Knockout Rats Research Articles

Trim46 knockout impaired neuronal architecture and caused hypoactive behavior in rats.

Tripartite motif (TRIM46) is a relatively novel protein that belongs to tripartite motif family. TRIM46 organizes parallel microtubule arrays on the axons, which are important for neuronal polarity and axonal function. TRIM46 is highly expressed in the brain, but its biological function in adults has not yet been determined. Trim46 knockout (KO) rat line was established using CRISPR/cas9. Trim46 KO rats had smaller hippocampus sizes, fewer neuronal dendritic arbors and dendritic spines, and shorter and more distant axon initial segment. Furthermore, the protein interaction between endogenous TRIM46 and FK506 binding protein 5 (FKBP5) in brain tissues was determined; Trim46 KO increased hippocampal FKBP5 protein levels and decreased hippocampal protein kinase B (Akt) phosphorylation, gamma-aminobutyric acid type A receptor subunit alpha1 (GABRA1) and glutamate ionotropic receptor NMDA type subunit 1 (NMDAR1) protein levels. Trim46 KO rats exhibited hypoactive behavioral changes such as reduced spontaneous activity, social interaction, sucrose preference, impaired prepulse inhibition (PPI), and short-term reference memory. These results demonstrate the significant impact of Trim46 KO on brain structure and behavioral function. This study revealed a novel potential association of TRIM46 with dendritic development and neuropsychiatric behavior, providing new insights into the role of TRIM46 in the brain.

Vof16-miR-185-5p-GAP43 network improves the outcomes following spinal cord injury via enhancing self-repair and promoting axonal growth.

Self-repair of spinal cord injury (SCI) has been found in humans and experimental animals with partial recovery of neurological functions. However, the regulatory mechanisms underlying the spontaneous locomotion recovery after SCI are elusive. This study was aimed at evaluating the pathological changes in injured spinal cord and exploring the possible mechanism related to the spontaneous recovery. Immunofluorescence staining was performed to detect GAP43 expression in lesion site after spinal cord transection (SCT) in rats. Then RNA sequencing and gene ontology (GO) analysis were employed to predict lncRNA that correlates with GAP43. LncRNA smart-silencing was applied to verify the function of lncRNA vof16 invitro, and knockout rats were used to evaluate its role in neurobehavioral functions after SCT. MicroRNA sequencing, target scan, and RNA22 prediction were performed to further explore the underlying regulatory mechanisms, and miR-185-5p stands out. A miR-185-5p site-regulated relationship with GAP43 and vof16 was determined by luciferase activity analysis. GAP43-silencing, miR-185-5p-mimic/inhibitor, and miR-185-5p knockout rats were also applied to elucidate their effects on spinal cord neurite growth and neurobehavioral function after SCT. We found that a time-dependent increase of GAP43 corresponded with the limited neurological recovery in rats with SCT. CRNA chip and GO analysis revealed lncRNA vof16 was the most functional in targeting GAP43 in SCT rats. Additionally, silencing vof16 suppressed neurite growth and attenuated the motor dysfunction in SCT rats. Luciferase reporter assay showed that miR-185-5p competitively bound the same regulatory region of vof16 and GAP43. Our data indicated miR-185-5p could be a detrimental factor in SCT, and vof16 may function as a ceRNA by competitively binding miR-185-5p to modulate GAP43 in the process of self-recovery after SCT. Our study revealed a novel vof16-miR-185-5p-GAP43 regulatory network in neurological self-repair after SCT and may underlie the potential treatment target for SCI.

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.

Myelin lesion in the aspartoacylase (Aspa) knockout rat, an animal model for Canavan disease.

Canavan disease (CD) is a fatal hereditary neurological disorder caused by a mutation in the aspartoacylase (ASPA) gene and characterized by neurological signs and vacuolation in the central nervous system (CNS). The mutation inhibits the hydrolysis of N-acetyl-aspartate (NAA) resulting in accumulation of NAA in the CNS. A new Aspa-knockout rat was generated by transcription activator-like effector nuclease (TALEN) technology. Herein we describe the pathological and morphometrical findings in the brain and spinal cords of Aspa-knockout rats. Although Aspa-knockout rats did not show any neurological signs, vacuolation with swollen axons, hypomyelination, and activated swollen astrocytes were observed mainly in the brainstem reticular formation, ascending and descending motor neuron pathway, and in the olfactory tract. Morphometrical analysis revealed no obvious change in the number of neurons. These changes in the CNS are similar to human CD, suggesting that this animal model would be useful for further study of treatment and understanding the pathophysiology of human CD.

