Transgenic Rats Research Articles

Construction and characterization of a humanized SLCO1B1 rat model with its application in evaluating the uptake of different statins

Organic anion-transporting polypeptides 1B1 (OATP1B1) plays a crucial role in the transport of statins. However, there are too few animal models related to OATP1B1, especially humanized animal models. In this study, the human SLCO1B1 cDNA was inserted into the second exon of the rat Slco1b2 gene using CRISPR/Cas9 technology. Pharmacokinetic characteristics of statins were conducted in wild-type (WT), humanized OATP1B1 (hOATP1B1), and OATP1B2 knockout (OATP1B2 KO) rats, respectively. The results showed that human OATP1B1 was successfully expressed in rat liver and exhibited transport function. Furthermore, the pharmacokinetic results revealed that OATP1B1 exhibited varying uptake levels of pivastatin, rosuvastatin, and fluvastatin, leading to different levels of exposure within the body. These results were consistent with those obtained from in vitro experiments using overexpressed cell lines. In conclusion, we established a novel humanized SLCO1B1 transgenic rat model to assess the role of human OATP1B1 in the uptake of different statins. The different uptake mediated by OATP1B1 may be an important reason for the different efficacy of statins. The hOATP1B1 rat is a promising model for improving the prediction of human drug transport.

Hexane insoluble fraction from purple rice extract improves steatohepatitis and fibrosis via inhibition of NF-κB and JNK signaling.

Nonalcoholic fatty liver disease (NAFLD) is fatty liver mainly related to metabolic syndrome. NAFLD with inflammation and hepatocellular damage is defined as nonalcoholic steatohepatitis (NASH), which can progress to cirrhosis and hepatocellular carcinoma. We have previously reported that a hexane insoluble fraction from an anthocyanin-rich purple rice ethanolic extract (PRE-HIF) can suppress prostate carcinogenesis. However, the extract's effect on NASH has not yet been established. In the present study, we investigated the chemopreventive effect of a PRE-HIF on NASH and liver fibrosis using a connexin 32 (Cx32) dominant negative transgenic (Cx32ΔTg) rat NASH model. Seven-week-old male Cx32ΔTg rats were fed a control diet, a high-fat diet (HFD), or an HFD with 1% PRE-HIF and intraperitoneal administration of dimethylnitrosamine for 17 weeks. Histological findings of NASH such as fat deposition, lobular inflammation, hepatocyte ballooning injury, and bridging fibrosis were observed in the HFD group but not in the control group, and all histological parameters were significantly improved by PRE-HIF treatment. Corresponding to the histological changes, increased expression of inflammatory cytokine mRNAs (TNF-α, IL-6, IL-18, IFN-γ, IL-1β, TGF-β1, TIMP1, TIMP2, COL1A1), along with and activation of nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) signaling were observed in the HFD group, which was significantly decreased by PRE-HIF. The number and area of hepatic precancerous glutathione S-transferase placental form-positive foci tended to be decreased by PRE-HIF. These results indicate that intake of purple rice as a dietary supplement may reduce steatohepatitis, liver injury, and fibrosis in NASH by inactivation of NF-κB or JNK.

Lipid Droplets Accumulation in the Brain of HIV Transgenic Rat: Implication in the Accelerated Aging of HIV Infected Individuals.

Abnormal microglial activation has been suggested as "driven force" promoting brain aging. Lipid droplets accumulating microglia (LDAM), identified as a novel inflammatory phenotype, elevate neuroinflammation and exaggerate neuronal injuries in aging and multiple neurodegenerative diseases. Since chronic HIV (human immunodeficiency virus) (+) individuals show an accelerated brain aging and higher incidence of neurological symptoms compared to age-matched HIV (-) population, we hypothesize that LDAM are also involved in such phenomenon. For validating the hypothesis, we employed HIV transgenic (HIV-Tg) and wilt type (WT) rats to check lipid droplets (LDs) accumulation in the brains at mature (6 months) and middle age (12 months). Our results showed that HIV-Tg rats possess higher levels of LDs formation in the hippocampus (HP) and prefrontal cortex (PFc) than controls at middle age. Increased LDs are mainly presented in microglia in the HP but largely co-localized with astrocytes in the PFc. Interestingly, increased LDs are associated with upregulation on Iba1 but not with GFAP levels. HIV-Tg rats reveal an accelerated LDs accumulation during normal aging. Purified microglia from HIV-Tg rats (12 month) show higher expression of neuroimmune signaling than microglia from controls. HIV-Tg rats showed dysregulation on cholesterol synthesis in the brain HP as well as deficiency on locomotion coordination compared to controls. Overall, our results demonstrate substantial LDs accumulation in the brains of HIV-Tg rats which is associated with abnormal microglial activation and accelerated decline on locomotion coordination during aging. Dysregulation on lipid metabolism might underlie accelerated brain aging in the context of chronic HIV infection.

