Control Wistar Rats Research Articles

Compromised Neurotrophic and Angiogenic Regenerative Capability during Tendon Healing in a Rat Model of Type-II Diabetes.

Metabolic diseases such as diabetes mellitus type-II (DM-II) may increase the risk of suffering painful connective tissue disorders and tendon ruptures. The pathomechanisms, however, by which diabetes adversely affects connective tissue matrix metabolism and regeneration, still need better definition. Our aim was to study the effect of DM-II on expressional changes of neuro- and angiotrophic mediators and receptors in intact and healing Achilles tendon. The right Achilles tendon was transected in 5 male DM-II Goto-Kakizaki (GK) and 4 age-matched Wistar control rats. The left Achilles tendons were left intact. At week 2 post-injury, NGF, BDNF, TSP, and receptors TrkA, TrkB and Nk1 gene expression was studied by quantitative RT-PCR (qRT-PCR) and their protein distribution by immunohistochemistry in intact and injured tendons. The expression of tendon-related markers, Scleraxis (SCX) and Tenomodulin (TNMD), was evaluated by qRT-PCR in intact and injured tendons. Injured tendons of diabetic GK rats exhibited significantly down-regulated Ngf and Tsp1 mRNA and corresponding protein levels, and down-regulated Trka gene expression compared to injured Wistar controls. Intact tendons of DM-II GK rats displayed reduced mRNA levels for Ngf, Tsp1 and Trkb compared to corresponding intact non-diabetic tendons. Up-regulated Scx and Tnmd gene expression was observed in injured tendons of normal and diabetic GK rats compared to intact Wistar controls. However, these molecules were not up-regulated in injured DM-II GK rats compared to their corresponding controls. Our results suggest that DM-II has detrimental effects on neuro- and angiotrophic pathways, and such effects may reflect the compromised repair seen in diabetic Achilles tendon. Thus, novel approaches for regeneration of injured, including tendinopathic, and surgically repaired diabetic tendons may include therapeutic molecular modulation of neurotrophic pathways such as NGF and its receptors.

Relationship between PDK1 and contraction in carotid arteries in Goto-Kakizaki rat, a spontaneous type 2 diabetic animal model.

We studied the relationship between 3-phosphoinositide-dependent protein kinase 1 (PDK1) and contractions induced by serotonin, phenylephrine, and thromboxane A2 mimetic (U46619) in the presence of nitric oxide synthase inhibitor in the carotid arteries of Goto-Kakizaki (GK) rats, a spontaneous type 2 diabetic model, in the chronic stage of disease. Serotonin-induced contraction was greater in the GK rats than in the control Wistar rats. A specific PDK1 inhibitor (GSK2334470) decreased the serotonin-induced contraction in the GK rats but not in the Wistar rats, and the difference in such contraction was abolished with this treatment. In GK rats, phenylephrine-induced contraction exhibited a leftward shift and U46619-induced contraction was greater still. Phenylephrine- and U46619-induced contractions were reduced by GSK2334470 in both groups. These results suggest, for the first time, that the contribution of PDK1 is different among 3 vasoconstrictors and that PDK1 contributed to increased serotonin-induced contraction in the carotid arteries of GK rats.

Compensatory role of the NBCn1 sodium/bicarbonate cotransporter on Ca2+-induced mitochondrial swelling in hypertrophic hearts.