AT1R autoantibody promotes phenotypic transition of smooth muscle cells by activating AT1R-OAS2

Phenotypic transition of vascular smooth muscle cells (VSMCs) is an early event in the onset and progression of several cardiovascular diseases. As an important mediator of the renin-angiotensin system (RAS), activation of the angiotensin II type 1 receptor (AT1R) induces phenotypic transition of VSMCs. AT1R autoantibodies (AT1-AAs), which are agonistic autoantibodies of AT1R, have been detected in the sera of patients with a variety of cardiovascular diseases associated with phenotypic transition. However, the effect of AT1-AA on phenotypic transition is currently unknown. In this study, AT1-AA-positive rat model was established by active immunization to detect markers of VSMCs phenotypic transition. The results showed that AT1-AA-positive rats showed phenotypic transition of VSMCs, which was evidenced by the decrease of contractile markers, while the increase of synthetic markers in the thoracic aorta. However, in AT1-AA-positive AT1R knockout rats, the phenotypic transition-related proteins were not altered. In vitro, after stimulating human aortic smooth muscle cells with AT1-AA for 48 h, 2′-5′ oligoadenylate synthase 2 (OAS2) was identified as the key differentially expressed gene by RNA sequencing and bioinformatics analysis. Furthermore, high expression of OAS2 was found in aorta of AT1-AA-positive rats; knockdown of OAS2 by siRNA can reverse the phenotypic transition of VSMCs induced by AT1-AA. In summary, this study suggests that AT1-AA can promote phenotypic transition of VSMCs through AT1R-OAS2 pathway, and OAS2 might serve as a potential therapeutic target to prevent pathological phenotypic transition of smooth muscle cells.

Comparison of the effects of BDNF/TRKB signalling on metabolic biomarkers in the liver of sedentary and trained rats with normal and knockout BDNF genotypes.

Introduction: The effect of brain-derived neurotrophic factor (BDNF) on the modulation of metabolic processes in the liver is poorly understood. Therefore, the aim of this study was to investigate whether hepatic concentrations or activities of metabolic biomarkers depend on altered BDNF/TrkB content in the liver, resulting from different BDNF genotypes of rats. In addition, it was assessed whether 5-week moderate endurance training modifies the levels of BDNF/Trk-B signaling and studied hepatic markers. Methods: Experiments were performed on wild-type and heterozygous BDNF knockout (HET, SD-Bdnf) rats, which were divided into four groups: control with normal genotype (Bdnf+/+), control with BDNF knockout genotype (Bdnf+/-), trained with normal genotype (Bdnf+/+T) and trained with BDNF knockout genotype (Bdnf +/-T). BDNF/TrkB concentrations as well as selected metabolic biomarkers including lipids-total cholesterol (CHOL), low-density lipoprotein (LDL), triglycerides (TG); enzymes-alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP); hormones-insulin (INS) and leptin (LEPT) as well as interleukin-6 (IL-6) as regeneration indicator were measured directly in liver homogenates. Results and Discussion: The study showed that Bdnf+/- rats exhibited reduced BDNF/TrkB signaling (BDNF, p < 0.0001; Trk-B, p = 0.0005), altered lipid levels (CHOL, p < 0.0001; LDL, p < 0.0001; TG, p = 0.0006) and reduced hepatic ALAT (p = 0.0004) and GGT (p < 0.0001) activity, which may contribute to hepatic steatosis and obesity, as well as indicate impairment of specific metabolic pathways in the liver. Interestingly, endurance training did not alter hepatic BDNF and TrkB content, but improved ALAT (p = 0.0366) and ASAT (p = 0.0191) activities and increased hepatic IL-6 (p = 0.0422) levels in Bdnf +/- rats, suggesting enhanced liver regeneration in animals with BDNF allele loss.

Rescue of sharp wave-ripples and prevention of network hyperexcitability in the ventral but not the dorsal hippocampus of a rat model of fragile X syndrome.

