Transgenic Rat Model Research Articles

Abstract 65: Postpartum Alterations of Middle Cerebral Artery Reactivity and Hemodynamics in a Transgenic Rat Model of Preeclampsia

Evidence suggests that cerebrovascular disease is a leading cause of morbidity and mortality in women with preeclampsia (PE), however the mechanisms are unknown. In a transgenic rat displaying PE-like syndrome (TGA-PE: female transgenic for angiotensinogen mated to male transgenic for renin), we have shown postpartum alterations in cerebrovascular hemodynamics suggesting dysfunctional autoregulation. We hypothesize that impaired middle cerebral artery (MCA) reactivity contributes to the cerebrovascular dysfunction in this model. High frequency ultrasound was used to determine the pulsatility index (PI) of left MCA (LMCA). Vascular responses were studied in isolated segments of LMCA from SD (n= 12) and TGA-PE (n=8) two months post-delivery. Arteries were mounted on a Multi-Wire Myograph for determination of isometric force (USA-DMT). The contractile responses to K + 75 mM and endothelin-1 (ET-1, 10 -11 -10 -7 M) were measured in basal tone. Vasodilatory responses to acetylcholine (ACh, 10 -11 -10 -4 M) were determined in arteries pre-constricted with the thromboxane agonist U46619 (10 -7 M); the maximal response ( R MAX ) and sensitivity (pD 2 =-Log[EC 50 ]) were analyzed. Our data show that systolic blood pressures were elevated in TGA-PE versus SD at 2 months post-delivery (144.9±2.7 vs. 118.4±2.5, p<0.05; n=10-19). However, the PI was lower in TGA-PE vs. SD (0.96±0.05 vs. 1.31±0.06; p<0.05, n=8-13). No differences in basal tone of MCA segments were observed, whereas TGA-PE arteries displayed a lower optimal diameter (175±4 μm vs. 217±4 μm; p<0.05) and a lower response to K + (0.85±0.11 vs. 1.29±0.1 mN/mm; p<0.05) compared to SD. Vasodilatation to acetylcholine was similar (ACh MAX : 79±8 vs. 70±9%) with a lower sensitivity (pD 2 : 5.2±0.1 vs. 6.7±0.4; p<0.05) in TGA-PE. The contractile response to ET-1 was increased in TGA-PE arteries (ET-1 MAX : 159±7 vs. 129±6 % K MAX ; p<0.05) with no differences in sensitivity (pD 2 : 8.8±0.1 vs. 8.9±0.1). Our data demonstrate structural and functional differences in MCA from TGA-PE vs. SD at 2 months postpartum. In addition to a smaller functional diameter, a lower non-receptor dependent contraction may explain the lower resistance of MCA observed in the preeclamptic model. We conclude that attenuated vasodilatory and increased contractile responses of MCA could promote cerebrovascular dysfunction in this model, and that these adaptations may contribute to cerebrovascular pathology in postpartum preeclamptic state. Supported by NIH 1R01HL155420

Impaired Dynamics of Positional and Contextual Neural Coding in an Alzheimer's Disease Rat Model.

The hippocampal representation of space, formed by the collective activity of populations of place cells, is considered as a substrate of spatial memory. Alzheimer's disease (AD), a widespread severe neurodegenerative condition of multifactorial origin, typically exhibits spatial memory deficits among its early clinical signs before more severe cognitive impacts develop. To investigate mechanisms of spatial memory impairment in a double transgenic rat model of AD. In this study, we utilized 9-12-month-old double-transgenic TgF344-AD rats and age-matched controls to analyze the spatial coding properties of CA1 place cells. We characterized the spatial memory representation, assessed cells' spatial information content and direction-specific activity, and compared their population coding in familiar and novel conditions. Our findings revealed that TgF344-AD animals exhibited lower precision in coding, as evidenced by reduced spatial information and larger receptive zones. This impairment was evident in maps representing novel environments. While controls instantly encoded directional context during their initial exposure to a novel environment, transgenics struggled to incorporate this information into the newly developed hippocampal spatial representation. This resulted in impairment in orthogonalization of stored activity patterns, an important feature directly related to episodic memory encoding capacity. Overall, the results shed light on the nature of impairment at both the single-cell and population levels in the transgenic AD model. In addition to the observed spatial coding inaccuracy, the findings reveal a significantly impaired ability to adaptively modify and refine newly stored hippocampal memory patterns.

