Postnatal Rat Research Articles

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells. The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

Chronic SSRI Treatment, but Not Norepinephrine Reuptake Inhibitor Treatment, Increases Neurogenesis in Juvenile Rats

There has been growing recognition that major depressive disorder is a serious medical disorder that also affects children. This has been accompanied by an increased use of antidepressant drugs in adolescents; however, not all classes of antidepressants are effective in children and adolescents. There is an increasing need to understand the differences in antidepressant action in different developmental stages. There are some data indicating that the behavioral effect of chronic antidepressant treatment in adult rodents is dependent on hippocampal neurogenesis; however, it is not known which classes of antidepressant drugs induce hippocampal neurogenesis in adolescent rodents. Three classes of antidepressant drugs were tested in two age groups of Sprague Dawley rats, pre-adolescent (postnatal days 11–24) and adolescent (postnatal days 21–34): monoamine oxidase inhibitors (MAOIs); selective serotonin reuptake inhibitors (SSRIs); serotonin norepinephrine reuptake inhibitors (SNRIs); and tricyclic antidepressants (TCAs). To address which classes of antidepressant drugs might alter the rate of mitogenesis in neural progenitor cells in an adolescent rodent model, adolescent Sprague Dawley rats were treated with the thymidine analog 5-bromo-deoxy-2′-uridine (BrdU) on postnatal days 21 and 22 and antidepressant drugs or vehicle for 14 days (postnatal days 21–34). To address which classes of antidepressant drugs might alter the rate of neurogenesis, postnatal day-21 Sprague Dawley rats were treated with antidepressant drugs or vehicle for 14 days (postnatal days 21–34) and BrdU on postnatal days 33 and 34. In both experimental paradigms, BrdU-positive cells in the subgranular zone and the granule cell layer were counted. Newborn neurons were identified in the neurogenic paradigm by identifying cells expressing both the neuronal specific marker NeuN and BrdU using confocal microscopy. Only the SSRI fluoxetine significantly altered the basal mitogenic and neurogenic rates in adolescent rats. Treatment with the monoamine oxidase inhibitor (MAOI) tranylcypromine (TCP) and the TCA desipramine did not alter the rate of hippocampal neurogenesis in the adolescent rats. This is consistent with human clinical observations, where only SSRIs have efficacy for treatment of depression in patients under the age of 18. In pre-adolescent rats, postnatal days 11–24, none of the drugs tested significantly altered the basal mitogenic or neurogenic rates. All of the classes of antidepressant drugs are known to induce hippocampal neurogenesis in adult rats. The mechanisms of action underlying this developmental difference in antidepressant drug action between juveniles and adults are not known.

AP-1 activation mediates post-natal cardiomyocyte maturation.

Post-natal maturation of mammalian cardiomyocytes proceeds rapidly after birth, with most of the myocytes exiting cell cycle, becoming binucleated, and adopting oxidative phosphorylation as the primary metabolic route. The triggers and transcriptional programmes regulating cardiomyocyte maturation have not been fully understood yet. We performed single-cell RNA-Seq in post-natal rat hearts in order to identify the important factors for this process. Single-cell RNA-Seq profiling was performed of post-natal Day 1 and Day 7 rat hearts, and we found that members of the activating protein 1 (AP-1) transcription factors showed a transient up-regulation in the maturing cardiomyocytes, suggesting their functional involvement in the process. Activating members of the AP-1 family by palmitate or adrenergic stimulation inhibited cardiomyocyte cytokinesis and promoted cardiomyocyte maturation. In contrast, knocking down AP-1 members Atf3 and Jun promoted cardiomyocyte cytokinesis, reduced polyploidy, and inhibited maturation. Mechanistically, RNA-Seq results and rescue experiments indicated that AP-1 members activate the expression of fatty acid metabolic genes to promote cardiomyocyte maturation. Finally, intraperitoneal injection of AP-1 inhibitor T-5224 in neonatal mice inhibits cardiomyocyte maturation in vivo. Our results are the first evidence implicating AP-1 transcription factors in post-natal cardiomyocyte maturation both in vitro and in vivo, which expand our understanding of the molecular mechanism of cardiomyocyte maturation, and may lead to novel therapies to treat congenital heart diseases.