NBC Na+/HCO3- cotransporter (NBCn1) and NHE1 Na+/H+ exchanger have been associated with cardiac disorders and recently located in coronary endothelial cells (CEC) and cardiomyocytes mitochondria, respectively. Mitochondrial NHE1 blockade delays permeability transition pore (MPTP) opening and reduces superoxide levels, two critical events exacerbated in cells of diseased hearts. Conversely, activation of NBCn1 prevented apoptosis in CEC subjected to ischemic stress. We characterized the role of the NHE1 and NBCn1 transporters in heart mitochondria from hypertrophic (SHR) and control (Wistar) rats. Expression of NHE1 was analyzed in left ventricular mitochondrial lysates (LVML), by immunoblots. NHE1 expression increased by ~40% in SHR compared to control (P<0.05, n=4). To examine NHE1-mediated Na+/H+ exchange activity in cardiac hypertrophy, mitochondria were loaded with BCECF-AM dye and the maximal rate of pHm change measured after the addition of 50mM NaCl. SHR mitochondria had greater changes in pHm compared to Wistar, 0.10±0.01 vs. 0.06±0.01, respectively (P<0.05, n=5). In addition, mitochondrial suspensions from SHR and control myocardium were exposed to 200μM CaCl2 to induce MPTP opening (light-scattering decrease, LSD) and swelling. Surprisingly, SHR rats showed smaller LSD and a reduction in mitochondrial swelling, 67±10% (n=15), compared to control, 100±8% (n=13). NBC inhibition with S0859 (1μM) significantly increased swelling in both control 139±10% (n=8) and SHR 115±10% (n=4). Finally, NBCn1 Na+/HCO3- cotransporter increased by twofold its expression in SHR LVML, compared to normal (P<0.05, n=5). We conclude that increased NBCn1 activity may play a compensatory role in hypertrophic hearts, protecting mitochondria from Ca2+-induced MPTP opening and swelling.

Possible mechanism for the polychlorinated bipheny-linduced liver-selective accumulation of thyroxine in rats.

We have previously reported that decrease in level of serum thyroxine T4 by Kanechor 500 (KC500) in rats would occur through the increase in hepatic T4 accumulation rather than the increase in hepatic T4-glucuronyl transferase activity. In the present study, to understand the mechanism underlying the KC500-mediated increase in hepatic T4 accumulation, we examined the relationship between the KC500-mediated changes in hepatic T4 accumulation and the expression levels of mRNAs of hepatic transporters including T4 transporters. [125I]T4 was intravenously injected into KC500-pretreated and control (KC500-untreated) Wistar rats, and [125I]T4 uptake levels of liver parenchymal cells were comparatively examined. The amount of [125I]T4 uptake by hepatic cells increased in a time-dependent manner up to 96 hr after KC500 treatment. Following KC500 treatment, a time-dependent increase in the mRNA level of hepatic T4 influx transporter LAT1 was observed up to 96 hr later, while a significant increase in hepatic T4 influx transporter Oatp2 mRNA occurred only at 96 hr later. No KC500-mediated increases in the mRNAs of other hepatic transporters (Oatp1, Oatp3, Oatp4, Ntcp, LAT2, and Mrp2) were observed at any timepoints, although the mRNA expression of the T4 conjugate(s) efflux transporter Mrp3 significantly increased in a time-dependent manner 24-96 hr following KC500 treatment. The present findings suggest that KC500-mediated increase in hepatic T4 accumulation occurs, at least in part, through the increase in the expression of hepatic T4-transporters, such as LAT1 and Oatp2.

A developmental study of glutamatergic neuron populations in the ventrobasal and the lateral geniculate nucleus of the thalamus: Comparing Genetic Absence Rats from Strasbourg (GAERS) and normal control wistar rats

An imbalance of GABAergic inhibition and glutamatergic excitation is suspected to be the cause of absence epileptic seizures. Absence seizures are known to be generated in thalamocortical circuitry. In the present study we used light microscopy immunohistochemistry to quantify the density of glutamate+ve neurons at two developmental stages (P10 and P60) in two thalamic nuclei, the ventrobasal (VB) and lateral geniculate nucleus (LGN) in Wistar rats and compared the results with similar data obtained from genetic absence epilepsy rats from Strasbourg (GAERS).Rats were perfused transcardially with glutaraldehyde and paraformaldehyde fixative, then samples from VB and LGN were removed from each animal and sectioned. The glutamatergic neurons were labelled using light-microscopic glutamate immunohistochemistry. The disector method was used to quantify the glutamate+ve neurons in VB and LGN of GAERS and Wistar rats. The data were statistically analyzed.The distribution of the glutamate+ve neurons in the VB thalamic nucleus showed a significant reduction in the neuronal profiles per unit thalamic area from P10 to P60 in both Wistar and GAERS. The decrease was greater in the GAERS compared to the Wistar animals. However, in the LGN no reduction was observed either in the Wistar or in the GAERS. Comparing the density of glutamate+ve neurons in the VB thalamic nucleus of P10 of Wistar animals with of P10 GAERS showed statistically significant greater densities of these neurons in GAERS than in the Wistar rats. However no significant difference was present at P60 between the Wistar and GAERS animals.The disproportional decrease in GAERS may be related to the onset of absence seizures or may be related to neurogenesis of absence epilepsy.