Differential effect of an evolving amyloid and tau pathology on brain phospholipids and bioactive lipid mediators in rat models of Alzheimer-like pathology

BackgroundBrain inflammation contributes significantly to the pathophysiology of Alzheimer’s disease, and it is manifested by glial cell activation, increased production of cytokines/chemokines, and a shift in lipid mediators from a pro-homeostatic to a pro-inflammatory profile. However, whether the production of bioactive lipid mediators is affected at earlier stages, prior to the deposition of Aβ plaques and tau hyperphosphorylation, is unknown. The differential contribution of an evolving amyloid and tau pathology on the composition and abundance of membrane phospholipids and bioactive lipid mediators also remains unresolved.MethodsIn this study, we examined the cortical levels of DHA- and AA-derived bioactive lipid mediators and of membrane phospholipids by liquid chromatography with tandem mass spectrometry in transgenic rat models of the Alzheimer’s-like amyloid and tau pathologies at early and advanced pathological stages.ResultsOur findings revealed a complex balance between pro-inflammatory and pro-resolving processes in which tau pathology has a more pronounced effect compared to amyloid pathology. At stages preceding tau misfolding and aggregation, there was an increase in pro-resolving lipid mediators (RVD6 and NPD1), DHA-containing phospholipids and IFN-γ levels. However, in advanced tau pathology displaying NFT-like inclusions, neuronal death, glial activation and cognitive deficits, there was an increase in cytokine and PGD2, PGE2, and PGF2α generation accompanied by a drop in IFN-γ levels. This pathology also resulted in a marked increase in AA-containing phospholipids. In comparison, pre-plaque amyloid pathology already presented high levels of cytokines and AA-containing phospholipids together with elevated RVD6 and NPD1 levels. Finally, Aβ plaque deposition was accompanied by a modest increase in prostaglandins, increased AA-containing phospholipids and reduced DHA-containing phospholipids.ConclusionsOur findings suggest a dynamic trajectory of inflammatory and lipid mediators in the evolving amyloid and tau pathologies and support their differing roles on membrane properties and, consequentially, on signal transduction.

Adolescent alcohol exposure alters age-related progression of behavioral and neurotrophic dysfunction in the TgF344-AD model in a sex-specific manner.

Alzheimer's Disease (AD) and heavy alcohol use are widely prevalent and lead to brain pathology. Both alcohol-related brain damage (ABRD) and AD result in cholinergic dysfunction, reductions in hippocampal neurogenesis, and the emergence of hippocampal-dependent cognitive impairments. It is still unknown how ARBD caused during a critical developmental timepoint, such as adolescence, interacts with AD-related pathologies to accelerate disease progression later in life. The current study utilized a longitudinal design to characterize behavioral and pathological changes in a transgenic rat model of AD (TgF344-AD) following adolescent intermittent ethanol (AIE) exposure. We found that AIE accelerates cognitive decline associated with AD transgenes in female rats at 6 months of age, and male AD-rats are impaired on spatial navigation by 3-months with no additional deficits due to AIE exposure. Protein levels of various AD-pathological markers were analyzed in the dorsal and ventral hippocampus of male and female rats. The data suggests that AIE-induced alterations of the tropomyosin-related kinase A receptor (TrkA) / p75 neurotrophin receptor (p75NTR) ratio creates a brain that is vulnerable to age- and AD-related pathologies, which leads to an acceleration of cognitive decline, particularly in female rats.