Long-term amelioration of an early-onset familial atrial fibrillation model with AAV-mediated in vivo gene therapy

Atrial fibrillation (AF) is a common cardiac disease with high prevalence in the general population. Despite a mild manifestation at the onset stage, it causes serious consequences, including sudden death, when the disease progresses to the late stage. Most available treatments of AF focus on symptom management or alleviation, due to a lack of fundamental knowledge and the fact that considerable variations of AF exist. With the popularisation of the next-generation sequencing technology, several causal genetic factors, including MYL4, have been discovered to contribute to AF, giving hope to developing its gene therapies. In this study, we attempted to treat a previously established rat AF model, which carried Myl4E11K/E11K loss of function mutation, via overexpression of exogenous wild-type Myl4 by AAV9 vectors. Our results showed that delivery of Myl4 expressing AAV9 to postnatal rat models rescued the symptoms of AF, indicating the therapeutic potential that early gene therapy intervention can achieve long-term effects in treating cardiac arrhythmias caused by gene mutations.

Metabolic alterations in the visual pathway of retinitis pigmentosa rats: A longitudinal multimodal magnetic resonance imaging study with histopathological validation.

Because retinitis pigmentosa (RP) has been shown to cause degenerative changes in the entire visual pathway, there is an urgent need to perform longitudinal assessments of RP-induced degeneration and identify imaging protocols to detect this degeneration as early as possible. In this study, we assessed a transgenic rat model of RP by using complementary noninvasive magnetic resonance imaging techniques, namely, proton magnetic resonance spectroscopy (1 H-MRS), to investigate the metabolic changes in RP. Our study demonstrated decreased concentrations and ratios to creatine (Cr) of N-acetylaspartate (NAA), glutamate (Glu), γ-aminobutyric acid (GABA), and taurine (Tau), whereas myo-inositol (Ins) and choline (Cho) were increased in the visual cortex of Royal College of Surgeons (RCS) rats compared with control rats (p < 0.05). Furthermore, with the progression of RP, the concentrations of NAA, Glu, GABA, and Tau, and the ratios of GABA/Cr and Tau/Cr significantly decreased over time, whereas the concentrations of Ins and Cho and the ratio of Ins/Cr significantly increased over time (p < 0.05). In addition, in RCS rats, NAA/Cr decreased significantly from 3 to 4 months postnatal (p < 0.001), and Cho/Cr increased significantly from 4 to 5 months postnatal (p = 0.005). Meanwhile, the 1 H-MRS indicators in 5-month postnatal RCS rats could be confirmed by immunohistochemical staining. In conclusion, with the progression of RP, the metabolic alterations in the visual cortex indicated progressive reprogramming with the decrease of neurons and axons, accompanied by the proliferation of gliocytes.

Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina.

Identified as a member of the secretin/glucagon/VIP superfamily, pituitary adenylate cyclase-activating polypeptide (PACAP1-38) has been recognized as a hormone, neurohormone, transmitter, trophic factor, and known to be involved in diverse and multiple developmental processes. PACAP1-38 was reported to regulate the production of important morphogens (Fgf1, Bmp4, Gdf3) through PAC1-receptor in the newborn rat retina. To follow up, we aimed to reveal the identity of retinal cells responsible for the production and secretion of Fgf1, Bmp4, and Gdf3 in response to PACAP1-38 treatment. Newborn (P1) rats were treated with 100 pmol PACAP1-38 intravitreally. After 24 h, retinas were dissected and processed for immunohistochemistry performed either on flat-mounted retinas or cryosections. Brn3a and PAC1-R double labeling revealed that 90% of retinal ganglion cells (RGCs) expressed PAC1-receptor. We showed that RGCs were Fgf1, Bmp4, and Gdf3- immunopositive and PAC1-R was co-expressed with each protein. To elucidate if RGCs release these secreted regulators, the key components for vesicle release were examined. No labeling was detected for synaptophysin, Exo70, or NESP55 in RGCs but an intense Rab3a-immunoreactivity was detected in their cell bodies. We found that the vast majority of RGCs are responsive to PACAP, which in turn could have a significant impact on their development or/and physiology. Although Fgf1, Bmp4, and Gdf3 were abundantly expressed in PAC1-positive RGCs, the cells lack synaptophysin and Exo70 in the newborn retina thus unable to release these proteins. These proteins could regulate postnatal RGC development acting through intracrine pathways.