ISH NIA OS-04 P2X3 RECEPTOR ACTIVITY IN THE CAROTID BODY (CB) OF SPONTANEOUSLY HYPERTENSIVE (SH) RATS CONTRIBUTES TO INCREASED CHEMOREFLEX HYPERSENSITIVITY AND HYPERTENSION

Objective: Arterial hypertension is associated with increases in both peripheral chemoreceptor reflex sensitivity and sympathetic nerve activity (SNA). We have shown that CB resection in SH rats reduces arterial pressure (AP; McBryde et al. 2013). Since P2X3 receptors exist in the rat CB and P2X3 receptor activity is associated with afferent hyperreflexia (Ford et al. 2015), we hypothesized that P2X3 receptor would be upregulated in the CB of SH vs normotensive (Wistar) control rats and that a selective P2X3 receptor antagonist (AF-219) would reduce chemoreflex hypersensitivity, AP and SNA in SH rats. Design and Method: SH and Wistar rats were implanted with radio-telemetry devices to record AP and renal SNA (RSNA) in conscious animals. Animals were infused with AF-219 (8 mg/kg/h) or vehicle i.v. for 1 h. The CB was stimulated with sodium cyanide (120 μg/kg i.v.). Western blotting and immunohistochemistry revealed the level of expression and localisation of P2X3 receptor protein in the CB, respectively. Results: In the CB, P2X3 receptor protein was upregulated four fold in SH vs Wistar rat (P < 0.05). Glomus cells of SH rat (identified as expressing tyrosine hydroxylase) and afferent nerve fibres were immunopositive for P2X3 receptors. In contrast, in Wistar rats, glomus cells expressing P2X3 receptors were fewer and P2X3 was not detected in nerve fibres. Infusion of AF-219 reduced: CB hyperreflexia (P < 0.05); AP (systolic −28 ± 3mmHg; P < 0.001) and RSNA (34%; P < 0.01). No changes in chemoreflex sensitivity, AP or RSNA were detected after vehicle or AF-219 infusion in Wistar rats. Conclusions: We surmise that upregulation of P2X3 receptors in the CB of SH rats contributes to chemoreflex hypersensitivity. Further, antagonism of P2X3 receptors lowers both AP and RSNA substantially in SH rats. Thus, P2X3 receptor antagonists may provide a novel way to attenuate CB over-activity and offer a novel approach for controlling hypertension. Supported by: Afferent Pharmaceuticals

Mitochondrial function assessed by 31P MRS and BOLD MRI in non-obese type 2 diabetic rats.

The study aims to characterize age‐associated changes in skeletal muscle bioenergetics by evaluating the response to ischemia‐reperfusion in the skeletal muscle of the Goto‐Kakizaki (GK) rats, a rat model of non‐obese type 2 diabetes (T2D). 31P magnetic resonance spectroscopy (MRS) and blood oxygen level‐dependent (BOLD) MRI was performed on the hindlimb of young (12 weeks) and adult (20 weeks) GK and Wistar (control) rats. 31P‐MRS and BOLD‐MRI data were acquired continuously during an ischemia and reperfusion protocol to quantify changes in phosphate metabolites and muscle oxygenation. The time constant of phosphocreatine recovery, an index of mitochondrial oxidative capacity, was not statistically different between GK rats (60.8 ± 13.9 sec in young group, 83.7 ± 13.0 sec in adult group) and their age‐matched controls (62.4 ± 11.6 sec in young group, 77.5 ± 7.1 sec in adult group). During ischemia, baseline‐normalized BOLD‐MRI signal was significantly lower in GK rats than in their age‐matched controls. These results suggest that insulin resistance leads to alterations in tissue metabolism without impaired mitochondrial oxidative capacity in GK rats.

