages. The most common animal models of diabetes are described, and in addition to non-obese diabetic mice, biobreeding diabetes-prone (BB-DP) rats, streptozotocin-induced models, or high-fat diet-induced diabetic C57Bl/6J mice, new valuable models, such as dogs and cats with spontaneous diabetes, are described.

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.

Purpose: Immunosuppression is frequently employed to enhance survival of xenografted human cells as part of translational proof-of-concept studies. However, the potential effects of this treatment are easily overlooked. Methods: As part of baseline testing in the dark-eyed variant of the dystrophic Royal College of Surgeons (RCS) rat, we documented the time course of retinal degenerative changes versus Long Evans controls using bright field retinal imaging, fluorescein angiography, and histology and examined the impact of immunosuppression on visual function. Rats received either no treatment or systemic immunosuppression with oral cyclosporine A and injectable dexamethasone and subsequently underwent functional evaluation by optomotor response testing and electroretinography (ERG) at multiple intervals from P45 to P180. Results: Immunosuppressed RCS animals demonstrated poorer performance on functional tests than age-matched untreated rats during the earlier stages of degeneration, including significantly lower spatial acuities on optomotor threshold testing and significantly lower b-wave amplitudes on scotopic and photopic ERGs. Retinal imaging documented the progression of degenerative changes in the RCS fundus and histologic evaluation of the RCS retina confirmed progressive thinning of the outer nuclear layer. Conclusions: A standard regimen of cyclosporine A plus dexamethasone, administered to RCS rats, results in demonstrable systemic side effects and depressed scores on behavioral and electrophysiological testing at time points before P90. The source of the functional impairment was not identified. This finding has implications for the interpretation of data generated using this commonly used translational model.

The Foxn1 gene is known as a critical factor for the differentiation of thymic and skin epithelial cells. This study was designed to examine the phenotype of Foxn1-modified rats generated by the CRISPR/Cas9 system. Guide-RNA designed for first exon of the Foxn1 and mRNA of Cas9 were co-injected into the pronucleus of Crlj:WI zygotes. Transfer of 158 injected zygotes resulted in the birth of 50 offspring (32 %), and PCR identified five (10 %) as Foxn1-edited. Genomic sequencing revealed the deletion of 44 or 60 bp from and/or insertion of 4 bp into the Foxn1 gene in a single allele. The number of T-cells in the peripheral blood lymphocytes of mutant rats decreased markedly. While homozygous deleted mutant rats had no thymus, the mutant rats were not completely hairless and showed normal performance in delivery and nursing. Splicing variants of the indel-mutation in the Foxn1 gene may cause hypomorphic allele, resulting in the phenotype of thymus deficiency and incomplete hairless. In conclusion, the mutant rats in Foxn1 gene edited by the CRISPR/Cas9 system showed the phenotype of thymus deficiency and incomplete hairless which was characterized by splicing variants.

Hawthorn is a widely used herbal alternative medicine for the treatment of various cardiovascular diseases. However, the attributed health benefits, purported to be due to the presence of phenolic compounds, may depend on both the specific species and plant part. Studies to date investigating effects of hawthorn on heart disease(s) have used well-described European and/or Asian species, while little is known regarding the bioactivity of species native to North America. Six weeks of supplementation of both fireberry hawthorn berry (native Crataegus chrysocarpa) and English hawthorn leaf (C. monogyna, naturalized in North America) in the JCR:LA-cp rat, resulted in a significant reduction in heart weight, fasting LDL-C and improved heart function (p < 0.05). Fasting triglyceride and myocardial fibrosis were also reduced, but only by the berry extract. We demonstrate that both of the Canadian-sourced hawthorn extracts (introduced leaf and native berry) have cardioprotective benefits, likely via increased availability of nitric oxide.

Author(s): Lin, Bin; McLelland, Bryce T; Aramant, Robert B; Thomas, Biju B; Nistor, Gabriel; Keirstead, Hans S; Seiler, Magdalene J | Abstract: PurposeTo study if human embryonic stem cell-derived photoreceptors could survive and function without the support of retinal pigment epithelium (RPE) after transplantation into Royal College of Surgeons rats, a rat model of retinal degeneration caused by RPE dysfunction.MethodsCSC14 human embryonic stem cells were differentiated into primordial eye structures called retinal organoids. Retinal organoids were analyzed by quantitative PCR and immunofluorescence and compared with human fetal retina. Retinal organoid sheets (30-70 day of differentiation) were transplanted into immunodeficient RCS rats, aged 44 to 56 days. The development of transplant organoids in vivo in relation to the host was examined by optical coherence tomography. Visual function was assessed by optokinetic testing, electroretinogram, and superior colliculus electrophysiologic recording. Cryostat sections were analyzed for various retinal, synaptic, and donor markers.ResultsRetinal organoids showed similar gene expression to human fetal retina transplanted rats demonstrated significant improvement in visual function compared with RCS nonsurgery and sham surgery controls by ERGs at 2 months after surgery (but not later), optokinetic testing (up to 6 months after surgery) and electrophysiologic superior colliculus recordings (6-8 months after surgery). The transplanted organoids survived more than 7 months; developed photoreceptors with inner and outer segments, and other retinal cells; and were well-integrated within the host.ConclusionsThis study, to our knowledge, is the first to show that transplanted photoreceptors survive and function even with host's dysfunctional RPE. Our findings suggest that transplantation of organoid sheets from stem cells may be a promising approach/therapeutic for blinding diseases.

Taiep rat is a myelin mutant with a progressive motor syndrome characterized by tremor, ataxia, immobility episodes, epilepsy and paralysis of the hindlimbs. Taiep had an initial hypomyelination followed by a progressive demyelination associated with an increased expression of some interleukins and their receptors. The pathology correlated with an increase in nitric oxide activity and lipoperoxidation. In base of the above evidences taiep rat is an appropriate model to study neuroimmune interactions. The aim of this study was to analyze the immune responses in male taiep rats after acute infection with Trichinella spiralis. Our results show that there is an important decrease in the number of intestinal larvae in the taiep rat with respect to Sprague-Dawley control rats. We also found differences in the percentage of innate and adaptive immune cell profile in the mesenteric lymphatic nodes and the spleen that correlated with the demyelination process that took place on taiep subjects. Finally, a clear pro-inflammatory cytokine pattern was seen on infected taiep rats, that could be responsible of the decrement in the number of larvae number. These results sustain the theory that neuroimmune interaction is a fundamental process capable of modulating the immune response, particularly against the parasite Trichinella spiralis in an animal model of progressive demyelination due to tubulinopathy, that could be an important mechanism for the clinical course of autoimmune diseases associated with parasite infection.