Synergistic effects of mechanical stimulation and crimped topography to stimulate natural collagen development for tendon engineering

Suitable scaffold structures and mechanical loading are essential for functional tendon engineering. However, the bipolar fibril structure of native tendon collagen is yet to be recaptured in engineered tendons. This study compared the development of Achilles tendons of postnatal rats with and without (via surgical section) mechanical loading to define the mechanism of mechanical stimulation-mediated tendon development. The results demonstrated that the severed tendons weakened mechanically and exhibited disorganization without a bipolar fibril superstructure. Proteomic analysis revealed differentially expressed key regulatory molecules related to the collagen assembly process, including decreased fibromodulin, keratocan, fibroblast growth factor-1, and increased lumican and collagen5a1 in the severed tendons with immunohistochemical verification. Additionally, a complex regulatory network of mechanical stimulation-mediated collagen assembly in a spatiotemporal manner was also revealed using bioinformatics analysis, wherein PI3K–Akt and HDAC4 may be the predominant signaling pathways. A wavy microgrooved surface (Y = 5.47sin(0.015x)) that biomimics tendon topography was observed to enhance the expression of collagen assembly molecules under mechanical loading, and the aforementioned pathways are particularly involved and verified with their respective inhibitors of LY-294002 and LMK-235. Furthermore, an electrospun crimped nanofiber scaffold (approximately 2 μm fiber diameter and 0.12 crimpness) was fabricated to biomimic the tenogenic niche environment; this was observed to be more effective on enhancing collagen production and assembly under mechanical stimulation. In conclusion, the synergistic effect between topographical niche and mechanical stimulation was observed to be essential for collagen assembly and maturation and should be applied to functional tendon engineering in the future. Statement of significanceIn biomaterial-mediated tendon regeneration, mechanical stimulation is essential for tendon collagen assembly. However, the underlying mechanisms remain not fully defined, leading to the failure of the native-like collagen regeneration. In this study, a mechanical stimulation deprivation model of rat tendon was established to reveal the mechanisms in tendon development and define the key regulatory molecules including small leucine-rich proteoglycans, lysyl oxidase and collagen V. After ensuring the importance of biomimetic structure in tendon remodeling, crimped nanofibers were developed to verify these regulatory molecules, and demonstrated that mechanical stimulation significantly enhanced collagen assembly via PIK3 and HDAC4 pathways in biomaterial-regulated tendon regeneration. This study provides more insightful perspectives in the physiologically remodeling progression of tendon collagen and design of tendon scaffolds.

AAV-CRISPR/Cas9 Gene Editing Preserves Long-Term Vision in the P23H Rat Model of Autosomal Dominant Retinitis Pigmentosa.

Retinitis pigmentosa (RP) consists of a group of inherited, retinal degenerative disorders and is characterized by progressive loss of rod photoreceptors and eventual degeneration of cones in advanced stages, resulting in vision loss or blindness. Gene therapy has been effective in treating autosomal recessive RP (arRP). However, limited options are available for patients with autosomal dominant RP (adRP). In vivo gene editing may be a therapeutic option to treat adRP. We previously rescued vision in neonatal adRP rats by the selective ablation of the Rhodopsin S334ter transgene following electroporation of a CRISPR/Cas9 vector. However, the translational feasibility and long-term safety and efficacy of ablation therapy is unclear. To this end, we show that AAV delivery of a CRISPR/Cas9 construct disrupted the Rhodopsin P23H transgene in postnatal rats, which rescued long-term vision and retinal morphology.

Deficiency of Thyroid Hormone Reduces Voltage-Gated Na+ Currents as Well as Expression of Na+/K+-ATPase in the Mouse Hippocampus.