Fragile X syndrome (FXS) is a genetic neurodevelopmental disorder characterized by intellectual disability and is related to autism. FXS is caused by mutations of the fragile X messenger ribonucleoprotein 1 gene (Fmr1) and is associated with alterations in neuronal network excitability in several brain areas including hippocampus. The loss of fragile X protein affects brain oscillations, however, the effects of FXS on hippocampal sharp wave-ripples (SWRs), an endogenous hippocampal pattern contributing to memory consolidation have not been sufficiently clarified. In addition, it is still not known whether dorsal and ventral hippocampus are similarly affected by FXS. We used a Fmr1 knock-out (KO) rat model of FXS and electrophysiological recordings from the CA1 area of adult rat hippocampal slices to assess spontaneous and evoked neural activity. We find that SWRs and associated multiunit activity are affected in the dorsal but not the ventral KO hippocampus, while complex spike bursts remain normal in both segments of the KO hippocampus. Local network excitability increases in the dorsal KO hippocampus. Furthermore, specifically in the ventral hippocampus of KO rats we found an increased effectiveness of inhibition in suppressing excitation and an upregulation of α1GABAA receptor subtype. These changes in the ventral KO hippocampus are accompanied by a striking reduction in its susceptibility to induced epileptiform activity. We propose that the neuronal network specifically in the ventral segment of the hippocampus is reorganized in adult Fmr1-KO rats by means of balanced changes between excitability and inhibition to ensure normal generation of SWRs and preventing at the same time derailment of the neural activity toward hyperexcitability.

ALKBH5-Mediated RNA m6 A Methylation Regulates the Migration, Invasion, and Proliferation of Rheumatoid Fibroblast-Like Synoviocytes.

Fibroblast-like synoviocytes (FLSs) are critical for promoting joint damage in rheumatoid arthritis (RA). N6 -methyladenosine (m6 A) modification plays key roles in various diseases, but its role in the pathogenesis of RA is largely unknown. Here, we investigate increased demethylase ALKBH5 promotion of proliferation, migration, and invasion of RA FLSs via regulating JARID2 expression. ALKBH5 expression in FLSs was evaluated using real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot. 5-ethynyl-2'-deoxyuridine, scratch wound healing, and transwell assays were implemented to determine the role of ALKBH5 on RA FLS proliferation, mobility, and migration. Then, m6 A sequencing combined with RNA sequencing was performed to identify the potential targets of ALKBH5. RNA immunoprecipitation and RNA pulldown were then used to validate the interaction between the protein and messenger RNA (mRNA). Collagen-induced arthritis (CIA) and delayed-type hypersensitivity arthritis (DTHA) models were further established to assess the therapeutic potency of ALKBH5 in vivo. We demonstrated that ALKBH5 expression was increased in FLSs and synovium from RA. Functionally, ALKBH5 knockdown inhibited the proliferation, migration, and invasion of RA FLSs, whereas overexpression of ALKBH5 displayed the opposite effect. Mechanistically, ALKBH5 mediated m6 A modification in the JARID2 mRNA and enhanced its mRNA stability in cooperation with IGF2BP3. Intriguingly, the severity of arthritis was attenuated in mice with DTHA and ALKBH5 knockout or rats with CIA and intra-articular injection of ALKBH5 short hairpin RNA. Our findings suggest that ALKBH5-mediated m6 A modification is crucial for synovial hyperplasia and invasion in RA. ALKBH5 might be a potential therapeutic target for RA and even for dysregulated fibroblasts in a wide range of diseases.

Discovery of Guanfacine as a Novel TAAR1 Agonist: A Combination Strategy through Molecular Modeling Studies and Biological Assays.