Cerebral Proteomic Changes in the rTg-D Rat Model of Cerebral Amyloid Angiopathy Type-2 With Cortical Microhemorrhages and Cognitive Impairments.

Cerebral amyloid angiopathy (CAA) is a common disorder of the elderly, a prominent comorbidity of Alzheimer's disease, and causes vascular cognitive impairment and dementia. Previously, we generated a novel transgenic rat model (rTg-D) that produces human familial CAA Dutch E22Q mutant amyloid β-protein (Aβ) in brain and develops arteriolar CAA type-2. Here, we show that deposition of fibrillar Aβ promotes arteriolar smooth muscle cell loss and cerebral microhemorrhages that can be detected by magnetic resonance imaging and confirmed by histopathology. Aged rTg-D rats also present with cognitive deficits. Cerebral proteomic analyses revealed 241 proteins that were significantly elevated with an increase of >50% in rTg-D rats presenting with CAA compared to wild-type rats. Fewer proteins were significantly decreased in rTg-D rats. Of note, high temperature requirement peptidase A (HTRA1), a proteinase linked to transforming growth factor beta 1 (TGF-β1) signaling, was elevated and found to accumulate in cerebral vessels harboring amyloid deposits. Pathway analysis indicated elevation of the TGF-β1 pathway and increased TGF-β1 levels were detected in rTg-D rats. In conclusion, the present findings provide new molecular insights into the pathogenesis of CAA and suggest a role for interactions between HTRA1 and TGF-β1 in the disease process.

P2X receptor- and postsynaptic NMDA receptor-mediated long-lasting facilitation of inhibitory synapses in the rat insular cortex

Adenosine triphosphate (ATP) changes the efficacy of synaptic transmission. Despite recent progress in terms of the roles of purinergic receptors in cerebrocortical excitatory synaptic transmission, their contribution to inhibitory synaptic transmission is unknown. To elucidate the effects of α,β-methylene ATP (αβ-mATP), a selective agonist of P2X receptors (P2XRs), on inhibitory synaptic transmission in the insular cortex (IC), we performed whole-cell patch-clamp recording from IC pyramidal neurons (PNs) and fast-spiking neurons (FSNs) in either sex of VGAT-Venus transgenic rats. αβ-mATP increased the amplitude of miniature IPSCs (mIPSCs) under conditions in which NMDA receptors (NMDARs) are recruitable. αβ-mATP-induced facilitation of mIPSCs was sustained even after the washout of αβ-mATP, which was blocked by preincubation with fluorocitrate. The preapplication of NF023 (a P2X1 receptor antagonist) or AF-353 (a P2X3 receptor antagonist) blocked αβ-mATP-induced mIPSC facilitation. Intracellular application of the NMDAR antagonist MK801 blocked the facilitation. d-serine, which is an intrinsic agonist of NMDARs, mimicked αβ-mATP-induced mIPSC facilitation. The intracellular application of BAPTA a Ca2+ chelator, or the bath application of KN-62, a CaMKII inhibitor, blocked αβ-mATP-induced mIPSC facilitation, thus indicating that mIPSC facilitation by αβ-mATP required postsynaptic [Ca2+]i elevation through NMDAR activation. Paired whole-cell patch-clamp recordings from FSNs and PNs demonstrated that αβ-mATP increased the amplitude of unitary IPSCs without changing the paired-pulse ratio. These results suggest that αβ-mATP-induced IPSC facilitation is mediated by postsynaptic NMDAR activations through d-serine released from astrocytes. Subsequent [Ca2+]i increase and postsynaptic CaMKII activation may release retrograde messengers that upregulate GABA release from presynaptic inhibitory neurons, including FSNs. (250/250 words)

Inactivation of ERK1/2 Signaling in Dopaminergic Neurons by Map Kinase Phosphatase MKP3 Regulates Dopamine Signaling and Motivation for Cocaine.