Integrator of Stress Responses Calmodulin Binding Transcription Activator 1 (Camta1) Regulates miR-212/miR-132 Expression and Insulin Secretion

Altered microRNA profiles have been demonstrated in experimental models of type 2 diabetes, including in islets of the diabetic Goto-Kakizaki (GK) rat. Our bioinformatic analysis of conserved sequences in promoters of microRNAs, previously observed to be up-regulated in GK rat islets, revealed putative CGCG-core motifs on the promoter of the miR-212/miR-132 cluster, overexpression of which has been shown to increase insulin secretion. These motifs are possible targets of calmodulin binding transcription activators Camta1 and Camta2 that have been recognized as integrators of stress responses. We also identified putative NKE elements, possible targets of NK2 homeobox proteins like the essential islet transcription factor Nkx2-2. As Camtas can function as co-activators with NK2 proteins in other tissues, we explored the role of Camta1, Camta2, and Nkx2-2 in the regulation of the miR-212/miR-132 cluster and insulin secretion. We demonstrate that exposure of control Wistar or GK rat islets to 16.7 mm glucose increases miR-212/miR-132 expression but significantly less so in the GK rat. In addition, Camta1, Camta2, and Nkx2-2 were down-regulated in GK rat islets, and knockdown of Camta1 reduced miR-212/miR-132 promoter activity and miR-212/miR-132 expression, even under cAMP elevation. Knockdown of Camta1 decreased insulin secretion in INS-1 832/13 cells and Wistar rat islets but increased insulin content. Furthermore, knockdown of Camta1 reduced K(+)-induced insulin secretion and voltage-dependent Ca(2+) currents. We also demonstrate Camta1 and Nkx2-2 protein interaction. These results indicate that Camta1 is required not only for expression of the miR-212/miR-132 cluster but at multiple levels for regulating beta cell insulin content and secretion.

The influence of changes in expression of redox-sensitive genes on the development of retinopathy in rats

Age-related macular degeneration (AMD) is a complex multifactorial disease of the elderly, with unclear pathogenesis; AMD is the leading cause of blindness. One of the destructive processes in AMD is oxidative stress, which leads to an imbalance in the processes responsible for production and detoxification of reactive oxygen species. The aryl hydrocarbon receptor (AhR) signaling pathway can participate in the development of oxidative stress, but the main regulator of antioxidant defense is nuclear factor, erythroid derived 2 (Nrf2). AhR-dependent oxidative stress can be attenuated by activation of Nrf2, and defects in the Nrf2 signaling pathway can increase sensitivity of the cell to oxidative stress. Our aim was to determine the role of the pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems in the pathogenesis of AMD using rats of OXYS strain and of OXYSb substrain with signs of AMD-like retinopathy of varying severity. We compared the retinal levels of mRNA expression of Nrf2- and AhR-dependent redox-sensitive systems between 1-, 3-, and 12- month-old senescence-accelerated OXYS rats (have been shown to be a valid experimental model of AMD) and the rat substrain OXYSb, which shows low morbidity of AMD. We uncovered interstrain differences in the expression of Nrf2 and Nrf2-dependent genes (glutathione S-reductase [Gsr] and heme oxygenase 1 [Hmox1]), in the expression of AhR-dependent genes (cytochrome P450 1A2 [Cyp1a2] and cytochrome P450 1B1 [Cyp1b1]), and in the NADPH-quinone oxidoreductase (Nqo1) expression, which is controlled by both AhR and Nrf2. Binding of AhR and Nrf2 proteins to the regulatory regions of AhR and Nrf2 genes, respectively, was detected by chromatin immunoprecipitation in the retina of 1-, 3-, and 12-month-old OXYS, OXYSb, and Wistar (control) rats. We compared the strength of DNA-protein interactions of AhR and Nrf2 with regulatory sequences and found that the level of autoupregulation of the AhR gene was higher in the retina of 1-month-old OXYSb rats in comparison with OXYS rats. An imbalance between pro-oxidant (AhR-dependent) and antioxidant (Nrf2-dependent) systems may play a crucial role in the onset and/or progression of AMD.