Early oxidative stress and DNA damage in Aβ-burdened hippocampal neurons in an Alzheimer’s-like transgenic rat model

Oxidative stress is a key contributor to AD pathology. However, the earliest role of pre-plaque neuronal oxidative stress, remains elusive. Using laser microdissected hippocampal neurons extracted from McGill-R-Thy1-APP transgenic rats we found that intraneuronal amyloid beta (iAβ)-burdened neurons had increased expression of genes related to oxidative stress and DNA damage responses including Ercc2, Fancc, Sod2, Gsr, and Idh1. DNA damage was further evidenced by increased neuronal levels of XPD (Ercc2) and γH2AX foci, indicative of DNA double stranded breaks (DSBs), and by increased expression of Ercc6, Rad51, and Fen1, and decreased Sirt6 in hippocampal homogenates. We also found increased expression of synaptic plasticity genes (Grin2b (NR2B), CamkIIα, Bdnf, c-fos, and Homer1A) and increased protein levels of TOP2β. Our findings indicate that early accumulation of iAβ, prior to Aβ plaques, is accompanied by incipient oxidative stress and DSBs that may arise directly from oxidative stress or from maladaptive synaptic plasticity.

Stable long-term germline transmission of GFP transgenic rat via PiggyBac transposon mediated gene transfer

Transgene silencing provides a significant challenge in animal model production via gene engineering using viral vectors or transposons. Selecting an appropriate strategy, contingent upon the species is crucial to circumvent transgene silencing, necessitating long-term observation of in vivo gene expression. This study employed the PiggyBac transposon to create a GFP rat model to address transgene silencing in rats. Surprisingly, transgene silencing occurred while using the CAG promoter, contrary to conventional understanding, whereas the Ef1α promoter prevented silencing. GFP expression remained stable through over five generations, confirming efficacy of the Ef1α promoter for long-term protein expression in rats. Additionally, GFP expression was consistently maintained at the cellular level in various cellular sources produced from the GFP rats, thereby validating the in vitro GFP expression of GFP rats. Whole-genome sequencing identified a stable integration site in Akap1 between exons 1 and 2, mitigating sequence-independent mechanism-mediated transgene silencing. This study established an efficient method for producing transgenic rat models using PiggyBac transposon. Our GFP rats represent the first model to exhibit prolonged expression of foreign genes over five generations, with implications for future research in gene-engineered rat models.

Acute nicotine vapor normalizes sensorimotor gating and reduces locomotor activity deficits in HIV-1 transgenic rats.

Despite improved life expectancy of people with HIV (PWH), HIV-associated neurocognitive impairment (NCI) persists, alongside deficits in sensorimotor gating and neuroinflammation. PWH exhibit high smoking rates, possibly due to neuroprotective, anti-inflammatory, and cognitive-enhancing effects of nicotine, suggesting potential self-medication. Here, we tested the effects of acute nicotine vapor exposure on translatable measures of sensorimotor gating and exploratory behavior in the HIV-1 transgenic (HIV-1Tg) rat model of HIV. Male and female HIV-1Tg and F344 control rats (n=57) were exposed to acute nicotine or vehicle vapor. Sensorimotor gating was assessed using prepulse inhibition (PPI) of the acoustic startle response, and exploratory behavior was evaluated using the behavioral pattern monitor (BPM). Vehicle-treated HIV-1Tg rats exhibited PPI deficits at low prepulse intensities compared to F344 controls, as seen previously. No PPI deficits were observed in nicotine-treated HIV1-Tg rats, however. HIV-1Tg rats were hypoactive in the BPM relative to controls, whilst nicotine vapor increased activity and exploratory behavior across genotypes. Cotinine analyses confirmed comparable levels of the primary metabolite of nicotine across genotypes. Previous findings of PPI deficits in HIV-1Tg rats were replicated and, importantly, attenuated by acute nicotine vapor. Evidence for similar cotinine levels suggest a nicotine-specific effect in HIV-1Tg rats. HIV-1Tg rats had reduced exploratory behavior compared to controls, attenuated by acute nicotine vapor. Therefore, acute nicotine may be beneficial for remediating sensorimotor and locomotor activity deficits in PWH. Future studies should determine the long-term effects of nicotine vapor on similar HIV/NCI-relevant behaviors.