Trace amine-associated receptor 1 (TAAR1) is an attractive target for the design of innovative drugs to be applied in diverse pharmacological settings. Due to a non-negligible structural similarity with endogenous ligands, most of the agonists developed so far resulted in being affected by a low selectivity for TAAR1 with respect to other monoaminergic G protein-coupled receptors, like the adrenoreceptors. This study utilized comparative molecular docking studies and quantitative-structure activity relationship (QSAR) analyses to unveil key structural differences between TAAR1 and alpha2-adrenoreceptor (α2-ADR), with the aim to design novel TAAR1 agonists characterized by a higher selectivity profile and reduced off-target effects. While the presence of hydrophobic motives is encouraged towards both the two receptors, the introduction of polar/positively charged groups and the ligand conformation deeply affect the TAAR1 or α2-ADR putative selectivity. These computational methods allowed the identification of the α2A-ADR agonist guanfacine as an attractive TAAR1-targeting lead compound, demonstrating nanomolar activity in vitro. In vivo exploration of the efficacy of guanfacine showed that it is able to decrease the locomotor activity of dopamine transporter knockout (DAT-KO) rats. Therefore, guanfacine can be considered as an interesting template molecule worthy of structural optimization. The dual activity of guanfacine on both α2-ADR and TAAR1 signaling and the related crosstalk between the two pathways will deserve more in-depth investigation.

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.

Sleeve Gastrectomy Provides Cardioprotection from Oxidative Stress In Vitro Due to Reduction of Circulating Myeloperoxidase.

Bariatric surgery, including sleeve gastrectomy (SG), improves systolic and diastolic function, which is independent of weight loss in rodent models. The cause of weight loss-independent improvements in cardiac function are unknown but may originate from the gastrointestinal tract. In this study, we investigated whether a circulating blood factor is a mechanism for acute cardioprotection after SG by testing the utility of rodent SG plasma to reduce metabolic stress in vitro. For the initial experiment, obese male Zucker rats underwent SG, ad lib sham, or pair-fed sham surgeries (n = six SG, n = eight SH, n = eight PF). For all other studies, a second group of Zucker rats underwent SG or ad lib sham surgeries (n = eight SH, n = six SG). Six weeks following surgery, plasma was collected from each group, both in the fasting and post-prandial (pp) state. This plasma was then pooled per surgical group and nutrient state and tested in multiple in vitro cell culture and extra-cellular assays to determine the effect of SG on myotubular metabolic stress compared to the sham surgeries. Post-prandial SG plasma (ppSG), but not fasting SG, pp, or fasting sham plasma, reduced the metabolic stress of the H9c2 cells as measured by lactate dehydrogenase (LDH) release (p < 0.01). Unlike SG, weight reduction through pair-feeding did not prevent H9c2 metabolic stress. The PpSG plasma had the slowest rate of extracellular hydrogen peroxide consumption and peroxidatic activity compared to the pp sham, fasting SG, and fasting sham groups. Redox testing of plasma with aminiobenzoic acid hydrazide and edaravone suggested a pattern supporting myeloperoxidase (MPO), or other peroxidases, as the primary component responsible for reduced metabolic stress with ppSG plasma. The PpSG plasma contained 35% less circulating MPO protein as compared to the pp sham and fasting SG plasma. The plasma from an MPO global knockout rat also prevented metabolic stress of the H9c2 cells, compared to the significant increase in LDH release from the plasma of the WT controls (p < 0.01). The MPO global knockout plasma also had a rate of extracellular hydrogen peroxide consumption and peroxidatic activity comparable to the ppSG plasma. These studies suggest that one of the weight loss-independent mechanisms by which SG improves myocellular function could be a reduced pro-oxidative environment due to lower circulating levels of MPO. It appears that the gastrointestinal tract is of critical importance to these findings, as the MPO levels were only lowered after enteral, nutrient stimulation in the SG rats. If this surgical effect is confirmed in humans, SG may be a unique surgical treatment for multiple diseases with a pathogenesis of inflammation and oxidative damage, including obesity-associated heart failure with preserved ejection fraction.

