Mechanical reperfusion is associated with post-ischemic hemorrhage in rat brain

A major complication of recanalization therapy after an acute arterial occlusion in brain is hemorrhagic transformation (HT). Although it is known that prolonged ischemia is important in the development of HT, the role of reperfusion in ischemia–reperfusion induced HT is less well studied. To address the effect of reperfusion on HT, we assessed the incidence and severity of hemorrhage in rats after 5 h of middle cerebral artery occlusion (MCAO) followed by 19-hour reperfusion compared to rats with permanent occlusion (PMCAO) at the same 24-hour time point. The incidence and amount of hemorrhage, neurological function, and mortality rates were measured. MCAO (5 h) with 19-hour reperfusion was associated with a significantly higher incidence of cortical hemorrhage compared to PMCAO (81.8% vs 18.2%, p < 0.05). Hemorrhage scores were higher in the 5-hour MCAO/reperfusion group compared to PMCAO rats (17.6 ± 11.5 vs 2.4 ± 5.3 in cortex, 20.4 ± 4.6 vs 9.7 ± 4.5 in striatum, p < 0.01). Neurological function was worse in the ischemia–reperfusion group compared to PMCAO (p < 0.05) and mortality rates were insignificantly higher in the 5-hour MCAO/reperfusion group vs PMCAO group (54.5% vs18.1%; p < 0.08). The results suggest that reperfusion after prolonged ischemia is associated with increased hemorrhagic transformation and neurological deterioration as compared to permanent ischemia. Whether pharmacological treatments prior to reperfusion attenuate post-ischemic HT requires further study.     

Effects of short-term training on heart rate dynamics in individuals with spinal cord injury

Increased risk of cardiovascular diseases and autonomic dysregulation are common health concerns in individuals with spinal cord injury (SCI). Two therapies that may help improve cardiovascular control are body-weight supported treadmill training (BWSTT) and head-up tilt training (HUTT). The purpose of this study was to examine the effects of short-term BWSTT and HUTT on cardiac autonomic function. Seven participants (6 male, 37.1 ± 7.7 years) with SCI (C5-T10, ASIA A-C; 5.0 ± 4.4 years post-injury) completed the study protocol. In this randomized cross-over design, participants were required to complete 4 weeks of thrice-weekly BWSTT and HUTT (i.e. 12 sessions each), separated by a 4 week detraining period. Cardiac autonomic function was assessed at rest, before and after, each 4 week training period using linear and non-linear measures (sample entropy and detrended fluctuation analysis (α₁)) of heart rate dynamics. Participants completed equivalent amounts of time performing BWSTT and HUTT (453.7 ± 27.3 min vs. 471.6 ± 19.7 min, p = 0.24). There were no significant differences in linear heart rate variability following BWSTT or HUTT (p > 0.05). In contrast, there was a significant change in sample entropy following BWSTT (1.05 ± 0.14 to 1.42 ± 0.12, p < 0.05). Due to the bi-directional pattern of α₁ values, distance scores were calculated (│1 − α₁│) and demonstrated a significant reduction following BWSTT (0.54 ± 0.06 to 0.26 ± 0.05, p = 0.001). In conclusion, 4 weeks of BWSTT but not HUTT training are sufficient to increase sample entropy and reduce the fractal scaling distance score in participants with SCI.     

Post-ischemic leakiness of the blood–brain barrier: A quantitative and systematic assessment by Patlak plots

The Patlak plot analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows estimation of blood–brain barrier (BBB) leakage following temporary focal cerebral ischemia. Thus far, a systematic and quantitative in vivo evaluation of post-ischemic BBB leakage is lacking. Here, using DCE-MRI and the Patlak plot method, we quantitatively assessed BBB leakage in rats at the following time-points after reperfusion: 25 min, 2, 4, 6, 12, 18, 24, 36, 48, and 72 h, and 1, 2, 3, 4, and 5 weeks. Sham-operated animals served as controls. Data collected for each time-point were: the blood-to-brain transfer rate constant (K_i) of the contrast agent gadolinium, distribution volume (V_p), ischemic lesion volume, and apparent diffusion coefficient (ADC) values. Compared to controls, K_i, measured at all time-points, except for 5 weeks, appeared significantly different (p < 0.001). At several time-points (25 min, 48 and 72 h, 4 and 5 weeks), V_p was similar compared to that of controls, but for the remaining groups the difference was significant (p < 0.001). Analyzing the relationship of K_i values to time-points, we observed a trend towards a decrease over time (r = − 0.61, p = 0.014). Both ADC values (r = − 0.58, p = 0.02) and ischemic lesion volumes (r = 0.75, p = 0.0015) correlated with K_i values. These results suggest that after ischemia–reperfusion in rats, BBB leakage is continuous during a 4-week period. Its magnitude diminishes over time and correlates with severity and extent of ischemic injury.     