Mice lacking functional thyroid follicular cells, Pax8−/− mice, die early postnatally, making them suitable models for extreme hypothyroidism. We have previously obtained evidence in postnatal rat neurons, that a down-regulation of Na+-current density could explain the reduced excitability of the nervous system in hypothyroidism. If such a mechanism underlies the development of coma and death in severe hypothyroidism, Pax8−/− mice should show deficits in the expression of Na+ currents and potentially also in the expression of Na+/K+-ATPases, which are necessary to maintain low intracellular Na+ levels. We thus compared Na+ current densities in postnatal mice using the patch-clamp technique in the whole-cell configuration as well as the expression of three alpha and two beta-subunits of the Na+/K+-ATPase in wild type versus Pax8−/− mice. Whereas the Na+ current density in hippocampal neurons from wild type mice was upregulated within the first postnatal week, the Na+ current density remained at a very low level in hippocampal neurons from Pax8−/− mice. Pax8−/− mice also showed significantly decreased protein expression levels of the catalytic α1 and α3 subunits of the Na+/K+-ATPase as well as decreased levels of the β2 isoform, with no changes in the α2 and β1 subunits.

Adjusting the neuron to astrocyte ratio with cytostatics in hippocampal cell cultures from postnatal rats: A comparison of cytarabino furanoside (AraC) and 5-fluoro-2'-deoxyuridine (FUdR).

Cell culture studies offer the unique possibility to investigate the influence of pharmacological treatments with quantified dosages applied for defined time durations on survival, morphological maturation, protein expression and function as well as the mutual interaction of various cell types. Cultures obtained from postnatal rat brain contain a substantial number of glial cells that further proliferate with time in culture leading to an overgrowth of neurons with glia, especially astrocytes and microglia. A well-established method to decrease glial proliferation in vitro is to apply low concentrations of cytosine arabinoside (AraC). While AraC primarily effects dividing cells, it has been reported repeatedly that it is also neurotoxic, which is the reason why most protocols limit its application to concentrations of up to 5 μM for a duration of 24 h. Here, we investigated 5-fluoro-2'-deoxyuridine (FUdR) as a possible substitute for AraC. We applied concentrations of both cytostatics ranging from 4 μM to 75 μM and compared cell composition and cell viability in cultures prepared from 0-2- and 3-4-day old rat pups. Using FUdR as proliferation inhibitor, higher ratios of neurons to glia cells were obtained with a maximal neuron to astrocyte ratio of up to 10:1, which could not be obtained using AraC in postnatal cultures. Patch-clamp recordings revealed no difference in the amplitudes of voltage-gated Na+ currents in neurons treated with FUdR compared with untreated control cells suggesting replacement of AraC by FUdR as glia proliferation inhibitor if highly neuron-enriched postnatal cultures are desired.

Neuroprotective effects of tumor necrosis factor-α inhibitor on rat hippocampal organotypic slice cultures treated with the 1-42 β–amyloid

Background: Alzheimer disease (AD) is a chronic degenerative neuronal disease. Tumor necrosis factor (TNF) inhibitors involved in neuroinflammation in AD pathogenesis can be a potential treatment strategy for AD. Therefore, we performed organotypic hippocampal slice culture to investigate neuroprotective effects by TNF-α inhibitor from β-amyloid (Aβ)-induced neurotoxicity.Methods: We cultured the hippocampus of a 7 days postnatal Sprague-Dawley rats with 450 µm thick slices. The tissue slices had been exposed with 100 µM Aβ at an interval of 2 days since 14 days in vitro. Following co-treatment of the tissue slice with 100 µg/ml adalimumab and Aβ, we evaluated adalimumab effect on Aβ induced neurotoxicity by morphological observation of the hippocampus slice cultures with 1 µg/ml propidium iodide (PI) and measuring the expression levels of NeuN, Bcl2, and tau proteins.Results: 100 µM Aβ induced increase of PI-positive fluorescence in the hippocampal slice. The adalimumab as a TNF-α inhibitor showed protective effects for Aβ-induced neurotoxicity, and reduced cell death of the CA1 region in hippocampal slice. The expression of NeuN and Bcl2 proteins was significantly decreased by 100 µM Aβ, and increased by co-treatment of 100 µg/ml adalimumab. Phosphorylation of tau protein increased by 100 µM Aβ and decreased by co-treatment of 100 µg/ml adalimumab.Conclusions: Adalimumab reduced the Aβ-induced neurotoxicity of the hippocampal slice culture, inhibited expression of NeuN, Bcl2 proteins and increasing of phophorylated tau protein. These findings would be useful as baseline data in the field of AD research and development of noble drugs for neurodegenerative diseases.