Abstract 14597: Beyond Sex Bias: The Uncharted Role of Rage Signaling in Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a life-threatening disease that initiates in response to pulmonary artery endothelial cells (PAEC) damage followed by inadequate repair. The Receptor for Advanced Glycation Endproducts (RAGE) triggered upon the damage, plays a crucial role in vascular regeneration. However, its specific impact on PAH remains incompletely explored. Hypothesis: In this study, we investigated the sex-specific outcome of RAGE deletion on PAEC homeostasis. Methods: The effect of the global RAGE knockout (KO) on pulmonary hemodynamics, cardiac and endothelial function was studied disaggregated by sex in WT and RAGE KO rats. Endothelial integrity was evaluated via the expression of endothelial junction proteins, the activity of barrier disruptive signaling pathways, and in vivo pulmonary vascular permeability assay. To understand the significance of diminished RAGE signaling in male PAH, we measured membrane RAGE levels in untreated and Sugen/Hypoxia treated rats of both sexes and examined the publicly available single-cell transcriptomic profiles of human PAECs. Results: Male but not female RAGE KO rats developed a spontaneous PAH characterized by a significant increase in the right ventricle (RV) systolic pressure and RV hypertrophy. The PAH phenotype in males corresponded with a male-specific decrease in the expression of CD31 (WT vs. RAGE KO: 1.0 ± 0.07 vs. 0.1 ± 0.02, p &lt;0001, N=6), activation of p38/RhoA signaling axis (p-p38 in WT vs. RAGE KO: 1.0 ± 0.2 vs. 3.2 ± 0.6, p=0.01, N=4), significant PAEC cytoskeletal rearrangement, and increased FITC-dextran extravasation, while female RAGE KOs remained unaffected. We also discovered that Sugen/Hypoxia treatment induced a substantial loss of membrane RAGE, specifically in males. Additionally, human PAEC transcriptomic data revealed that RAGE is expressed primarily in young females but not in males or older females. Conclusions: We conclude that the male sex associates with a reduced RAGE expression and signaling in PAEC, which is further diminished in PAH. The disruption in RAGE signaling, in turn, impairs the pulmonary endothelial barrier, instigates spontaneous PAH, and may contribute to the more severe phenotype observed in male PAH patients.

Assessing the Cntnap2 knockout rat prepulse inhibition deficit through prepulse scaling of the baseline startle response curve

Many neurodevelopmental disorders, including autism spectrum disorder (ASD), are associated with changes in sensory processing and sensorimotor gating. The acoustic startle response and prepulse inhibition (PPI) of startle are widely used translational measures for assessing sensory processing and sensorimotor gating, respectively. The Cntnap2 knockout (KO) rat has proven to be a valid model for ASD, displaying core symptoms, including sensory processing perturbations. Here, we used a novel method to assess startle and PPI in Cntnap2 KO rats that allows for the identification of separate scaling components: startle scaling, which is a change in startle amplitude to a given sound, and sound scaling, which reflects a change in sound processing. Cntnap2 KO rats show increased startle due to both an increased overall response (startle scaling) and a left shift of the sound/response curve (sound scaling). In the presence of a prepulse, wildtype rats show a reduction of startle due to both startle scaling and sound scaling, whereas Cntnap2 KO rats show normal startle scaling, but disrupted sound scaling, resulting in the reported PPI deficit. These results validate that startle and sound scaling by a prepulse are indeed two independent processes, with only the latter being impaired in Cntnap2 KO rats. As startle scaling is likely related to motor output and sound scaling to sound processing, this novel approach reveals additional information on the possible cause of PPI disruptions in preclinical models.

Treatment with the dual-incretin agonist DA-CH5 demonstrates potent therapeutic effect in a rat model of Wolfram Syndrome.

Wolfram Syndrome (WS) is a rare condition caused by mutations in Wfs1, with a poor prognosis and no cure. Mono-agonists targeting the incretin glucagon-like-peptide 1 (GLP-1) have demonstrated disease-modifying potential in pre-clinical and clinical settings. Dual agonists that target GLP-1 and glucose-dependent insulinotropic polypeptide (GIP-1) are reportedly more efficacious; hence, we evaluated the therapeutic potential of dual incretin agonism in a loss-of-function rat model of WS. Eight-month-old Wfs1 knock-out (KO) and wild-type control rats were continuously treated with either the dual agonist DA-CH5 or saline for four months. Glycemic profile, visual acuity and hearing sensitivity were longitudinally monitored pre-treatment, and then at 10.5 and 12 months. Pancreata and retina were harvested for immunohistological analysis. DA-CH5 therapy reversed glucose intolerance in KO rats and provided lasting anti-diabetogenic protection. Treatment also reversed intra-islet alterations, including reduced endocrine islet area and β-cell density, indicating its regenerative potential. Although no rescue effect was noted for hearing loss, visual acuity and retinal ganglion cell density were better preserved in DA-CH5-treated rats. We present preclinical evidence for the pleiotropic therapeutic effects of long-term dual incretin agonist treatment; effects were seen despite treatment beginning after symptom-onset, indicating reversal of disease progression. Dual incretins represent a promising therapeutic avenue for WS patients.