The mesolimbic dopamine system is a crucial component of reward and reinforcement processing, including the psychotropic effects of drugs of abuse such as cocaine. Drugs of abuse can activate intracellular signaling cascades that engender long-term molecular changes to brain reward circuitry, which can promote further drug use. However, gaps remain about how the activity of these signaling pathways, such as ERK1/2 signaling, can affect cocaine-induced neurochemical plasticity and cocaine-associated behaviors specifically within dopaminergic cells. To enable specific modulation of ERK1/2 signaling in dopaminergic neurons of the ventral tegmental area, we utilize a viral construct that Cre dependently expresses Map kinase phosphatase 3 (MKP3) to reduce the activity of ERK1/2, in combination with transgenic rats that express Cre in tyrosine hydroxylase (TH)-positive cells. Following viral transfection, we found an increase in the surface expression of the dopamine transporter (DAT), a protein associated with the regulation of dopamine signaling, dopamine transmission, and cocaine-associated behavior. We found that inactivation of ERK1/2 reduced post-translational phosphorylation of the DAT, attenuated the ability of cocaine to inhibit the DAT, and decreased motivation for cocaine without affecting associative learning as tested by conditioned place preference. Together, these results indicate that ERK1/2 signaling plays a critical role in shaping the dopamine response to cocaine and may provide additional insights into the function of dopaminergic neurons. Further, these findings lay important groundwork toward the assessment of how signaling pathways and their downstream effectors influence dopamine transmission and could ultimately provide therapeutic targets for treating cocaine use disorders.

Association between plasma inflammatory biomarkers and [18F]FDG‐PET signal in a transgenic amyloid rat model

AbstractBackgroundNeuroinflammation is thought to play an important role in the pathogenesis of Alzheimer’s disease (AD). It has recently been demonstrated that [18F]FDG positron emission tomography (PET) signal is sensitive to central inflammatory changes in AD. However, it is unknown whether blood cytokines, which are important signaling molecules that regulate the peripheral inflammatory process, are associated with the cerebral [18F]FDG‐PET signal. In this work, we aimed to investigate whether plasma cytokines are associated with brain glucose metabolism in a transgenic amyloid rat model.MethodBrain metabolism of 4‐ and 10‐month‐old wild‐type (WT) and APP/PS1 rats (TgF344‐AD) were assessed with [18F]FDG‐PET imaging. Plasma inflammatory markers (IFN‐γ, IL‐1α, IL‐1β, IL‐6, IL‐10 and TNF‐α) were measured by multiplex immunoassay. [18F]FDG‐SUVr was calculated with pons as the reference region. Correlation between inflammatory markers and [18F]FDG‐PET signal was conducted at the voxel level (RMINC; p<0.05 and t>2).ResultAt 4 months, IL‐10 levels significantly associate with [18F]FDG‐PET signal in the frontal and temporoparietal cortices (FCx and TPCx) and hippocampus (Fig1A), whereas other biomarkers present only small clusters associated with [18F]FDG‐PET signal (Fig1A). At 10 months, a positive association with [18F]FDG‐PET was found between IL‐1α and IL‐1β at FCx and TPCx, while IL‐10 showed a positive correlation at the TPCx (Fig1B). Additionality, IFN‐γ and TNF‐α presented small correlation clusters at the TPCx, and IL‐6 at the TPCx and CxF (Fig1B).ConclusionAt the pre‐amyloid plaque stage, only an anti‐inflammatory cytokine, IL‐10, presented positive associations with brain glucose metabolism. However, multiple pro‐inflammatory cytokines correlated with brain glucose metabolism at the amyloid plaque stage. These findings suggest a dual peripheral inflammatory response impacting brain metabolism, which can be associated with amyloid species and deposition.

Transcriptomic and proteomic characterization of the locus coeruleus in a rat model of Alzheimer’s disease