Impairment of synaptic development in the hippocampus of diabetic Goto-Kakizaki rats

Insulin receptor signaling has been shown to regulate essential aspects of CNS function such as synaptic plasticity and neuronal survival. To elucidate its roles during CNS development in vivo, we examined the synaptic and cognitive development of the spontaneously diabetic Goto-Kakizaki (GK) rats in the present study. GK rats are non-obese models of type 2 diabetes established by selective inbreeding of Wistar rats based on impaired glucose tolerance. Though they start exhibiting only moderate hyperglycemia without changes in plasma insulin levels from 3 weeks postnatally, behavioral alterations in the open-field as well as significant impairments in memory retention compared with Wistar rats were observed at 10 weeks and were worsened at 20 weeks. Alterations in insulin receptor signaling and signs of insulin resistance were detected in the GK rat hippocampus at 3 weeks, as early as in other insulin-responsive peripheral tissues. Significant reduction of an excitatory postsynaptic scaffold protein, PSD95, was found at 5w and later in the hippocampus of GK rats due to the absence of a two-fold developmental increase of this protein observed in Wistar control rats between 3 and 20w. In the GK rat hippocampus, NR2A which is a NMDA receptor subunit selectively anchored to PSD95 was also reduced. In contrast, both NR2B and its anchoring protein, SAP102, showed similar developmental profiles in Wistar and GK rats with expression peaks at 2 and 3w. The results suggest that early alterations in insulin receptor signaling in the GK rat hippocampus may affect cognitive performance by suppressing synaptic maturation.

Effect of electroacupuncture stimulation on expression of angiotensinogen, angiotensin II type 1 receptor, endothelin-1, and endothelin a receptor mRNA in spontaneously hypertensive rat aorta

To observe the effect of electroacupuncture (EA) stimulation on the expressions of angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R), endothelin-1 (ET1), and endothelin A receptor (ETAR) mRNA in spontaneously hypertensive rat (SHR) aorta. Eighteen male SHRs were randomly divided into three groups, an SHR group, an SHR Baihui (DU 20) and Zusanli (ST 36) acupoint (SHR-AP) group, and an SHR non-acupoint (SHR-NAP) group, with 6 rats in each group. Six Wistar rats were used as a control. Rats in the SHR-AP group were stimulated by DU 20 and ST 36 acupoints, both of which were connected with EA. EA was handled one time every Monday, Wednesday and Friday, for total 24 times (8 weeks). SHRNAP rats were acupointed at a 15°angle flat into 0.5 cm to two points, which were 1 and 2 cm from rail tip separately. EA parameters were the same as the SHR-AP rats. SHR control rats and Wistar rats were fixed without EA. Real-time quantitative polymerase chain reaction (PCR) was used to measure AGT, AT1R, ET1, and ETAR mRNA expression in rat aorta. EA stimulation significantly reduced rat aorta vascular AGT, ET1, ETAR and AT1R mRNA expressions in the SHR-AP and SHR-NAP groups (P <0.01). Among these four genes, AT1R mRNA expression was significantly lower in the SHR-AP than in the SHR-NAP group (P <0.01). EA could reduce the AT1R mRNA expression in SHR-AP rat aorta, indicating a potential mechanism for the hypotensive effects of EA.

Effects of dopamine agents on a schedule-induced polydipsia procedure in the spontaneously hypertensive rat and in Wistar control rats.

The spontaneously hypertensive rat (SHR) has been proposed as an animal model for attention deficit hyperactivity disorder (ADHD), and typically develops excessive patterns of response under most behavioural protocols. Schedule-induced polydipsia (SIP) is the excessive water consumption that occurs as a schedule effect when food is intermittently delivered and animals are partially food- but not water-deprived. SIP has been used as a model of excessive behaviour, and considerable evidence has involved the dopaminergic system in its development and maintenance. The aim of this study was to evaluate the effects of the most common psychostimulants used in ADHD treatment on SIP, comparing their effects in SHRs with rats from control populations. SHR, Wistar Kyoto (WKY) and Wistar rats were submitted to a multiple fixed time (FT) food schedule with two components: 30 s and 90 s. The acute effects of different dopaminergic compounds were evaluated after 40 sessions of SIP acquisition. All animals showed higher adjunctive drinking under FT 30 s than FT 90 s, and SHRs displayed higher asymptotic SIP levels in FT 90 s compared to WKY and Wistar rats. SHRs were less sensitive to dopaminergic agents than control rats in terms of affecting rates of adjunctive drinking. These differences point to an altered dopaminergic system in the SHR and provide new insights into the neurobiological basis of ADHD pharmacological treatments.