Evidence for altered post-receptor modulation of the serotonin 2a receptor in schizophrenia

We have shown a decrease in cortical serotonin_2A receptors using tissue sections, but not with washed membranes, from the same cohort of subjects. These discrepant findings led us to determine if we could obtain similar results using samples from the same tissue block. Our studies used single-point saturation analyses to estimate the total number of [³H]ketanserin binding sites in tissue sections, crude homogenate, membrane-enriched and cytosol-enriched tissue samples from Brodmann's area 9. There were significant decreases in the levels of [³H]ketanserin binding using tissue sections (mean ± SD: 38 ± 16 vs. 56 ± 16 fmol/mg ETE; p = 0.008) and crude tissue homogenates (131 ± 53 vs. 168 ± 38 fmol/mg protein; p < 0.05) from subjects with schizophrenia compared to that in controls. By contrast, there was no significant difference in radioligand binding to the membrane-enriched (155 ± 95 vs. 145 ± 48 fmol/mg protein; p = 0.72) or cytosol-enriched (8.6 ± 14 vs. 7.5 ± 10 mol/mg protein; p = 0.85) tissue fraction. Significantly, adding 10^{− 5} M risperidone or chlorpromazine, as surrogates for residual antipsychotic drugs in the CNS, to crude homogenate from control subjects did not alter [³H]ketanserin binding. Our data therefore is consistent with the hypothesis that apparent decreases in serotonin_2A receptors in schizophrenia are due to altered levels of a regulatory factor(s) that modulates the binding of ligands to the serotonin_2A receptor and that separating the membrane and cytosol removes this regulatory control.     

Communicating hydrocephalus in adult rats with kaolin obstruction of the basal cisterns or the cortical subarachnoid space

Communicating hydrocephalus (CH) occurs frequently, but clinically-relevant animal models amenable to diagnostic imaging and cerebrospinal fluid shunting are not available. In order to develop and characterize models of subarachnoid space (SAS) obstruction at the basal cisterns (BC) or cerebral convexities (CX), 25% kaolin was injected in adult female Sprague–Dawley rats following halothane anesthesia; intact- or saline-injected animals served as controls. For BC animals (n = 28 hydrocephalics, n = 20 controls), an anterior approach to the C1-clivus interval was employed and 30 μl of kaolin or saline was injected. For CX injections (n = 13 hydrocephalics, n = 3 controls), 50–60 μl of kaolin was injected bilaterally after separating the partitions in the SAS. In BC-injected rats, kaolin was observed grossly in the basal cisterns but not in the cisterna magna or at the foramina of Luschka, indicating that communicating (or extra-ventricular) – not obstructive – hydrocephalus had been induced. Following ketamine/xylazine anesthesia, magnetic resonance imaging (MRI) of gadolinium injected into the lateral ventricle also demonstrated CSF flow from the foramina of Luschka. MRI also revealed that ventriculomegaly progressed steadily in BC animals and by 2 weeks post-kaolin the mean Evan's ratio (frontal horn) increased significantly (mean 0.45 compared to 0.31 in intact- and 0.34 in saline-injected controls; p < 0.001 for each). CX animals exhibited kaolin deposits covering approximately 80% of the cerebral hemispheres and developed noticeable ventriculomegaly (mean Evan's ratio 0.40), which was significant relative to intact animals (p = 0.011) but not saline-injected controls. Surprisingly, ventriculomegaly following CX injections was less severe and much more protracted, requiring 3–4 months to develop compared to ventriculomegaly produced by BC obstruction. No hydrocephalic animals demonstrated obvious neurological deficits, but BC-injected animals that subsequently developed more severe ventriculomegaly exhibited nasal discharges and “coughing” for several days following kaolin injection. The new BC model is relevant because the clinical presentation of CH in children is often associated with obstruction at this site, while the CX model may be more representative of late adult onset normal pressure hydrocephalus.     