AbstractBackgroundThe noradrenergic locus coeruleus (LC) is the first region to accumulate hyperphosphorylated tau pathology (p‐tau) in Alzheimer’s disease (AD). Using TgF344‐AD transgenic rats, which develop endogenous p‐tau in the LC prior to pathology in forebrain regions, we have recently demonstrated age‐dependent effects of p‐tau on LC firing rates. Specifically, LC neurons exhibit early hyperactivity followed by late hypoactivity. These changes in LC firing rates are coincident with the emergence of behavioral phenotypes that characterize different stages of AD (neuropsychiatric symptoms followed by cognitive deficits). Here, we sought to identify molecular mechanisms that could contribute to these changes using RNAscope and proteomics in TgF344‐AD rats.Methods32 male and female TgF344‐AD rats and wild‐type (WT) littermates aged 6 and 15 months were used for this study. Brains were flash‐frozen in isopentane on dry ice and stored at ‐80°C until being sectioned. At the level of the LC, the left hemisphere LC was collected with a 1 mm tissue punch. Punches of the cerebellum were taken as a control to verify LC enrichment in the brainstem punches. The right LC hemisphere was sectioned at 16 µm for processing with fluorescent in situ hybridization using the RNAscope Multiplex Fluorescent V2 Assay according to the manufacturer’s protocol. Probes included noradrenergic markers (tyrosine hydroxylase, norepinephrine transporter) and those that influence LC activity (α2‐adrenergic receptors, GRIA2, GRIN, ADCYAP1R1). Separately, tissue punches were submitted to the Emory Integrated Proteomics Core for label‐free quantification of proteins.ResultsRNAscope revealed no differences in transcript levels of noradrenergic markers at either age. Similarly, there were no differences in transcript levels of α2‐adrenergic receptors, GRIA, or ADCYAP1R1 at either age, or GRIN in 6‐month animals. We observed an increase of GRIN mRNA in 15‐month TgF344‐AD animals, suggesting an upregulation of excitatory neurotransmission.ConclusionsNoradrenergic neuronal identity remains in‐tact up to 15 months of age, a timepoint when there is no LC cell death in these rats. We have also uncovered a potential compensatory mechanism to maintain LC activity in late stages of disease. Proteomics and RNAscope analysis of other markers that may affect LC activity are ongoing.

Targeted metabolomics reveals progression of early Alzheimer’s disease in TgF344 transgenic rats

AbstractBackgroundAlzheimer’s disease (AD) is an aging‐related neurodegenerative disease, leading to the progressive loss of memory and other cognitive functions. As there is still no cure for AD, the growth in the number of susceptible individuals represents a major emerging threat to public health. Current AD diagnosis methods can only identify patients in the advanced stage of the disease, that means when the dementia is already present. This highlights the need for new approaches for the diagnosis of AD at earlier stages, especially before the symptoms appear. In addition, AD is reported to be closely linked with abnormal lipid metabolism. The objective of this study is to identify blood‐based lipid biomarkers to gain a more comprehensive understanding of what causes AD and its subsequent development.MethodA longitudinal study with 4 different ages (12 weeks, 25 weeks, 50 weeks, and 85 weeks) was conducted in TgF344 transgenic (AD) and wild‐type (WT) rats. Metabolomics profiling of EDTA plasma yielded 811 metabolites after quality control. Metabolites profiled included medium and apolar lipids covering 27 lipid classes, signaling lipids including oxylipins, isoprostanes, eicosanoids, bile acids, endocannabinoids. All data was log2 transformed. All statistical analysis and visualizations were performed in R.ResultThe sphingomyelins (SM) were decreased at 25 weeks with AD having more SM expressed than the WT group, while this was reversed at 50 weeks. In 12 weeks, the volcano plot of AD/WT showed more significant lipids on female (p<0.05:15) than male (p<0.05:2). Whereas, in 25 weeks, male (p<0.05:56) showed more significant lipids than female (p<0.05:7). The ratios of an eicosanoid to its seven products were first decreased, then increased over time in both WT and AD group. However, the WT group reached its minimum at 50 weeks, while the AD group reached its minimum at 25 weeks. Sex differences were found in AD/WT among all ages.ConclusionLipidomics data on longitudinal course of potential lipid biomarkers in plasma of WT and AD rats show some effects of AD and/or aging. It is anticipated that this information will contribute to developing lipid biomarker fingerprints for diagnosis and monitoring AD progression distinct from normal aging.

Age‐ and sex‐dependent differences in cognitive decline in rat models of Alzheimer’s disease