Early postnatal handling alters social behavior, learning, and memory of pre- and postnatal VPA-induced rat models of autism in a context-based manner

Early life events are known to greatly affect brain development, cortical neurogenesis, and Hypothalamic-pituitary-adrenal activity. Mainly characterized by impairment in social communication, language, and cognitive development, autism spectrum disorder (ASD) refers to a class of neuropsychiatric disorders with numerous genetic and environmental risk factors. In the early handling (EH) method, daily separation of infants from their mother, physical touching, and exposure to a new environment occur. Here, we studied the effect of EH on Social interaction, learning, and memory in rats exposed pre-or post-natally to valproic acid (VPA). Gestational VPA exposure (600 mg/kg) led to some severe autistic-like traits, more notable in the social behavior of the male sex, along with unchanged to partially altered spatial learning and memory function and reduced avoidance memory. In comparison, while causing a sex-dependent increase in spatial memory, subcutaneous injection of VPA (400 mg/kg) in infancy resulted in limited adverse autistic features, including a decrease in males’ social preference, as well as reduced avoidance memory. The results indicated that neonatal handling significantly improved multiple social behavior and memory deficits in prenatally injected rats. In contrast, EH in rats receiving postnatal VPA elicited a restricted advantage on social novelty tendency; while negatively affecting some other social behavior criteria and spatial learning of males and encouraging sex-dependent repetitive behaviors in the social setting. The controversial influence of postnatal handling on juvenile rats of postnatal VPA treatment vs. prenatal VPA treatment opens up the potential for future research on the context-based consequence of early-life handling stress using different behavioral tasks and to benefit therapeutic procedures through understanding the sex- and age-specific neurobiology of short-term environmental manipulation in animal models of autism.

Protective effect of luteolin against oxidative stress during hypoxic ischemia-induced neuronal damage in neonatal rats

&#x0D; &#x0D; &#x0D; &#x0D; Purpose: To investigate the neuroprotective efficacy of plant polyphenol luteolin against hypoxic ischemia (HI)-induced neuronal injury in neonatal rat pups.&#x0D; Methods: Postnatal rat pups (aged 7 days) were subjected to HI insult, and then treated with luteolin at a dose of 1.2 mg/kg body weight (bwt) for up to 4 days after HI injury. Following the sacrifice of the pups, the brain tissues were subjected to histological examination (H &amp; E staining), as well as biochemical and antioxidant assays. Moreover, levels of cell death regulator proteins were determined by enzyme-linked immunosorbent assay (ELISA).&#x0D; Results: There was significant increase in the tissue levels of reactive oxygen species (ROS) and malondialdehyde (MDA), but marked decrease in mitochondrial membrane potential (ΔΨm) in the HI rat pups (p &lt; 0.05). Furthermore, HI decreased the brain tissue levels of the antioxidants, SOD, CAT, GPX and GSH, in the postnatal pups. However, luteolin treatment significantly reversed the abnormal increase in the levels of ROS and MDA, but reduced the loss in mitochondrial membrane potential (ΔΨm), while increasing the levels of SOD, CAT GPX and GSH (p &lt; 0.05). Furthermore, HI significantly increased the brain expression levels of the pro-apoptotic markers, i.e., cytosolic cytochrome C (cyt c), caspase-3 and caspase-9 in the pups exposed to HI, indicating neuronal cell death. However, the HI- induced increase in the expression levels of these pro-apoptotic factors was reduced by luteolin treatment.&#x0D; Conclusion: These results suggest that luteolin protects postnatal rat pups from hypoxic ischemic- induced brain damage (neuronal cell death) due to its antioxidant and free radical-scavenging activities. Therefore, luteolin may be a potential neuroprotective agent in the management of HI-associated complications.&#x0D; &#x0D; &#x0D; &#x0D;

GPR68 Improves Nerve Damage and Myelination in an Immature Rat Model Induced by Sevoflurane Anesthesia by Activating cAMP/CREB to Mediate BDNF.

Ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) is a member of proton-sensing G-protein-coupled receptors, involved in cardiovascular physiology, tumor biology, and asthma, and exerts a neuroprotective effect against brain ischemia. The effects of GPR68 on anesthesia-induced nerve damage and myelination were investigated in this study. First, 2-day old postnatal rats were exposed to 4.9% sevoflurane for 2 h. Data from hematoxylin and eosin staining and Nissl staining showed that sevoflurane induced pathological changes in the hippocampus with a reduced number of neurons. GPR68 was downregulated in the hippocampus of sevoflurane-induced rats. Second, sevoflurane-induced rats were injected with adeno-associated virus (AAV)-mediated overexpression of GPR68, and overexpression of GPR68 ameliorated sevoflurane-induced pathological changes, enhanced the number of neurons, and improved the learning and memory function. Moreover, overexpression of GPR68 increased the number of BrdU-positive and Olig2-positive cells and enhanced protein expression of Olig2 in sevoflurane-induced rats. Third, the number of myelin basic protein (MBP) positive cells and protein expression of MBP in sevoflurane-induced rats were also enhanced by injection with AAV-GPR68. Overexpression of GPR68 attenuated sevoflurane-induced neuronal apoptosis and oxidative stress in rats. Lastly, overexpression of GPR68 upregulated protein expression of the brain-derived neurotrophic factor (BDNF) by increasing cAMP and phosphorylated cAMP response element-binding protein (CREB). In conclusion, GPR68 alleviated sevoflurane-induced nerve damage and myelination through BDNF-mediated activation of the cAMP/CREB pathway.

Curcumin alleviates hepatic steatosis by improving mitochondrial function in postnatal overfed rats and fatty L02 cells through the SIRT3 pathway.

Postnatal overfeeding could increase the risk of non-alcoholic fatty liver disease (NAFLD) in adulthood. This study investigated the effects of curcumin (CUR) on hepatic steatosis in postnatal overfed rats and elucidated potential mechanisms in mitochondrial functions. Male rats were adjusted to ten (normal litter, NL) or three (small litter, SL) at postnatal day 3. After weaning, NL rats were fed with normal diet (NL) or a high-fat diet (NH) for 10 weeks. SL rats were fed with normal diet (SL), a high-fat diet (SH), a normal diet supplemented with 2% CUR (SL-CUR) or a high-fat diet supplemented with 2% CUR (SH-CUR). At week 13, compared with NL rats, SL and NH rats showed increased body weight, glucose intolerance, dyslipidemia and hepatic lipid accumulation, and these changes were more obvious in SH rats. The opposite trends were observed in SL-CUR and SH-CUR rats. Moreover, CUR could preserve mitochondrial biogenesis and antioxidant response in postnatal overfed rats, and upregulated the mRNA and protein levels of SIRT3. In vitro, L02 cells were exposed to free fatty acids and/or CUR. CUR decreased the levels of cellular lipids and mitochondrial reactive oxygen species, and increased the mitochondrial DNA copy number and superoxide dismutase activity in fatty L02 cells. However, these effects were blocked after SIRT3 silencing. It was concluded that postnatal overfeeding damaged mitochondrial biogenesis and antioxidant response, and increased hepatic lipids and the severity of high-fat-induced NAFLD, while CUR alleviated hepatic steatosis, at least partially, by enhancing mitochondrial function through SIRT3.

Transient receptor potential vanilloid type 4 (TRPV4) in urinary bladder structure and function.

Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a urologic, chronic pelvic pain syndrome characterized by pelvic pain, pressure, or discomfort with urinary symptoms. Symptom exacerbation (flare) is common with multiple, perceived triggers including stress. Multiple transient receptor potential (TRP) channels (TRPA1, TRPV1, TRPV4) expressed in the bladder have specific tissue distributions in the lower urinary tract (LUT) and are implicated in bladder disorders including overactive bladder (OAB) and BPS/IC. TRPV4 channels are strong candidates for mechanosensors in the urinary bladder and TRPV4 antagonists are promising therapeutic agents for OAB. In this perspective piece, we address the current knowledge of TRPV4 distribution and function in the LUT and its plasticity with injury or disease with an emphasis on BPS/IC. We review our studies that extend the knowledge of TRPV4 in urinary bladder function by focusing on (i) TRPV4 involvement in voiding dysfunction, pelvic pain, and non-voiding bladder contractions in NGF-OE mice; (ii) distention-induced luminal ATP release mechanisms and (iii) involvement of TRPV4 and vesicular release mechanisms. Finally, we review our lamina propria studies in postnatal rat studies that demonstrate: (i) the predominance of the TRPV4+ and PDGFRα+ lamina propria cellular network in early postnatal rats; (ii) the ability of exogenous mediators (i.e., ATP, TRPV4 agonist) to activate and increase the number of lamina propria cells exhibiting active Ca2+ events; and (iii) the ability of ATP and TRPV4 agonist to increase the rate of integrated Ca2+ activity corresponding to coupled lamina propria network events and the formation of propagating wavefronts.