Gad1 knock-out rats exhibit abundant spike-wave discharges in EEG, exacerbated with valproate treatment.

To elucidate the functional role of gamma-aminobutyric acid (GABA)-ergic inhibition in suppressing epileptic brain activities such as spike-wave discharge (SWD), we recorded electroencephalogram (EEG) in knockout rats for Glutamate decarboxylase 1 (Gad1), which encodes one of the two GABA-synthesizing enzymes in mammals. We also examined how anti-epileptic drug valproate (VPA) acts on the SWDs present in Gad1 rats and affects GABA synthesis in the reticular thalamic nucleus (RTN), which is known to play an essential role in suppressing SWD. Chronic EEG recordings were performed in freely moving control rats and homozygous knockout Gad1 (-/-) rats. Buzzer tones (82 dB) were delivered to the rats during EEG monitoring to test whether acoustic stimulation could interrupt ongoing SWDs. VPA was administered orally to the rats, and the change in the number of SWDs was examined. The distribution of GABA in the RTN was examined immunohistochemically. SWDs were abundant in EEG from Gad1 (-/-) rats as young as 2 months old. Although SWDs were universally detected in older rats irrespective of their Gad1 genotype, SWD symptom was most severe in Gad1 (-/-) rats. Acoustic stimulation readily interrupted ongoing SWDs irrespective of the Gad1 genotype, whereas SWDs were more resistant to interruption in Gad1 (-/-) rats. VPA treatment alleviated SWD symptoms in control rats, however, counterintuitively exacerbated the symptoms in Gad1 (-/-) rats. The immunohistochemistry results indicated that GABA immunoreactivity was significantly reduced in the somata of RTN neurons in Gad1 (-/-) rats but not in their axons targeting the thalamus. VPA treatment greatly increased GABA immunoreactivity in the RTN neurons of Gad1 (-/-) rats, which is likely due to the intact GAD2, another GAD isozyme, in these neurons. Our results revealed two opposing roles of GABA in SWD generation: suppression and enhancement of SWD. To account for these contradictory roles, we propose a model in which GABA produced by GAD1 in the RTN neuronal somata is released extrasynaptically and mediates intra-RTN inhibition.

Effect of transcutaneous auricular vagus nerve stimulation on the splenic α7nAchR/JAK2/STAT3 signaling pathway in LPS-induced depressive-like behavior rats

To observe the effect of transcutaneous auricular vagus nerve stimulation (taVNS) on the improvement of depressive-like behavior and the splenic α7 nicotinic acetylcholine receptor (α7nAchR) / Janus kinase 2 (JAK2 / signal transducer and activator of transcription 3 (STAT3) signaling pathway in lipopolysaccharide (LPS)-induced depressive-like behavior rats, so as to investigate the antidepressant mechanism of taVNS. SD rats were randomly divided into SD control group, SD model group and SD taVNS group, and α7nAchR knockout rats were also randomly divided into α7 control group, α7 model group and α7 taVNS group, with 6 rats in each group. Rat model of depressive-like behavior was established by intraperitoneal injection of LPS (1 mg/kg). Rats in both SD taVNS and α7 taVNS groups received taVNS intervention once a day (2 Hz/15 Hz, 2 mA, 30 min) from 7 days before LPS injection to 2 days after LPS injection, respectively. The mean speed, activity time and side immobility time in the open field test were recorded after taVNS. The contents of interleukin 10 (IL-10) and chemokine (C-X-C motif) ligand 1 (CXCL1) in serum were detected by electrochemiluminescence multifactorial method. The splenic phosphorylated (p)-JAK2 and p-STAT3 protein expressions were detected by Western blot. Compared with their respective control groups, the mean speed and active time were reduced (P<0.01, P<0.05, P<0.001) and the side immobility time was increased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels were up-regulated (P<0.01, P<0.05, P<0.001), and splenic p-JAK2 protein expressions were down-regulated (P<0.05, P<0.01) in SD and α7nAchR knockout rats, and splenic p-STAT3 protein expression were down-regulated (P<0.05) in SD rats after LPS injection. Following taVNS intervention and in comparison with the model group , the mean speed and active time were increased (P<0.01) and the side immobility time was decreased (P<0.001) in the open field test, serum IL-10 and CXCL1 levels down-regulated (P<0.05), while splenic p-JAK2 and p-STAT3 protein expressions were up-regulated (P<0.01, P<0.001) in the SD taVNS group rather than in the α7 taVNS group. Compared with SD taVNS group, the α7 taVNS group showed increased (P<0.001, P<0.05) side immobility time in the open field test and serum IL-10, decreased splenic p-JAK2 and p-STAT3 protein expressions (P<0.01, P<0.05). taVNS may exert anti-inflammatory effects through modulating the splenic α7nAchR/JAK2/STAT3 signaling pathway, thereby ameliorating LPS-induced depressive-like behavior in rats.