AbstractBackgroundMemory loss has been a central focus of Alzheimer’s disease (AD) research but executive dysfunction may be one of the earliest indicators of AD (Baudic et al., 2006). Executive function refers to a group of cognitive processes that are required for goal directed behaviours, such as working memory and cognitive flexibility. Amyloid‐β (Aβ) plaques and neurofibrillary tangles are considered the pathological hallmarks of AD, but evidence suggests that these are not the earliest pathologies to appear in the AD brain (Sperling et al., 2011). This highlights the importance of assessing both the prodromal and more advanced phases of AD in preclinical modelling. This study aimed to characterize age‐ and sex‐related changes in executive function, learning, and memory across normal aging in rats as well as in two aging transgenic rat strains of AD: TgAPP21 (Aβ plaque‐negative) and TgAPP/PS1 (Aβ plaque‐positive).MethodMale and female wildtype Fischer 344, TgAPP21, and TgAPP/PS1 rats were used in this study. The TgAPP21 rat overexpresses the mutant human amyloid precursor protein but does not spontaneously develop Aβ plaques, serving as an important model of prodromal AD. The TgAPP/PS1 rat overexpresses mutant human forms of both the amyloid precursor protein and Presenilin‐1 and does develop age‐dependent Aβ plaques. These strains allowed us to assess cognition under three conditions (i) normal aging, (ii) increased Aβ without plaque deposition, and (iii) age‐dependent Aβ plaque deposition. At 3, 9, or 15 months of age the rats were tested for spatial learning and memory, working memory, and cognitive flexibility using the Morris water maze, the 8‐arm radial water maze, and a T‐maze set‐shifting task.ResultAge‐dependent cognitive decline was observed across all genotypes but was exacerbated in both transgenic models. Effects of age, sex, and genotype varied across the cognitive tasks, emphasizing the importance of assessing multiple cognitive domains.ConclusionThese findings expand our understanding of the roles of sex, age and Aβ plaque‐positive/negative environments in cognitive decline in preclinical rat strains of AD. Overall, this work supports further investigation into the pathologies driving these cognitive changes and potential biomarkers for earlier detection.

Investigating the role of Donepezil in promoting Oligodendrocyte precursor cell (OPC) differentiation and Therapeutic Potential in Alzheimer’s Disease (AD)

AbstractBackgroundStatistical analysis of myelin basic protein (MBP) cells generated from Neural progenitor cell (NPC)‐derived Oligodendrocyte precursor cells (OPCs) treated with various AChEIs, including donepezil, huperzine A, rivastigmine and tacrine, revealed that only donepezil is capable of facilitating OPC‐to‐OL formation, indicating that donepezil’s effect in promoting OPC differentiation is not dependent on the inhibition of AChE. So, it can be possible that OPC‐to‐OL formation is why donepezil is considered leading in Alzheimer’s disease (AD) treatment.MethodTrial will include transgenic rat line Tg6590 that expresses human APP with the Swedish mutation driven by the ubiquitin promoter that doesn’t develop extracellular beta‐amyloid pathology before 15 months at least. Controlling their genome influence, the cognitive characteristics of mice can be related to neurobiological characteristics only. Excluding a standard (S), group of control animals was subjected to a CPZ‐free diet; rat (+MBP) will be injected with OPCs, and rat (‐MBP) will be supplemented with 0.6% cuprizone (CPZ) for 2 weeks, which facilitates demyelination. This study enables us to observe the myelination effect on its behavioral impairment (n = 3). Luxol fast blue (LFB) test or MBP tracking will be held monthly to observe OPCs‐to‐OL process. It was reported that Tg6590 rats display learning and memory deficits in the Morris water maze at the age of 9 months and altered spontaneous behavior measured in open‐field. So, with continuous behavioral observations, by procedure for a redundant place/cued version of Morris water maze task, memory‐learning assessment (MLA) will depend upon duration it takes to observe obvious behavioral impairment.ResultThis clinical trial is now under process and it is set to start by late 2024.ConclusionOPCs implantation may lead to axonal adaptation to physical stress with less beta‐amyloid and may also maintain the myelination density in CNS, especially in hippocampal‐neocortical neural connections, which can be therapeutically efficient for memory‐learning deficits.

The Effect of Fat Intake with Increased Omega-6-to-Omega-3 Polyunsaturated Fatty Acid Ratio in Animal Models of Early and Late Alzheimer’s Disease-like Pathogenesis

This work aims to clarify the effect of dietary polyunsaturated fatty acid (PUFA) intake on the adult brain affected by amyloid pathology. McGill-R-Thy1-APP transgenic (Tg) rat and 5xFAD Tg mouse models that represent earlier or later disease stages were employed. The animals were exposed to a control diet (CD) or an HFD based on corn oil, from young (rats) or adult (mice) ages for 24 or 10 weeks, respectively. In rats and mice, the HFD impaired reference memory in wild-type (WT) animals but did not worsen it in Tg, did not cause obesity, and did not increase triglycerides or glucose levels. Conversely, the HFD promoted stronger microglial activation in Tg vs. WT rats but had no effect on cerebral amyloid deposition. IFN-γ, IL-1β, and IL-6 plasma levels were increased in Tg rats, regardless of diet, while CXCL1 chemokine levels were increased in HFD-fed mice, regardless of genotype. Hippocampal 3-nitrotyrosine levels tended to increase in HFD-fed Tg rats but not in mice. Overall, an HFD with an elevated omega-6-to-omega-3 ratio as compared to the CD (25:1 vs. 8.4:1) did not aggravate the outcome of AD regardless of the stage of amyloid pathology, suggesting that many neurobiological processes relevant to AD are not directly dependent on PUFA intake.