Early introduction of exercise prevents insulin resistance in postnatal overfed rats.

Early childhood obesity increases the risk of developing metabolic diseases. We examined the early introduction of exercise in small-litter obese-induced rats (SL) on glucose metabolism in the epididymal adipose tissue (AT) and soleus muscle (SM). On day 3 post-birth, pups were divided into groups of ten or three (SL). On day 22, rats were split into sedentary (S and SLS) and exercise (E and SLE) groups. The rats swam three times/week carrying a load for 30 min. In the first week, they swam without a load; in the 2nd week, they carried a load equivalent to 2% of their body weight; from the 3rd week to the final week, they carried a 5% body load. At 85 days of age, an insulin tolerance test was performed in some rats. At 90 days of age, rats were killed, and blood was harvested for plasma glucose, cholesterol, and triacylglycerol measurements. Mesenteric, epididymal, retroperitoneal, and brown adipose tissues were removed and weighed. SM and AT were incubated in the Krebs-Ringer bicarbonate buffer, 5.5 mM glucose for 1 h with or without 10 mU/mL insulin. Comparison between the groups was performed by 3-way ANOVA followed by the Tukey post-hoc test. Sedentary, overfed rats had greater body mass, more visceral fat, lower lactate production, and insulin resistance. Early introduction of exercise reduced plasma cholesterol and contained the deposition of white adipose tissue and insulin resistance. In conclusion, the early introduction of exercise prevents the effects of obesity on glucose metabolism in adulthood in this rat model.

A comparative histomorphological and histochemical study for trachea in postnatal and adult rats Rattus

A comparative histomorphological and histochemical study for trachea in postnatal and adult rats Rattus

A comparative histomorphological and histochemical study for trachea in postnatal and adult rats Rattus

A comparative histomorphological and histochemical study for trachea in postnatal and adult rats Rattus

Ginsenoside Rg1 Inhibits Microglia Pyroptosis Induced by Lipopolysaccharide Through Regulating STAT3 Signaling.

PurposeNeuroinflammation runs through the whole process of nervous system diseases and brain injury. Inflammasomes are thought to be especially relevant to immune homeostasis, and their dysregulation contributes to pyroptosis. The natural compound Ginsenoside Rg1 has been shown to possess anti-inflammatory effects; however, its underlying mechanisms are not entirely clear. Therefore, this study was undertaken to investigate the role and mechanisms of Rg1 in regulating the production of inflammasomes and pyroptosis of microglia in vivo and in vitro.MethodsBV-2 microglial cells were pretreated with Rg1, stattic and interleukin-6 (IL-6), and then stimulated with lipopolysaccharide (LPS) (2μg/mL). Hoechst staining and Annexin V-FITC/PI assay were then carried out. The expression levels of cleaved-caspase-1, pro-caspase-1, interleukin-1β (IL-1β), mature-IL-1β, gasdermin D (GSDMD), activated NH(2)-terminal fragment of GSDMD (GSDMD-N), NOD-, LRR- and pyrin domain-containing 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), absent in melanoma 2 (AIM2), signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 in BV-2 were detected by Western blotting. Additionally, immunofluorescence staining was used to determine the expression of NLRP3 and p-STAT3 in postnatal rat brain and BV-2 microglia subjected to LPS stimulation and Rg1 pretreatment. The targets of transcription factor STAT3 were predicted by hTFtarget and chromatin immunoprecipitation (ChIP) was used to confirm the interaction between STAT3 and AIM2.ResultsWe showed here that Rg1 effectively inhibited the expression of inflammasomes and microglia pyroptosis induced by LPS. The targets predicted data of Rg1 from Swiss target prediction database showed STAT3 had the highest thresholds of probability score. Rg1 can regulate the phosphorylation of STAT3, which binds to the promoter region of inflammasome AIM2.ConclusionIt is suggested that STAT3 signaling can initiate the transcription activity of AIM2. Rg1 regulates microglia pyroptosis in neuroinflammation induced by LPS through targeting STAT3 signaling.