Fibre tract analysis using diffusion tensor imaging reveals aberrant connectivity in a rat model of depression

Objectives: Abnormal brain connectivity has been described in depressive disorder. However, these studies are correlational or cross-sectional and their design does not examine causal relationships. We aimed to investigate structural connectivity in a genetic rat model of depression.Methods: Using diffusion tensor imaging (DTI), we reconstructed white matter tracts and analysed fractional anisotropy (FA) and diffusivity indices (mean, axial and radial) to investigate structural connectivity in fibre tracts implicated in major depression: the corpus callosum, fornix, cingulum and anterior commissures.Results: Tractography-based analysis revealed that, compared to Wistar control rats, the Wistar–Kyoto strain (WKY) rat model of depression exhibited decreased connectivity, manifested by decreased FA in the corpus callosum, right and left anterior commissures. A statistical trend of decreased FA was observed in both the right and left cingulum. Increased diffusivity (mean diffusion) was detected in both the corpus callosum and the fornix of WKY rats compared to controls. Voxel-based analysis confirmed differences between WKY and controls in the regions investigated.Conclusions: Decreased connectivity in a genetic rat model of depression corroborates the findings in patients suffering from major depression suggesting that the vulnerability for developing depression is mainly polygenic and less likely to be due to childhood adversity per se.

Chronic intermittent hypoxia accelerates coronary microcirculatory dysfunction in insulin-resistant Goto-Kakizaki rats.

Chronic intermittent hypoxia (IH) induces oxidative stress and inflammation, which impair vascular endothelial function. Long-term insulin resistance also leads to endothelial dysfunction. We determined, in vivo, whether the effects of chronic IH and insulin resistance on endothelial function augment each other. Male 12-wk-old Goto-Kakizaki (GK) and Wistar control rats were subjected to normoxia or chronic IH (90-s N2, 5% O2 at nadir, 90-s air, 20 cycles/h, 8 h/day) for 4 wk. Coronary endothelial function was assessed using microangiography with synchrotron radiation. Imaging was performed at baseline, during infusion of acetylcholine (ACh, 5 μg·kg(-1)·min(-1)) and then sodium nitroprusside (SNP, 5 μg·kg(-1)·min(-1)), after blockade of both nitric oxide (NO) synthase (NOS) with N(ω)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg) and cyclooxygenase (COX, meclofenamate, 3 mg/kg), and during subsequent ACh. In GK rats, coronary vasodilatation in response to ACh and SNP was blunted compared with Wistar rats, and responses to ACh were abolished after blockade. In Wistar rats, IH blunted the ability of ACh or SNP to increase the number of visible vessels. In GK rats exposed to IH, neither ACh nor SNP were able to increase visible vessel number or caliber, and blockade resulted in marked vasoconstriction. Our findings indicate that IH augments the deleterious effects of insulin resistance on coronary endothelial function. They appear to increase the dependence of the coronary microcirculation on NO and/or vasodilator prostanoids, and greatly blunt the residual vasodilation in response to ACh after blockade of NOS/COX, presumably mediated by endothelium-derived hyperpolarizing factors.

Comparing glutamatergic neuron population in the mediodorsal thalamic nucleus of genetic absence epilepsy rats from strasbourg (GAERS) and normal control Wistar rats

An imbalance between GABAergic inhibition and glutamatergic excitation is suspected to play a role in the genesis of epileptic processes. In the present study we quantified the number of glutamate+ve neurons in the mediodorsal thalamic nucleus (MD) of genetic absence epilepsy rats from Strasbourg (GAERS) and compared these with values for normal Wistar rats.The MD thalamic nucleus was removed from each animal and the glutamatergic neurons were labelled using light-microscopy glutamate immunohistochemistry. The disector method was used to quantify the glutamate+ve neurons in the MD thalamic nucleus of GAERS and Wistar rats. The data were statistically analyzed.In the Wistar animals glutamate+ve neurons formed 89% and in GAERS 92.3% of the total neurons in 1000μm3 of MD thalamic nucleus. In GAERS glutamate+ve neurons showed statistically significant increase in the MD thalamic nucleus compared to Wistar animals. In Wistar animals the glutamate−ve neurons formed 11% and in GAERS 7.7% of the total neurons in 1000μm3 of MD thalamic. No significant difference was observed in glutamate−ve neurons between the two strains. The average diameter of glutamate+ve neurons showed no significance, while glutamate−ve neurons were significant between the two strains.The results of the present study, on genetic absence epilepsy model, GAERS, confirms the role of MD thalamic nucleus in chemically induced absence epilepsy.