Direct quantification of CSF alpha-synuclein by ELISA and first cross-sectional study in patients with neurodegeneration

Because accumulation of α-synuclein (αS) in the brain is a hallmark of Parkinson disease (PD) and related disorders, we examined its occurrence in human cerebrospinal fluid (CSF). Following affinity enrichment and trypsin digestion of CSF collected from a neurologically healthy donor, we identified several αS-derived peptides by mass spectrometry. The concentration of αS amounted to < 0.001% of the CSF proteome. We then built, validated and optimized a sandwich-type, enzyme-linked immunoadsorbent assay (ELISA) to measure total αS levels in unconcentrated CSF. In a cross-sectional study of 100 living donors, we examined cell-free CSF samples from subjects clinically diagnosed with advanced PD, dementia with Lewy bodies (DLB), Alzheimer disease (AD), and a group of non-neurodegenerative disease controls (NCO). In these four groups the CSF αS concentrations ranged from 0.8 to 16.2 pg/μl. Mean CSF αS values were lower in donors with a primary synucleinopathy (PD, DLB: n = 57) than in the other two groups (AD, NCO: n = 35; p = 0.025). By contrast, living Creutzfeldt–Jakob disease patients showed markedly elevated CSF αS levels (n = 8; mean, 300 pg/μl; p < 0.001). Our results unequivocally confirm the presence of αS in adult human CSF. In a first feasibility study employing a novel ELISA, we found relatively low CSF αS concentrations in subjects with parkinsonism linked to synucleinopathy, PD and DLB. In definite prion disease cases, we recorded a marked rise in total CSF αS resulting from rapid cell death. Our results will likely aid future biomarker explorations in neurodegenerative conditions and facilitate target validation studies.     

Altered dopaminergic profiles: Implications for the regulation of voluntary physical activity

The biological regulating factors of physical activity in animals are not well understood. This study investigated differences in the central mRNA expression of seven dopamine genes (Drd1, Drd2, Drd3, Drd4, Drd5, TH, and DAT) between high active C57/LJ (n = 17) male mice and low active C3H/HeJ (n = 20) male mice, and between mice with access to a running wheel and without running wheel access within strain. Mice were housed with running wheels interfaced with a computer for 21 days with distance and duration recorded every 24 h. On day 21, the striatum and nucleus accumbens were removed during the active period (9 pm) for dopaminergic analysis. On average, the C57L/J mice with wheels ran significantly farther (10.25 ± 1.37 km/day vs. 0.01 ± 0.09 km/day, p < 0.001), longer (329.73 ± 30.52 min/day vs. 7.81 ± 6.32 min/day, p < 0.001), and faster (31.27 ± 3.13 m/min vs. 11.81 ± 1.08 m/min, p < 0.001) than the C3H/HeJ mice with wheels over the 21 day period. No differences in gene expression were found between mice in either strain with wheels and those without wheels suggesting that access to running wheels did not alter dopaminergic expression. In contrast, relative expression for two dopamine genes was significantly lower in the C57L/J mice compared to the C3H/HeJ mice. These results indicate that decreased dopaminergic functioning is correlated with increased activity levels in C57L/J mice and suggests that D1-like receptors as well as tyrosine hydroxylase (an indicator of dopamine production), but not D2-like receptors may be associated with the regulation of physical activity in inbred mice.     

Pharmacokinetics of olanzapine in Chinese male schizophrenic patients with various smoking behaviors

