Norman E. Taylor

Renal Medullary MicroRNAs in Dahl Salt-Sensitive Rats. miR-29b Regulates Several Collagens and Related Genes

MicroRNAs are endogenous repressors of gene expression. We examined microRNAs in the renal medulla of Dahl salt-sensitive rats and consomic SS-13BN rats. Salt-induced hypertension and renal injury in Dahl salt-sensitive rats, particularly medullary interstitial fibrosis, have been shown previously to be substantially attenuated in SS-13BN rats. Of 377 microRNAs examined, 5 were found to be differentially expressed between Dahl salt-sensitive rats and consomic SS-13BN rats receiving a high-salt diet. Real-time PCR analysis demonstrated that high-salt diets induced substantial upregulation of miR-29b in the renal medulla of SS-13BN rats but not in SS rats. miR-29b was predicted to regulate 20 collagen genes, matrix metalloproteinase 2 (Mmp2), integrin &bgr;1 (Itgb1), and other genes related to the extracellular matrix. Expression of 9 collagen genes and Mmp2 was upregulated by a high-salt diet in the renal medulla of SS rats, but not in SS-13BN rats, an expression pattern opposite to miR-29b. Knockdown of miR-29b in the kidneys of SS-13BN rats resulted in upregulation of several collagen genes. miR-29b reduced expression levels of several collagen genes and Itgb1 in cultured rat renal medullary epithelial cells. Moreover, miR-29b suppressed the activity of luciferase when the reporter gene was linked to a 3′-untranslated segment of collagen genes Col1a1, Col3a1, Col4a1, Col5a1, Col5a2, Col5a3, Col7a1, Col8a1, Mmp2, or Itgb1 but not Col12a1. The result demonstrated broad effects of miR-29b on a large number of collagens and genes related to the extracellular matrix and suggested involvement of miR-29b in the protection from renal medullary injury in SS-13BN rats.

NADPH Oxidase in the Renal Medulla Causes Oxidative Stress and Contributes to Salt-Sensitive Hypertension in Dahl S Rats

Dahl salt-sensitive (SS) rats exhibit increased renal medullary oxidative stress and blood pressure salt-sensitivity compared with consomic, salt-resistant SS-13BN rats, despite highly similar genetic backgrounds. The present study examined potential sources of renal medullary superoxide in prehypertensive SS rats fed a 0.4% NaCl diet by assessing activity and protein levels of superoxide producing and scavenging enzymes. Superoxide production was nearly doubled in SS rats compared with SS-13BN rats as determined by urinary 8-isoprostane excretion and renal medullary oxy-ethidium microdialysate levels. Medullary superoxide production in tissue homogenates was greater in SS rats, and the NADPH oxidase inhibitor diphenylene iodonium preferentially reduced SS levels to those found in SS-13BN rats. Dinitrophenol, a mitochondrial uncoupler, eliminated the remaining superoxide production in both strains, whereas inhibition of xanthine oxidase, NO synthase, and cycloxygenase had no effect. l-arginine, NO synthase, superoxide dismutase, catalase, and glutathione peroxidase activities between SS and SS-13BN rats did not differ. Chronic blood pressure responses to a 4% NaCl diet were then determined in the presence or absence of the NADPH oxidase inhibitor apocynin (3.5 &mgr;g/kg per minute), chronically delivered directly into the renal medulla. Apocynin infusion reduced renal medullary interstitial superoxide from 1059±130 to 422±80 (oxyethidium fluorescence units) and mean arterial pressure from 175±4 to 157±6 mm Hg in SS rats, whereas no effects on either were observed in the SS-13BN. We conclude that excess renal medullary superoxide production in SS rats contributes to salt-induced hypertension, and NADPH oxidase is the major source of the excess superoxide.

Activation of D1 dopamine receptors induces emergence from isoflurane general anesthesia.