Deschloroclozapine exhibits an exquisite agonistic effect at lower concentration compared to clozapine-N-oxide in hM3Dq expressing chemogenetically modified rats

IntroductionWithin the realm of chemogenetics, a particular form of agonists targeting designer receptors exclusively activated by designer drugs (DREADDs) has emerged. Deschloroclozapine (DCZ), a recently introduced DREADDs agonist, demonstrates remarkable potency in activating targeted neurons at a lower dosage compared to clozapine-N-oxide (CNO).MethodsWe conducted a comparative analysis of the effects of subcutaneously administered CNO (1 mg/kg) and DCZ (0.1 mg/kg) in our transgenic rats expressing hM3Dq and mCherry exclusively in oxytocin (OXT) neurons.Results and DiscussionNotably, DCZ exhibited a swift and robust elevation of serum OXT, surpassing the effects of CNO, with a significant increase in the area under the curve (AUC) up to 3 hours post-administration. Comprehensive assessment of brain neuronal activity, using Fos as an indicator, revealed comparable effects between CNO and DCZ. Additionally, in a neuropathic pain model, both CNO and DCZ increased the mechanical nociceptive and thermal thresholds; however, the DCZ-treated group exhibited a significantly accelerated onset of the effects, aligning harmoniously with the observed alterations in serum OXT concentration following DCZ administration. These findings emphasize the remarkable efficacy of DCZ in rats, suggesting its equivalent or potentially superior performance to CNO at considerably lower dosages, thus positioning it as a promising contender among DREADDs agonists.

The treatment with trandolapril and losartan attenuates pressure and volume overload alternations of cardiac connexin-43 and extracellular matrix in Ren-2 transgenic rats

Heart failure (HF) is life-threatening disease due to electro-mechanical dysfunction associated with hemodynamic overload, while alterations of extracellular matrix (ECM) along with perturbed connexin-43 (Cx43) might be key factors involved. We aimed to explore a dual impact of pressure, and volume overload due to aorto-caval fistula (ACF) on Cx43 and ECM as well as effect of renin–angiotensin blockade. Hypertensive Ren-2 transgenic rats (TGR) and normotensive Hannover Sprague–Dawley rats (HSD) that underwent ACF were treated for 15-weeks with trandolapril or losartan. Blood serum and heart tissue samples of the right (RV) and left ventricles (LV) were used for analyses. ACF-HF increased RV, LV and lung mass in HSD and to lesser extent in TGR, while treatment attenuated it and normalized serum ANP, BNP-45 and TBARS. Cx43 protein and its ser368 variant along with PKCε were lower in TGR vs HSD and suppressed in both rat strains due to ACF but prevented more by trandolapril. Pro-hypertrophic PKCδ, collagen I and hydroxyproline were elevated in TGR and increased due to ACF in both rat strains. While SMAD2/3 and MMP2 levels were lower in TGR vs HSD and reduced due to ACF in both strains. Findings point out the strain-related differences in response to volume overload. Disorders of Cx43 and ECM signalling may contribute not only to HF but also to the formation of arrhythmogenic substrate. There is benefit of treatment with trandolapril and losartan indicating their pleiotropic anti-arrhythmic potential. It may provide novel input to therapy.

Reduction in Insulin Uncovers a Novel Effect of VEGFB on Cardiac Substrate Utilization.