Tobacco consumption has been recognized as a factor mediating the interindividual variations in olanzapine's pharmacokinetics and pharmacodynamics. The primary objective of this study was to describe the dose effect of smoking on the dose–plasma concentration relationship and the pharmacokinetics of oral olanzapine in male schizophrenic patients using high-performance liquid chromatography coupled with electrochemical detector. Twenty-seven male schizophrenic inpatients were recruited and were stratified into the following groups according to smoking behaviors: non-smokers (n = 9), light-smokers (1–4 cigarettes per day; n = 9), and heavy-smokers (≥ 5 cigarettes per day; n = 9). Plasma olanzapine concentrations were determined up to 120 h following a single oral dose of 10 mg olanzapine. The pharmacokinetic parameters were calculated by the non-compartment method using WinNonlin software. Results show that there was a significant correlation among non-smokers (n = 9; 0.79; p = 0.01) or combined with light-smokers (n = 18; 0.62; p < 0.01) between peak plasma olanzapine concentrations (C_max) and their individual dose-corrected by body weight, but this correlation did not appear in heavy-smokers. There were no significant differences between non-smokers and light-smokers except for significant decreased AUC_0→120 by 45.1% in light-smokers. The mean C_max and the mean area under the plasma concentration–time curve from time zero to 120 h (AUC_0→120) of the heavy-smoking patients was 9.3 ± 4.3 ng/ml (65.2% reduction compared to the non-smokers) and 302.4 ± 167.8 h ng/ml (67.6% reduction compared to the non-smokers), respectively. In summary, a daily consumption of 5 cigarettes is probably sufficient for induction of olanzapine metabolism. Smoking cessation is recommended for olanzapine therapy to have better prediction for therapeutic dosages particularly in heavy-smokers. Compared to non-smokers, heavy-smokers therefore require a 50–100% increase in olanzapine doses. Therapeutic drug monitoring will need to be considered when schizophrenic patients change their smoking behaviors.     

Focal nerve inflammation induces neuronal signs consistent with symptoms of early complex regional pain syndromes

Early forms of complex regional pain syndromes (CRPS) are characterized by severe pain and autonomic dysfunction in a limb, both of which seem out of proportion to the inciting event. While often caused by obvious nerve injury, the syndromes also occur following relatively trivial trauma. Persistent inflammation has been implicated in the etiology of CRPS. We hypothesized that inflammation of a nerve proximal to the symptoms could lead to neural changes consistent with clinical CRPS. Using a rat model of neuritis, the activity of sensory and autonomic neurons was recorded distal to the inflamed site using single fiber electrophysiological methods. In normal rats, no sensory neurons had ongoing activity. The discharge rate of sympathetic postganglionic neurons was 2.26 ± 1.33 Hz (mean ± SD). However, in rats with inflamed nerves, 27% of slowly conducting neurons had ongoing activity after 3–4 days, and 50% had such activity after 7–8 days. Other sensory neurons did not exhibit ongoing activity. Sympathetic postganglionic neurons had a significantly slower ongoing discharge rate during inflammation (1.96 ± 1.19 Hz after 3–4 days, 1.48 ± 1.23 Hz after 7–8 days). Additionally, none of the sympathetic axons in any group were mechanically sensitive. These findings support that focal nerve inflammation is sufficient to cause neuronal discharge changes that are consistent with clinical findings in early CRPS. Furthermore, the lack of axonal mechanical sensitivity in sympathetic axons rules out channels expressed in these neurons as possible mechano-electrical transducers.     

Thermoregulatory effects of centrally administered bombesin, bradykinin, and methionine-enkephalin

Bombesin (BO, 100 ng), bradykinin (BR, 10 μg), or methionine-enkephalin (EN, 10 μg) was administered intracerebroventricularly to adult male rats at an environmental temperature of 4°C, 22°C, or 35°C, and rectal (T_re) and tail-skin (T_sk) temperatures were monitored for 5 hours. At 4°C and 22°C BO-treated animals developed acute hypothermia (max ΔT_re = −3.25°Cand −2.71°C, respectively) which persisted for 2 hours (p<0.05). At 22°C and at 300 min post-injection, BO-treated animals became significantly (p<0.05) hyperthermic (ΔT_re = +1.28°C) when compared to controls. While BR had no effects at 22°C, EN-injected rats demonstrated a significant (p<0.05) hyperthermia from 180 min through 300 min (ΔT_re = +1.40°C). At 22°C both BO and, surprisingly, EN increased T_re (e.g. ΔT_re = +3.49°Cand +2.01°C at 60 min). At 35°C EN elicited hyperthermia which was significantly (p<0.05) increased from time 0 at all sampling times (mean ΔT_re = +1.85°C) and from control levels at 300 min (ΔT_re = +1.07°C, p<0.05). BO again caused a significant (p<0.05, BO vs control, 30 min) decrement (ΔT_re = −1.22°C followed by increments (p<0.05) from 120–300 min. We conclude that the hypothermic effect of BO is dependent upon environmental temperature, partially caused by vasodilation, and possibly biphasic in nature; EN treatment generally elicits hyperthermia under these conditions while BR produced no effects on thermoregulation.     