Background:A recent study showed that methylphenidate induces emergence from isoflurane anesthesia. Methylphenidate inhibits dopamine and norepinephrine reuptake transporters. The objective of this study was to test the hypothesis that selective dopamine receptor activation induces emergence from isoflurane anesthesia. Methods:In adult rats, we tested the effects of chloro-APB (D1 agonist) and quinpirole (D2 agonist) on time to emergence from isoflurane general anesthesia. We then performed a dose–response study to test for chloro-APB–induced restoration of righting during continuous isoflurane anesthesia. SCH-23390 (D1 antagonist) was used to confirm that the effects induced by chloro-APB are specifically mediated by D1 receptors. In a separate group of animals, spectral analysis was performed on surface electroencephalogram recordings to assess neurophysiologic changes induced by chloro-APB and quinpirole during isoflurane general anesthesia. Results:Chloro-APB decreased median time to emergence from 330 to 50 s. The median difference in time to emergence between the saline control group (n = 6) and the chloro-APB group (n = 6) was 222 s (95% CI: 77–534 s, Mann–Whitney test). This difference was statistically significant (P = 0.0082). During continuous isoflurane anesthesia, chloro-APB dose-dependently restored righting (n = 6) and decreased electroencephalogram &dgr; power (n = 4). These effects were inhibited by pretreatment with SCH-23390. Quinpirole did not restore righting (n = 6) and had no significant effect on the electroencephalogram (n = 4) during continuous isoflurane anesthesia. Conclusions:Activation of D1 receptors by chloro-APB decreases time to emergence from isoflurane anesthesia and produces behavioral and neurophysiologic evidence of arousal during continuous isoflurane anesthesia. These findings suggest that selective activation of a D1 receptor–mediated arousal mechanism is sufficient to induce emergence from isoflurane general anesthesia.

The neuroinflammatory component of gray matter pathology in multiple sclerosis

In multiple sclerosis (MS), using simultaneous magnetic resonance–positron emission tomography (MR‐PET) imaging with 11C‐PBR28, we quantified expression of the 18kDa translocator protein (TSPO), a marker of activated microglia/macrophages, in cortex, cortical lesions, deep gray matter (GM), white matter (WM) lesions, and normal‐appearing WM (NAWM) to investigate the in vivo pathological and clinical relevance of neuroinflammation.

Electrical Stimulation of the Ventral Tegmental Area Induces Reanimation from General Anesthesia

Background:Methylphenidate or a D1 dopamine receptor agonist induces reanimation (active emergence) from general anesthesia. The authors tested whether electrical stimulation of dopaminergic nuclei also induces reanimation from general anesthesia. Methods:In adult rats, a bipolar insulated stainless steel electrode was placed in the ventral tegmental area (VTA, n = 5) or substantia nigra (n = 5). After a minimum 7-day recovery period, the isoflurane dose sufficient to maintain loss of righting was established. Electrical stimulation was initiated and increased in intensity every 3 min to a maximum of 120 µA. If stimulation restored the righting reflex, an additional experiment was performed at least 3 days later during continuous propofol anesthesia. Histological analysis was conducted to identify the location of the electrode tip. In separate experiments, stimulation was performed in the prone position during general anesthesia with isoflurane or propofol, and the electroencephalogram was recorded. Results:To maintain loss of righting, the dose of isoflurane was 0.9% ± 0.1 vol%, and the target plasma dose of propofol was 4.4 ± 1.1 µg/ml (mean ± SD). In all rats with VTA electrodes, electrical stimulation induced a graded arousal response including righting that increased with current intensity. VTA stimulation induced a shift in electroencephalogram peak power from &dgr; (<4 Hz) to &thgr; (4–8 Hz). In all rats with substantia nigra electrodes, stimulation did not elicit an arousal response or significant electroencephalogram changes. Conclusions:Electrical stimulation of the VTA, but not the substantia nigra, induces reanimation during general anesthesia with isoflurane or propofol. These results are consistent with the hypothesis that dopamine release by VTA neurons, but not substantia nigra neurons, induces reanimation from general anesthesia.