The heart relies heavily on external fatty acid (FA) for energy production. VEGFB (vascular endothelial growth factor B) has been shown to promote endothelial FA uptake by upregulating FA transporters. However, its impact on LPL (lipoprotein lipase)-mediated lipolysis of lipoproteins, a major source of FA for cardiac use, is unknown. VEGFB transgenic (Tg) rats were generated by using the α-myosin heavy chain promoter to drive cardiomyocyte-specific overexpression. To measure coronary LPL activity, Langendorff hearts were perfused with heparin. In vivo positron emission tomography imaging with [18F]-triglyceride-fluoro-6-thia-heptadecanoic acid and [11C]-palmitate was used to determine cardiac FA uptake. Mitochondrial FA oxidation was evaluated by high-resolution respirometry. Streptozotocin was used to induce diabetes, and cardiac function was monitored using echocardiography. In Tg hearts, the vectorial transfer of LPL to the vascular lumen is obstructed, resulting in LPL buildup within cardiomyocytes, an effect likely due to coronary vascular development with its associated augmentation of insulin action. With insulin insufficiency following fasting, VEGFB acted unimpeded to facilitate LPL movement and increase its activity at the coronary lumen. In vivo PET imaging following fasting confirmed that VEGFB induced a greater FA uptake to the heart from circulating lipoproteins as compared with plasma-free FAs. As this was associated with augmented mitochondrial oxidation, lipid accumulation in the heart was prevented. We further examined whether this property of VEGFB on cardiac metabolism could be useful following diabetes and its associated cardiac dysfunction, with attendant loss of metabolic flexibility. In Tg hearts, diabetes inhibited myocyte VEGFB gene expression and protein secretion together with its downstream receptor signaling, effects that could explain its lack of cardioprotection. Our study highlights the novel role of VEGFB in LPL-derived FA supply and utilization. In diabetes, loss of VEGFB action may contribute toward metabolic inflexibility, lipotoxicity, and development of diabetic cardiomyopathy.

Role of nucleus accumbens D1-type medium spiny neurons in the expression and extinction of sign-tracking

While sign-tracking, also known as autoshaping, has been studied for many decades, only recently has the tendency to show sign-tracking behavior been linked to the development and persistence of addiction. Sign-tracking is dependent upon dopamine activity in the nucleus accumbens (NAc). The NAc is comprised predominantly of medium spiny projection neurons (MSN) that can be differentiated by their D1-like or D2-like dopamine receptor expression. Here we determined how reducing activity of D1-type MSNs in the NAc affects the expression and extinction of sign-tracking. To address this, we transfected the NAc of transgenic male and female rats that selectively express Cre recombinase in D1-type MSNs with a DIO viral vector expressing hM4Di. Cre- rats were given the same viral infusion but did not express the hM4Di receptor and therefore served as controls. Rats were then conditioned to associate lever presentations with pellet delivery. After sign-tracking was established, all rats were administered clozapine-n-oxide (CNO) prior to three additional conditioning sessions to assess the effects of NAc D1-MSNs inhibition on sign-tracking in the presence of reward. CNO treatment did not alter the expression of sign-tracking in Cre+ or Cre- rats. Next rats underwent extinction training where lever presentations occurred without pellet delivery and all rats received a CNO injection prior to each extinction session. In these extinction conditions, Cre+ rats exhibited robust extinction of sign-tracking across sessions, whereas Cre- rats did not. To determine if D1-MSN inhibition merely produced a temporary cessation of sign-tracking or instead had facilitated a persistent loss of sign-tracking, we evaluated the reemergence of sign-tracking in a test for reconditioning. During testing, reintroduction of the CS-US pairing did not promote the reemergence of sign-tracking in Cre+ rats, but restored sign-tracking in Cre- rats. Thus, chemogenetic inhibition of NAc D1-MSNs promoted extinction of sign-tracking. Collectively, these data suggest that D1-MSNs play an important role in resistance to extinction that typifies sign-tracking behavior.

Increased Inhibition May Contribute to Maintaining Normal Network Function in the Ventral Hippocampus of a Fmr1-Targeted Transgenic Rat Model of Fragile X Syndrome.

A common neurobiological mechanism in several neurodevelopmental disorders, including fragile X syndrome (FXS), is alterations in the balance between excitation and inhibition in the brain. It is thought that in the hippocampus, as in other brain regions, FXS is associated with increased excitability and reduced inhibition. However, it is still not known whether these changes apply to both the dorsal and ventral hippocampus, which appear to be differently involved in neurodegenerative disorders. Using a Fmr1 knock-out (KO) rat model of FXS, we found increased neuronal excitability in both the dorsal and ventral KO hippocampus and increased excitatory synaptic transmission in the dorsal hippocampus. Interestingly, synaptic inhibition is significantly increased in the ventral but not the dorsal KO hippocampus. Furthermore, the ventral KO hippocampus displays increased expression of the α1GABAA receptor subtype and a remarkably reduced rate of epileptiform discharges induced by magnesium-free medium. In contrast, the dorsal KO hippocampus displays an increased rate of epileptiform discharges and similar expression of α1GABAA receptors compared with the dorsal WT hippocampus. Blockade of α5GABAA receptors by L-655,708 did not affect epileptiform discharges in any genotype or hippocampal segment, and the expression of α5GABAA receptors did not differ between WT and KO hippocampus. These results suggest that the increased excitability of the dorsal KO hippocampus contributes to its heightened tendency to epileptiform discharges, while the increased phasic inhibition in the Fmr1-KO ventral hippocampus may represent a homeostatic mechanism that compensates for the increased excitability reducing its vulnerability to epileptic activity.