Effects of Intravenously Administered Plasma from Exercise-Trained Donors on Microglia and Cytokines in a Transgenic Rat Model of Alzheimer's Disease.

Microglia and inflammation play a significant role in Alzheimer's disease (AD). Physical exercise and peripheral signals can influence microglial activity in the brain. Modulating the inflammatory response in the brain may provide therapeutic approaches for AD. To assess the effects of intravenously administered blood plasma from exercise-trained donor rats on cognitive function, microglia, and cytokine levels in an AD rat model at two different pathological stages; an early pre-plaque stage and a later stage closer to the emergence of extracellular plaques. Male transgenic McGill-R-Thy1-APP rats aged 2 and 5 months received 14 injections over 6 weeks: 1) plasma from exercise-trained rats (ExPlas), 2) plasma from sedentary rats (SedPlas), or 3) saline. Cognitive function was evaluated in a novel object recognition task. Microglia count and morphology were analyzed in cornu ammonis, dentate gyrus, entorhinal cortex, and subiculum. Amyloid plaque number and size were assessed in the rats with the later treatment start. A multiplex assay was used to measure 23 cytokines in cornu ammonis. In rats treated from 2 months of age, ExPlas and SedPlas increased number and length of microglial branches in cornu ammonis and dentate gyrus compared to saline. Only ExPlas-treated rats exhibited similar changes in subiculum, while entorhinal cortex showed no differences across treatments. Microglia count remained unaffected. In rats treated from 5 months of age, there were no significant differences in microglia count or morphology or the number or size of amyloid plaques in any brain region. Compared to both other treatments in early pre-plaque stage rats, SedPlas increased TNF-α levels. ExPlas upregulated GM-CSF, IL-18, and VEGF, while SedPlas increased IL-10 compared to saline. In later-stage rats, ExPlas upregulated IL-17, and SedPlas upregulated TNF-α compared to saline. There were no effects of treatments on recognition memory. Intravenous injections of blood plasma from exercise-trained and sedentary donors differentially modulated microglial morphology and cytokine levels in the AD rat model at an early pre-plaque stage of pathology. Exercised plasma may reduce proinflammatory TNF-α signaling and promote microglial responses to early Aβ accumulation but the lack of treatment effects in the later-stage rats emphasizes the potential importance of treatment timing.

Endothelial DRP1 mediates microvascular dysfunction in the human and rat microcirculation independent of ROS production