Inflammatory atherosclerotic plaque identification by SPECT/CT imaging of LFA-1 using [111In] In-DANBIRT in a novel dyslipidemic rat model.

Atherosclerosis is prevalent globally, closely associated with dyslipidemia and other metabolic dysfunction. Early diagnosis of atherosclerosis is challenging due to limited diagnostic capabilities that need to be expanded with animal models with enhanced vascular biology like rats. Our previous research showed [111In] In-DANBIRT has potential as a diagnostic tool for detecting atherosclerosis in mice. The primary aim of the present study is to evaluate [111In] In-DANBIRT in a novel atherosclerotic rat with early- and late-stage atherosclerosis and metabolic disease. We characterized metabolic and body composition differences in these novel dyslipidemic rats under different diets using serum chemistry and dual-energy X-ray absorptiometry (DEXA) scan, respectively. We performed 1-h post-injection in vivo molecular imaging of ApoE knockout, lean Zucker (LZ) male rats at baseline and followed them into 10weeks of either normal or high-fat/cholesterol diet implementation (22weeks of age). We identified significant differences in body composition and metabolic changes in ApoE knockout rats compared to ApoE wildtype rats. Our findings indicate an increased uptake of [111In] In-DANBIRT in ApoE knockout, lean Zucker (LZ) rats, particularly in the descending aorta, a location where early-stage atherosclerosis is commonly found. Our findings, however, also revealed that the ApoE knockout, Zucker diabetic fatty (ZDF) model has high mortality rate, which may be attributed to alterations of critical enzymes involved in regulating metabolism and liver function. Our results are highly encouraging as they demonstrated the potential of [111In] In-DANBIRT to detect early-stage atherosclerosis in rats that might otherwise go unnoticed by other methods, showcasing the high sensitivity of [111In] In-DANBIRT. Our future studies will aim to establish a viable T2D atherosclerosis model in rats with more advanced stages of the disease to further demonstrate the reliability of [111In] In-DANBIRT as a diagnostic tool for patients in all stages of atherosclerosis.

Kallistatin Deficiency Induces the Oxidative Stress-Related Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells: A Novel Protagonist in Age-Related Macular Degeneration.

Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of age-related macular degeneration (AMD). Kallistatin (KAL) is a secreted protein with an antioxidative stress effect. However, the relationship between KAL and EMT in RPE has not been determined. Therefore we aimed to explore the impact and mechanism of KAL in oxidative stress-induced EMT of RPE. Sodium iodate (SI) was injected intraperitoneally to construct the AMD rat model and investigate the changes in RPE morphology and KAL expression. KAL knockout rats and KAL transgenic mice were used to explain the effects of KAL on EMT and oxidative stress. In addition, Snail overexpressed adenovirus and si-RNA transfected ARPE19 cells to verify the involvement of Snail in mediating KAL-suppressed EMT of RPE. AMD rats induced by SI expressed less KAL in the retina, and KAL knockout rats showed RPE dysfunction spontaneously where EMT and reactive oxygen species (ROS) production increased in RPE. In contrast, KAL overexpression attenuated EMT and ROS levels in RPE, even in TGF-β treatment. Mechanistically, Snail reversed the beneficial effect of KAL on EMT and ROS reduction. Moreover, KAL ameliorated SI-induced AMD-like pathological changes. Our findings demonstrated that KAL inhibits oxidative stress-induced EMT by downregulating the transcription factor Snail. Herein, KAL knockout rats may be an appropriate animal model for observing spontaneous RPE dysfunction for AMD-like retinopathy, and KAL may represent a novel therapeutic target for treating dry AMD.