Increase in hnRNPA1 Expression Suffices to Kill Motor Neurons in Transgenic Rats.

A dominant mutation in hnRNPA1 causes amyotrophic lateral sclerosis (ALS), but it is not known whether this mutation leads to motor neuron death through increased or decreased function. To elucidate the relationship between pathogenic hnRNPA1 mutation and its native function, we created novel transgenic rats that overexpressed wildtype rat hnRNPA1 exclusively in motor neurons. This targeted expression of wildtype hnRNPA1 caused severe motor neuron loss and subsequent denervation muscle atrophy in transgenic rats that recapitulated the characteristics of ALS. These findings demonstrate that the augmentation of hnRNPA1 expression suffices to trigger motor neuron degeneration and the manifestation of ALS-like phenotypes. It is reasonable to infer that an amplification of an as-yet undetermined hnRNPA1 function plays a pivotal role in the pathogenesis of familial ALS caused by pathogenic hnRNPA1 mutation.

Assessment of pain-related behaviors in HIV-1 transgenic rats as a model of HIV-associated chronic pain.

Human immunodeficiency virus-1 (HIV)-associated chronic pain is a debilitating comorbid condition that affects 25-85% of people with HIV. The use of opioids to alleviate pain has given rise to opioid dependency in this cohort. Therefore, there is an urgent need to understand mechanisms and identify novel therapeutics for HIV-associated chronic pain. Several animal models have been developed to study HIV-related comorbidities. HIV-1 transgenic (Tg) rats have been shown to serve as a reliable model that mimic the deficits observed in people with HIV, such as neurological and immune system alterations. However, pain-related behavior in these animals has not been extensively evaluated. In this study, we measured evoked and spontaneous behavior in HIV-1Tg male and female rats. The results indicated that HIV-1Tg rats exhibit similar behavior to those with HIV-1-related neuropathy, specifically, cold sensitivity. Consequently, HIV-1Tg rats can serve as a model of neuropathy to study pain-related mechanisms and therapeutics targeted toward individuals living with HIV-1.

Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat anterior cruciate ligament reconstruction model

When ruptured, ligaments and tendons have limited self-repair capacity and rarely heal spontaneously. In the knee, the Anterior Cruciate Ligament (ACL) often ruptures during sports activities, causing functional impairment and requiring surgery using tendon grafts. Patients with insufficient time to recover before resuming sports risk re-injury. To develop more effective treatment, it is necessary to define mechanisms underlying ligament repair. For this, animal models can be useful, but mice are too small to create an ACL reconstruction model. Thus, we developed a transgenic rat model using control elements of Scleraxis (Scx), a transcription factor essential for ligament and tendon development, to drive GFP expression in order to localize Scx-expressing cells. As anticipated, Tg rats exhibited Scx-GFP in ACL during developmental but not adult stages. Interestingly, when we transplanted the flexor digitorum longus (FDP) tendon derived from adult Scx-GFP+ rats into WT adults, Scx-GFP was not expressed in transplanted tendons. However, tendons transplanted from adult WT rats into Scx-GFP rats showed upregulated Scx expression in tendon, suggesting that Scx-GFP+ cells are mobilized from tissues outside the tendon. Importantly, at 4 weeks post-surgery, Scx-GFP-expressing cells were more frequent within the grafted tendon when an ACL remnant was preserved (P group) relative to when it was not (R group) (P vs R groups (both n = 5), p<0.05), and by 6 weeks, biomechanical strength of the transplanted tendon was significantly increased if the remnant was preserved (P vsR groups (both n = 14), p<0.05). Scx-GFP+ cells increased in remnant tissue after surgery, suggesting remnant tissue is a source of Scx+ cells in grafted tendons. We conclude that the novel Scx-GFP Tg rat is useful to monitor emergence of Scx-positive cells, which likely contribute to increased graft strength after ACL reconstruction.