Sipa1l1 is an early biomarker of liver fibrosis in CCl4-treated rats.

ABSTRACTAt present, several procedures are used for staging liver fibrosis. However, these methods may involve clinical complications and/or present diagnostic uncertainty mainly in the early stages of the disease. Thus, this study was designed to unveil new non-invasive biomarkers of liver fibrosis in an in vivo model of fibrosis/cirrhosis induction by CCl4 inhalation by using a label-free quantitative LC-MS/MS approach. We analyzed 94 serum samples from adult Wistar rats with different degrees of liver fibrosis and 36 control rats. Firstly, serum samples from 18 CCl4-treated rats were clustered into three different groups according to the severity of hepatic and the serum proteome was characterized by label-free LC-MS/MS. Furthermore, three different pooled serum samples obtained from 16 control Wistar rats were also analyzed. Based on the proteomic data obtained, we performed a multivariate analysis which displayed three main cell signaling pathways altered in fibrosis. In cirrhosis, more biological imbalances were detected as well as multi-organ alterations. In addition, hemopexin and signal-induced proliferation-associated 1 like 1 (SIPA1L1) were selected as potential serum markers of liver fibrogenesis among all the analyzed proteins. The results were validated by ELISA in an independent group of 76 fibrotic/cirrhotic rats and 20 controls which confirmed SIPA1L1 as a potential non-invasive biomarker of liver fibrosis. In particular, SIPA1L1 showed a clear diminution in serum samples from fibrotic/cirrhotic rats and a great accuracy at identifying early fibrotic stages. In conclusion, the proteomic analysis of serum samples from CCl4-treated rats has enabled the identification of SIPA1L1 as a non-invasive marker of early liver fibrosis.

0294 : Glucuronide metabolites of hydroxytyrosol are responsible for the antioxidative properties of olive oil

Hydroxytyrosol (HT) is known to be a strong polyphenolic antioxidant of olive oil. However, its in vivo effects are controversial since intestinal absorption is low and it is highly metabolized into glucuronides (GC), suggesting that these metabolites could be responsible of HT biological activity in vivo . Our objectives were to study the direct effects of HT and its glucuronides on vascular function and the oxidative stress vascular protection on healthy and diabetic rats The vascular function was assessed on aortas from diabetic-High Fat Sucrose diet (HFS) and age-matched (22 weeks) control fed with a standard diet (SD) Wistar rats, in comparison to young (9 weeks) control Wistar rats (ySD). The endothelium-dependent and independent vasorelaxing effects of these compounds were assessed via incremental doses of HT and GC (10 -9 to 10 -4 M) and compared to Resveratrol (Res). Expression and phosphorylation of Ser 1177 eNOS were measured by Western blot. To evaluate the anti-oxidative effects of these polyphenols (100 μM, 5min), vascular function was assessed in presence/absence of t -BOOH (500μM, 30min). HT and GC did not show any endothelium-dependent and independent vasorelaxation effects in ySD and HFS rats while Res produced vasorelaxing effect at 10 -4 M in all groups of rats and no vasorelaxant effects were noticed in SD rats. The overall absence of vasorelaxing effect can be explained by the lack eNOS phosphorylation. As for the antioxidative properties, HT, GC and Res increased significantly endothelium-dependent relaxation in comparison of aorta rings treated only with t -BOOH (79.9%±9.2, 74.1%±8.6, 71.0%±8.2, 48.4%±5.3 respectively). In addition, co-incubation of HT/GC and Res/GC increased even more the vasorelaxation under pro-oxydant conditions. In diabetic HFS rats, (i) the incubation for 5min with HT and GC did not improve neither vasorelaxation nor the contraction; (ii) however, a prolonged 30 min incubation resulted in more pronounced and significant contractile (64.8%±12.4 and 95.6%±16.3, respectively) and endothelium-dependent relaxation effects (30%±7.5 and 58.1%±10.3 respectively) for GC in comparison to HT. In conclusion, GC seems to be responsible for the anti-oxidative properties of olive oil but further experimentations have to be realized concerning its possible interconversion in HT by β-glucuronidase. The author hereby declares no conflict of interest