Introduction: Microvascular endothelial dysfunction is a strong, independent, and modifiable risk factor for the development of coronary artery disease (CAD). Under physiological conditions, an increase in blood flow stimulates endothelial cells (EC) to release nitric oxide (NO) which mediates microvascular dilation. Under pathological conditions, such as CAD, NO-mediated dilation is lost but dilator capacity is preserved by mitochondria-derived hydrogen peroxide (H2O2), a reactive oxygen species (ROS). Recent work in rodent models suggests that ATP is required for NO-mediated and increasing evidence suggests that the balance between mitochondrial ATP vs. ROS production contributes to this phenotypic switch. Mitochondrial fission, an autoregulatory process primarily mediated by DRP1, is associated with various cardiometabolic diseases and is a direct contributor to increased reactive oxygen species (ROS) formation and decreased production of ATP. However, the role of DRP1 in the endothelium and it impacts on vascular function is unknown. Hypothesis: We hypothesize that mitochondrial fission, mediated by DRP1, is a critical regulator of microvascular redox balance. Methods: Microvessels (~50-200μm) were obtained from discarded human adipose surgical tissue from individuals with and without CAD or from rat mesentery and subjected to pressure myography (flow-mediated dilation; FMD). A fluorescent mitochondrial-specific ATP probe was measured in vessels after 10 minutes at maximal shear stress. DRP1 overexpression was achieved by adeno-associated viral (AAV) gene transfer and downregulated via DRP1 targeted siRNA (si-DRP1). To show physiologic and translational relevance, a transgenic rat model with a cre-inducible endothelial-specific overexpression of DRP1 (DRP Tg) was created to test the in vivo effects of EC-DRP1 overexpression. To test whether reduction of ROS improved the observed phenotype, vessels were acutely treated ex vivo in the organ chamber with TEMPOL, a cytosolic antioxidant, and mito-tempo, a mitochondrial antioxidant. Two-way ANOVA, post-hoc analysis was performed using PRISM to best understand the effects of the treatment and pressure-gradient. T-test was performed for comparison analyses between patients with/without CAD. Results:Fluorescent analysis of showed that ATP is increased in vessels obtained from patients without CAD compared to those with CAD (6.3 vs 1.1, mean fluorescent intensity, N=3, p<0.05). In vessels obtained from patients without CAD, AAV-mediated DRP1 overexpression switches the mediator of FMD from NO to H2O2 (inhibitable by PEG-Catalase, max dilation 83.2% vs. 20.2%, N=6, p<0.05). Conversely, arterioles obtained from patients with CAD treated with si-DRP1 restores NO-mediated dilation (inhibitable by L-NAME, max dilation 94.6% vs. 41.7%, N=5, p<0.05). In the rat microcirculation, FMD is significantly impaired in EC-DRP1 rats compared to littermate controls (max dilation 41.2% vs. 80.3%, N=9, p<0.05). Scavenging of ROS (TEMPOL and mito-Tempo) does not restore the phenotype in both human and rat microcirculation suggesting DRP1 mediates dysfunction independent of ROS, which is in line with preliminary observations of reduced flow-induced ATP levels in vessels from patients with CAD. Significance: These data indicate that ROS may be secondary to mitochondrial fission rather than causal to the microvascular phenotype observed in patients with CAD. Therefore, targeting endothelial mitochondrial fission may be of therapeutic benefit to treat cardiovascular disease. AHA Diversity Research Pre-Doctoral Award (CGH), 5P30ES030283-04 (AJL), 5R01HL133029-5 (AB), 5R21OD018306-2 (AB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genetic modification of cardiac fibroblasts in adult rats using adeno-associated virus serotype 9

Cardiac fibrosis is a process involving pathological extracellular matrix remodeling as well as cardiac fibroblast (CF) activation in response to cardiac injury and stress, which is frequently associated with cardiac dysfunction. Therefore, understanding the molecular mechanisms underlying CF activation is critical to develop successful therapies for treating cardiac fibrosis. CF-specific gene modifications in mouse models have been reported as key tools for cardiac fibrosis research. Although rat cardiac disease models also offer many of the same advantages as the mouse models, there are no rat models that employ CF-specific conditional gene modification. Here, we attempted CF-specific gene introduction in rats using adeno-associated virus serotype 9 (AAV9), allowing us to develop fast and simple rat cardiac disease models rather than establishing transgenic rat lines. AAV9 carrying a CF-specific human TCF21 (hTCF21) promoter and a transgene of interest was generated and introduced into adult rats via tail vein injections. Since we previously reported in cultured CFs that protein kinase D (PKD) activation at the outer mitochondrial membrane (OMM) induces mitochondrial fragmentation via phosphorylation of a mitochondrial fission protein DRP1, we generated an OMM-targeted dominant-negative mutant of PKD (termed mt-PKD-DN) to specifically inhibit PKD activity at the OMM in CFs in vivo. In vivo cardiac function, gene expression, protein expression, and mitochondrial morphology were assessed 2-10 weeks after AAV9 injection by echocardiography, quantitative RT-PCR, immunohistochemistry, and transmission electron microscopy (TEM), respectively. AAV9-hTCF21-injected rats maintained normal cardiac dimensions and contractile function compared to aged-matched control rats. Using AAV9-hTCF21-EGFP, we confirmed organ-specific (i.e., heart) and cell type-specific (i.e., CF-specific) expression of GFP. Next, we injected AAV9-hTCF21-mt-PKD-DN or -luciferase (Luc) as a control in adult rats. TEM images from fixed rat ventricular tissues revealed a significant increase in mitochondrial aspect ratio and mitochondrial perimeter in CFs with AAV9-hTCF21-mt-PKD-DN compared to AAV9-hTCF21-Luc, indicating that PKD inhibition at the OMM induces mitochondrial elongation in CFs in vivo. In contrast, no significant change in mitochondrial morphology was observed in the cardiomyocyte area. In summary, we successfully achieved a CF-specific gene delivery to the adult rat hearts in vivo, demonstrating the potential of a simplified CF-specific rat transgenic model using the AAV9-hTCF21 system. NIH/NHLBI R01HL160699. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in lipid metabolism track with the progression of neurofibrillary pathology in tauopathies