NO Synthase Uncoupling in the Kidney of Dahl S Rats Role of Dihydrobiopterin

NO synthase (NOS) can paradoxically contribute to the production of reactive oxygen species when l-arginine or the cofactor R-tetrahydrobiopterin (BH4) becomes limited. The present study examined whether NOS contributes to superoxide production in kidneys of hypertensive Dahl salt-sensitive (SS) rats compared with an inbred consomic control strain (SS-13BN) and tested the hypothesis that elevated dihydrobiopterin (BH2) levels are importantly involved in this process. This was assessed by determining the effects of l-nitroarginine methyl ester (l-NAME) inhibition of NOS on superoxide production and by comparing tissue concentrations of BH4 and BH2. A reverse-phase high-performance liquid chromatography method was applied for direct measurements of BH4 and BH2 using (S)-tetrahydrobiopterin as an internal standard. Superoxide concentrations were measured in vivo from medullary microdialysis fluid using dihydroethidine and in vitro using lucigenin. The results indicate the following: (1) that superoxide levels were elevated in the outer medulla of SS rats fed a 4% salt diet and could be inhibited by l-NAME. In contrast, l-NAME resulted in elevated superoxide production in consomic SS-13BN rats because of higher NOS activity; (2) SS rats showed a reduced ratio of BH4/BH2 in the outer medulla that was driven by increased concentrations of BH2; and (3) lower superoxide dismutase and catalase activities contributed to elevated reactive oxygen species in SS samples. Based on the shift of BH4 to BH2 and the observation of l-NAME inhibitable superoxide production, we conclude that NOS uncoupling occurs in the renal medulla of hypertensive SS rats fed a high-salt diet.

Optogenetic activation of dopamine neurons in the ventral tegmental area induces reanimation from general anesthesia

Significance Although dopamine is known to promote wakefulness, the specific dopamine circuits in the brain that regulate arousal are not clear. Here we report that selective optogenetic stimulation of ventral tegmental area (VTA) dopamine neurons in mice produces a powerful arousal response sufficient to restore conscious behaviors, including the righting reflex, during continuous, steady-state general anesthesia. Although previous studies found that VTA dopamine neurons do not appear to play a central role in regulating sleep–wake transitions, our findings demonstrate that selective stimulation of these neurons is sufficient to induce the transition from an unconscious, anesthetized state to an awake state. These results suggest that VTA DA neurons play a critical role in promoting wakefulness. Dopamine (DA) promotes wakefulness, and DA transporter inhibitors such as dextroamphetamine and methylphenidate are effective for increasing arousal and inducing reanimation, or active emergence from general anesthesia. DA neurons in the ventral tegmental area (VTA) are involved in reward processing, motivation, emotion, reinforcement, and cognition, but their role in regulating wakefulness is less clear. The current study was performed to test the hypothesis that selective optogenetic activation of VTA DA neurons is sufficient to induce arousal from an unconscious, anesthetized state. Floxed-inverse (FLEX)-Channelrhodopsin2 (ChR2) expression was targeted to VTA DA neurons in DA transporter (DAT)-cre mice (ChR2+ group; n = 6). Optical VTA stimulation in ChR2+ mice during continuous, steady-state general anesthesia (CSSGA) with isoflurane produced behavioral and EEG evidence of arousal and restored the righting reflex in 6/6 mice. Pretreatment with the D1 receptor antagonist SCH-23390 before optical VTA stimulation inhibited the arousal responses and restoration of righting in 6/6 ChR2+ mice. In control DAT-cre mice, the VTA was targeted with a viral vector lacking the ChR2 gene (ChR2− group; n = 5). VTA optical stimulation in ChR2− mice did not restore righting or produce EEG changes during isoflurane CSSGA in 5/5 mice. These results provide compelling evidence that selective stimulation of VTA DA neurons is sufficient to induce the transition from an anesthetized, unconscious state to an awake state, suggesting critical involvement in behavioral arousal.

Rapid and Sustained Reductions in Current Suicidal Ideation Following Repeated Doses of Intravenous Ketamine: Secondary Analysis of an Open-Label Study.

BACKGROUND Ketamine rapidly reduces thoughts of suicide in patients with treatment-resistant depression who are at low risk for suicide. However, the extent to which ketamine reduces thoughts of suicide in depressed patients with current suicidal ideation remains unknown. METHODS Between April 2012 and October 2013, 14 outpatients with DSM-IV-diagnosed major depressive disorder were recruited for the presence of current, stable (≥ 3 months) suicidal thoughts. They received open-label ketamine infusions over 3 weeks (0.5 mg/kg over 45 minutes for the first 3 infusions; 0.75 mg/kg over 45 minutes for the last 3). In this secondary analysis, the primary outcome measures of suicidal ideation (Columbia-Suicide Severity Rating Scale [C-SSRS] and the Suicide Item of the 28-item Hamilton Depression Rating Scale [HDRS₂₈-SI]) were assessed at 240 minutes postinfusion and for 3 months thereafter in a naturalistic follow-up. RESULTS Over the course of the infusions (acute treatment phase), 7 of 14 patients (50%) showed remission of suicidal ideation on the C-SSRS Ideation scale (even among patients whose depression did not remit). There was a significant linear decrease in this score over time (P < .001), which approached significance even after controlling for severity of 6-item Hamilton Depression Rating Scale (HDRS₆) core depression items (P = .05). Similarly, there were significant decreases in the C-SSRS Intensity (P < .01) and HDRS₂₈-SI (P < .001) scores during the acute treatment phase. Two of the 7 patients who achieved remission during the acute treatment phase (29%) maintained their remission throughout a 3-month naturalistic follow-up. CONCLUSIONS In this preliminary study, repeated doses of open-label ketamine rapidly and robustly decreased suicidal ideation in pharmacologically treated outpatients with treatment-resistant depression with stable suicidal thoughts; this decrease was maintained for at least 3 months following the final ketamine infusion in 2 patients. TRIAL REGISTRATION ClinicalTrials.gov identifier: NCT01582945.