Cathepsin Localization and Activity on the Intestinal Epithelial Barrier of Rats following Hemorrhagic Shock and Reperfusion

Damage to the intestinal epithelial barrier during hemorrhagic shock results in tissue destruction and increased permeability. Although the downstream effects of damage to the intestinal epithelial barrier have been investigated, the mechanism behind the increase in permeability has not been elucidated. Previous studies have suggested a proteolytic source for the degradation, but the spectrum of proteolytic enzymes in the intestinal tissue is very broad. We employed mass‐spectroscopy based proteomic analysis of a jejunal intestinal sample obtained from a young adult male Wistar rat (flushed with PBS to remove luminal contents) and found that the enzymes with the greatest relative abundance among the proteolytic enzymes were cathepsin–B, –C, and –Z. We examined cathepsin localization profiles using rat tissue from normal control (NC) Wistar rats and rats that underwent ischemia (at a blood pressure of 35 mmHg) followed by 2 hours of reperfusion (IR). Immuno‐histochemistry (IHC) was used to determine the cathepsin localization profiles. In the case of NC, comparison of cathepsin–B, –C, and –Z localization profiles of the intestinal epithelial barrier between rats (in our study) and humans (Swedish Human Protein Atlas) revealed a significant degree of similarity. Western Blots and gel zymography were conducted to determine changes in cathepsin concentration and activity respectively. Western blots showed a decrease in cathepsin levels, while gel zymography showed no changes in cathepsin activity between NC and IR groups. However, cathepsin activity per mole of total cathepsin (expressed by the ratio between the gel zymography and western blot bands) was higher in the case of IR, supporting the potential role cathepsins play in causing damage to intestinal tissue during hemorrhagic shock, leading to increased intestinal permeability.Support or Funding InformationSupported by HL 10881.

Prohedonic Effect of Cannabidiol in a Rat Model of Depression

Background: Accumulating evidence suggests that cannabidiol (CBD) may be an effective and safe anxiolytic agent and potentially also an antidepressant. Aim: The objective of this study was to further examine these properties of CBD using the ‘depressive-like' Wistar-Kyoto (WKY) rat, focusing on the drug's effect on anhedonia-like behaviors. Methods: Forty-eight WKY and 48 control Wistar adult male rats were pretreated orally with CBD (15, 30 and 45 mg/kg) or vehicle. The saccharin preference test (SPT), the elevated plus maze (EPM) test and the novel object exploration (NOE) test were used. Results: CBD showed a prohedonic effect on the WKY rats at 30 mg/kg in the SPT. In the NOE, CBD increased exploration of the novel object and locomotion at 45 mg/kg and increased locomotion at 15 mg/kg, indicating an improvement in the characteristically low motivation of WKY rats to explore. There was no similar effect at any dose in the EPM or in open-field behavior in the habituation to the NOE. Conclusions: These findings extend the limited knowledge on the antidepressant effect of CBD, now shown for the first time in a genetic animal model of depression. These results suggest that CBD may be beneficial for the treatment of clinical depression and other states with prominent anhedonia.

Compensatory renal hypertrophy and the handling of an acute nephrotoxicant in a model of aging

Aging often results in progressive losses of functioning nephrons, which can lead to a significant reduction in overall renal function. Because of age-related pathological changes, the remaining functional nephrons within aged kidneys may be unable to fully counteract physiological and/or toxicological challenges. We hypothesized that when the total functional renal mass of aged rats is reduced by 50%, the nephrons within the remnant kidney do not fully undergo the functional and physiological changes that are necessary to maintain normal fluid and solute homeostasis. We also tested the hypothesis that the disposition and handling of a nephrotoxicant are altered significantly in aged kidneys following an acute, 50% reduction in functional renal mass. To test these hypotheses, we examined molecular indices of renal cellular hypertrophy and the disposition of inorganic mercury (Hg2+), a model nephrotoxicant, in young control, young uninephrectomized (NPX), aged control and aged NPX Wistar rats. We found that the process of aging reduces the ability of the remnant kidney to undergo compensatory renal growth. In addition, we found that an additional reduction in renal mass in aged animals alters the disposition of Hg2+ and potentially alters the risk of renal intoxication by this nephrotoxicant. To our knowledge, this study represents the first report of the handling of a nephrotoxicant in an aged animal following a 50% reduction in functional renal mass.