BackgroundAccumulation of tau leads to neuroinflammation and neuronal cell death in tauopathies, including Alzheimer’s disease. As the disease progresses, there is a decline in brain energy metabolism. However, the role of tau protein in regulating lipid metabolism remains less characterized and poorly understood.MethodsWe used a transgenic rat model for tauopathy to reveal metabolic alterations induced by neurofibrillary pathology. Transgenic rats express a tau fragment truncated at the N- and C-terminals. For phenotypic profiling, we performed targeted metabolomic and lipidomic analysis of brain tissue, CSF, and plasma, based on the LC-MS platform. To monitor disease progression, we employed samples from transgenic and control rats aged 4, 6, 8, 10, 12, and 14 months. To study neuron-glia interplay in lipidome changes induced by pathological tau we used well well-established multicomponent cell model system. Univariate and multivariate statistical approaches were used for data evaluation.ResultsWe showed that tau has an important role in the deregulation of lipid metabolism. In the lipidomic study, pathological tau was associated with higher production of lipids participating in protein fibrillization, membrane reorganization, and inflammation. Interestingly, significant changes have been found in the early stages of tauopathy before the formation of high-molecular-weight tau aggregates and neurofibrillary pathology. Increased secretion of pathological tau protein in vivo and in vitro induced upregulated production of phospholipids and sphingolipids and accumulation of lipid droplets in microglia. We also found that this process depended on the amount of extracellular tau. During the later stages of tauopathy, we found a connection between the transition of tau into an insoluble fraction and changes in brain metabolism.ConclusionOur results revealed that lipid metabolism is significantly affected during different stages of tau pathology. Thus, our results demonstrate that the dysregulation of lipid composition by pathological tau disrupts the microenvironment, further contributing to the propagation of pathology.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology invivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs invivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases invivo.

Human-induced pluripotent stem cell-derived neural stem/progenitor cell ex vivo gene therapy with synaptic organizer CPTX for spinal cord injury

The transplantation of neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs) has shown promise in spinal cord injury (SCI) model animals. Establishing a functional synaptic connection between the transplanted and host neurons is crucial for motor function recovery. To boost therapeutic outcomes, we developed an exvivo gene therapy aimed at promoting synapse formation by expressing the synthetic excitatory synapse organizer CPTX in hiPSC-NS/PCs. Using an immunocompromised transgenic rat model of SCI, we evaluated the effects of transplanting CPTX-expressing hiPSC-NS/PCs using histological and functional analyses. Our findings revealed a significant increase in excitatory synapse formation at the transplantation site. Retrograde monosynaptic tracing indicated extensive integration of transplanted neurons into the surrounding neuronal tracts facilitated by CPTX. Consequently, locomotion and spinal cord conduction significantly improved. Thus, exvivo gene therapy targeting synapse formation holds promise for future clinical applications and offers potential benefits to individuals with SCI.