Influence of Slco2b1-knockout and SLCO2B1-humanization on coproporphyrin I and III levels in rats.

Coproporphyrin (CP) I and III are byproducts of haem synthesis currently investigated as biomarkers for drug-drug interactions involving hepatic organic anion transporting polypeptide (OATP) 1B transporters. Another hepatically expressed OATP-member is OATP2B1. The aim of this study was to test the impact of OATP2B1, which specifically transports CPIII, on CP serum levels, applying novel rat models. CPIII transport kinetics and the interplay between OATP2B1 and multidrug resistance-associated proteins (MRPs) were determined in vitro using the vTF7 expression system. Novel rSlco2b1-/- and SLCO2B1+/+ rat models were characterized for physiological parameters and for CP serum levels. Hepatic and renal expression of transporters involved in CP disposition were determined by real-time qPCR, Western blot analysis, and immunohistochemistry. In vitro experiments revealed differences in transport kinetics comparing human and rat OATP2B1 and showed a consistent, species-specific interplay with hMRP3/rMRP3. Deletion of rOATP2B1 was associated with a trend towards lower CPI serum levels compared with wildtype rats, while CPIII remained unchanged. Comparing SLCO2B1+/+ with knockout rats revealed an effect of sex: only in females the genetic modification influenced CP serum levels. Analysis of hepatic and renal transporters revealed marginal, but in part, statistically significant differences in rMRP2 abundance, which may contribute to the observed changes in CP serum levels. Our findings support that factors other than OATP1B transporters are of relevance for basal CP levels. Only in female rats, humanization of SLCO2B1 affects basal CPI and CPIII serum levels, despite isomer selectivity of OATP2B1.

Abstract P137: Lack Of Atrial Natriuretic Peptide Aggravates Glomerular Disease In Type 1 Diabetic Nephropathy

Introduction: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease and DKD patients have a significantly higher risk of developing cardiovascular disease. Some studies have suggested the role of the Atrial Natriuretic Peptide (ANP) in DKD development. ANP is a cardiac hormone that regulates renal salt/water homeostasis and plays a major role in renal hemodynamics. We hypothesized here that ANP plays a critical role in T1DKD, playing a potential protective role in podocytes of the glomeruli. Methods: We used the ANP knock-out (SS NPPA-/- ) rat on the Dahl Salt-Sensitive (Dahl SS) background injected with streptozotocin (STZ; 75mg/kg) as a model of T1DKD. At 9-10 weeks of age, male SS NPPA-/- and Dahl SS wild-type (SS WT ) rats were i.p. injected with either STZ or vehicle. Baseline, midpoint, and endpoint blood glucose, urine, and body weights were collected. Glomerular filtration rate (GFR) was assessed before animals were. Glomerular injury was scored blindly on a scale from 0-4 (0 = healthy glomerulus, 4 = severely sclerosed) in Masson Trichrome stained kidneys. One-way ANOVA through Origin 2019b was used for statistical analysis. Results: STZ- SS WT and STZ-SS NPPA-/- groups exhibited similar body weight (237.8 ± 18.9 vs. 234.5 ± 10.03 g, respectively), two-kidney weight to body weight ratio (32.1 ± 1.6 vs. 29.4 ± 1.6 mg/g, respectively), and endpoint blood glucose levels (&gt;650 mg/dL). Interestingly, we observed a significantly higher heart to body weight ratio for the STZ-SS NPPA-/- vs STZ- SS WT rats (7.0 ± 0.33 vs. 5.1 ± 0.07 mg/g, respectively, p&lt;0.05). STZ- SS WT and STZ-SS NPPA-/- had similar diuresis (54.4 ± 2.8 vs. 52.5 ± 4.01 mL/24hr/100gTBW, respectively). Importantly, histological analysis displayed a more profound glomerular injury in the STZ-SS NPPA-/- when they were compared to STZ- SS WT (3.4 ± 0.02 vs. 3.2 ± 0.03 au, respectively, p&lt;0.05). Conclusion: Thus far the data suggest that lack of ANP has a detrimental impact on DKD-related renal tissue damage, specifically, glomerular disease progression. Future studies will provide mechanistic insight into the role of ANP in oxidative stress and assess the role of ANP in nitric oxide production and calcium transients in the podocyte.