Ketamine augmentation for outpatients with treatment-resistant depression: Preliminary evidence for two-step intravenous dose escalation

Objective: Preliminary evidence supports the safety and efficacy of subanesthetic ketamine as an experimental antidepressant, although its effects are often not sustained beyond one week. Studies are lacking that have examined the sustained effects of escalating ketamine doses as augmentation in outpatients with treatment-resistant depression. Therefore, the aims of this study were twofold: (1) to assess the safety and antidepressant efficacy of two-step, repeated-dose ketamine augmentation and (2) to assess the duration of ketamine’s antidepressant efficacy as augmentation to ongoing antidepressant pharmacotherapy for 3 months after the final infusion. Methods: Fourteen patients with treatment-resistant depression were eligible to receive augmentation with six open-label intravenous ketamine infusions over 3 weeks. For the first three infusions, ketamine was administered at a dose of 0.5 mg/kg over 45 minutes; the dose was increased to 0.75 mg/kg over 45 minutes for the subsequent three infusions. The primary outcome measure was response (as measured on Hamilton Depression Rating Scale–28 items). Results: After the completion of three ketamine infusions, 7.1% (1/14) responded; after all six ketamine infusions, 41.7% (5/12) completers responded and 16.7% (2/12) remitted. Intent-to-treat response and remission rates at the end of the final infusion were 35.7% (5/14) and 14.3% (2/14), respectively. However, all but one responder relapsed within 2 weeks after the final infusion. Conclusion: Repeated, escalating doses of intravenous ketamine augmentation were preliminarily found to be feasible, efficacious and well tolerated. Interaction with concomitant medications and elevated level of treatment resistance are possible factors for non-response.

Ageing delays emergence from general anaesthesia in rats by increasing anaesthetic sensitivity in the brain

BACKGROUND Little is known about ageing-related changes in the brain that affect emergence from general anaesthesia. We used young adult and aged Fischer 344 rats to test the hypothesis that ageing delays emergence from general anaesthesia by increasing anaesthetic sensitivity in the brain. METHODS Time to emergence was determined for isoflurane (1.5 vol% for 45 min) and propofol (8 mg kg(-1) i.v.). The dose of isoflurane required to maintain loss of righting (LOR) was established in young adult and aged rats. The efficacy of methylphenidate to reverse LOR from general anaesthesia was tested. Separate young adult and aged rats with implanted electroencephalogram (EEG) electrodes were used to test whether ageing increases sensitivity to anaesthetic-induced burst suppression. RESULTS Mean time to emergence from isoflurane anaesthesia was 47 s [95% CI 33, 60; young adult) compared with 243 s (95% CI 185, 308; aged). For propofol, mean time to emergence was 13.1 min (95% CI 11.9, 14.0; young adult) compared with 23.1 min (95% CI 18.8, 27.9; aged). These differences were statistically significant. When methylphenidate was administered after propofol, the mean time to emergence decreased to 6.6 min (95% CI 5.9, 7.1; young adult) and 10.2 min (95% CI 7.9, 12.3; aged). These reductions were statistically significant. Methylphenidate restored righting in all rats during continuous isoflurane anaesthesia. Aged rats had lower EEG power and were more sensitive to anaesthetic-induced burst suppression. CONCLUSIONS Ageing delays emergence from general anaesthesia. This is due, at least in part, to increased anaesthetic sensitivity in the brain. Further studies are warranted to establish the underlying causes.