Potential Alzheimer’s early biomarkers in a transgenic rat model and benefits of diazoxide/dibenzoylmethane co-treatment on spatial memory and AD-pathology

Alzheimer’s disease (AD) is the major form of dementia prevalent in older adults and with a high incidence in females. Identification of early biomarkers is essential for preventive intervention to delay its progression. Furthermore, due to its multifactorial nature, a multi-target approach could be therapeutically beneficial. Our studies included 4- (pre-pathology) and 11-month (mild-pathology) TgF344-AD rats, a transgenic Alzheimer’s model that exhibits age-dependent AD progression. We identified two potential early biomarker genes for AD, early growth response 2 (EGR2) and histone 1H2AA (HIST1H2AA), in the hippocampus of 4-month females. Out of 17,168 genes analyzed by RNA sequencing, expression of these two genes was significantly altered in 4-month TgF344-AD rats compared to wild-type littermates. We also evaluated co-treatment with diazoxide (DZ), a potassium channel activator, and dibenzoylmethane (DIB), which inhibits eIF2α-P activity, on TgF344-AD and wild-type rats. DZ/DIB-treatment mitigated spatial memory deficits and buildup of hippocampal Aβ plaques and tau PHF in 11-month TgF344-AD rats but had no effect on wild-type littermates. To our knowledge, this preclinical study is the first to report EGR2 and HIST1H2AA as potential AD biomarkers in females, and the benefits of DZ/DIB-treatment in AD. Evaluations across multiple AD-related models is warranted to corroborate our findings.

Early ultrasonic vocalization deficits and related thyroarytenoid muscle pathology in the transgenic TgF344-AD rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurologic disease and the most common cause of dementia. Classic pathology in AD is characterized by inflammation, abnormal presence of tau protein, and aggregation of β-amyloid that disrupt normal neuronal function and lead to cell death. Deficits in communication also occur during disease progression and significantly reduce health, well-being, and quality of life. Because clinical diagnosis occurs in the mid-stage of the disease, characterizing the prodrome and early stages in humans is currently challenging. To overcome these challenges, we use the validated TgF344-AD (F344-Tg(Prp-APP, Prp-PS1)19/Rrrc) transgenic rat model that manifests cognitive, behavioral, and neuropathological dysfunction akin to AD in humans. The overarching goal of our work is to test the central hypothesis that pathology and related behavioral deficits such as communication dysfunction in part manifest in the peripheral nervous system and corresponding target tissues already in the early stages. The primary aims of this study are to test the hypotheses that: (1) changes in ultrasonic vocalizations (USV) occur in the prodromal stage at 6 months of age and worsen at 9 months of age, (2) inflammation as well as AD-related pathology can be found in the thyroarytenoid muscle (TA) at 12 months of age (experimental endpoint tissue harvest), and to (3) demonstrate that the TgF344-AD rat model is an appropriate model for preclinical investigations of early AD-related vocal deficits. USVs were collected from male TgF344-AD (N = 19) and wildtype (WT) Fischer-344 rats (N = 19) at 6 months (N = 38; WT: n = 19; TgF344-AD: n = 19) and 9 months of age (N = 18; WT: n = 10; TgF344-AD: n = 8) and acoustically analyzed for duration, mean power, principal frequency, low frequency, high frequency, peak frequency, and call type. RT-qPCR was used to assay peripheral inflammation and AD-related pathology via gene expressions in the TA muscle of male TgF344-AD rats (n = 6) and WT rats (n = 6) at 12 months of age. This study revealed a significant reduction in mean power of ultrasonic calls from 6 to 9 months of age and increased peak frequency levels over time in TgF344-AD rats compared to WT controls. Additionally, significant downregulation of AD-related genes Uqcrc2, Bace2, Serpina3n, and Igf2, as well as downregulation of pro-inflammatory gene Myd88 was found in the TA muscle of TgF344-AD rats at 12 months of age. Our findings demonstrate early and progressive vocal deficits in the TgF344-AD rat model. We further provide evidence of dysregulation of AD-pathology-related genes as well as inflammatory genes in the TA muscles of TgF344-AD rats in the early stage of the disease, confirming this rat model for early-stage investigations of voice deficits and related pathology.

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.

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.

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.

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.

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.