Single unit “pauser” characteristics of the globus pallidus pars externa distinguish primary dystonia from secondary dystonia and Parkinson's disease

The presence of high frequency discharge neurons with long periods of silence or “pauses” in the globus pallidus pars externa (GPe) is a unique identifying feature of this nucleus. Prior studies have demonstrated that pause characteristics reflect synaptic inputs into GPe. We hypothesized that GPe pause characteristics should distinguish movement disorders whose basal ganglia network abnormalities are different. We examined pause characteristics in 224 GPe units in patients with primary generalized dystonia, Parkinson's disease (PD), and secondary dystonia, undergoing single unit microelectrode recording for DBS placement in the awake state. Pauses in neuronal discharge were identified using the Poisson surprise method. Mean pause length in primary dystonia (606.8373.3) was higher than in PD (557.4366.6) (p < 0.05). Interpause interval (IPI) was lower in primary dystonia (2331.63874.1) than PD (3646.45894.5) (p < 0.01), and mean pause frequency was higher in primary dystonia (0.140.10) than PD (0.070.12) (p < 0.01). Comparison of pause characteristics in primary versus secondary generalized dystonia revealed a significantly longer mean pause length in primary (606.8373.3) than in secondary dystonia (495.6236.5) (p < 0.01). IPI was shorter in primary (2331.6 ± 3874.1) than in secondary dystonia (3484.5 ± 3981.6) (p < 0.01). The results show that pause characteristics recorded in the awake human GPe distinguish primary dystonia from Parkinson's disease and secondary dystonia. The differences may reflect increased phasic input from striatal D2 receptor positive cells in primary dystonia, and are consistent with a recent model proposing that GPe provides capacity scaling for cortical input.     

Diabetes induces neural degeneration in nucleus ambiguus (NA) and attenuates heart rate control in OVE26 mice

Baroreflex sensitivity is impaired by diabetes mellitus. Previously, we found that diabetes induces a deficit of central mediation of baroreflex-mediated bradycardia. In this study, we assessed whether diabetes induces degeneration of the nucleus ambiguus (NA) and reduces heart rate (HR) responses to l-Glutamate (L-Glu) microinjection into the NA. FVB control and OVE26 diabetic mice (5–6 months) were anesthetized. Different doses of L-Glu (0.1–5 mM/l, 20 nl) were delivered into the left NA using a multi-channel injector. In other animals, the left vagus was electrically stimulated at 1–40 Hz (1 ms, 0.5 mA, 20 s). HR and mean arterial blood pressure (MAP) responses to L-Glu microinjections into the NA and to the electrical stimulation of the vagus were measured. The NA region was defined by tracer TMR-D injection into the ipsilateral nodose ganglion to retrogradely label vagal motoneurons in the NA. Brainstem slices at − 600, − 300, 0, + 300, and + 600 μm relative to the obex were processed using Nissl staining and the number of NA motoneurons was counted. Compared with FVB control, we found in OVE26 mice that: 1) HR responses to L-Glu injection into the NA at doses of 0.2–0.4 (mM/l, 20 nl) were attenuated (p  < 0.05), but MAP responses were unchanged (p  > 0.05). 2) HR responses to vagal stimulation were increased (p  < 0.05). 3) The total number of NA (left and right) motoneurons was reduced (p  < 0.05). Taken together, we concluded that diabetes reduces NA control of HR and induces degeneration of NA motoneurons. Degeneration of NA cardiac motoneurons may contribute to impairment of reflex-bradycardia in OVE26 diabetic mice.     

Restraint stress suppresses experimental allergic encephalomyelitis in Lewis rats

We studied the effect of restraint stress on experimental allergic encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS), in Lewis rats. Rats were subjected to 12 h restraint stress for 3 consecutive nights from the first (day 1) or the eighth day (day 8) after sensitization with the antigen (guinea-pig spinal cord). All controlled rats exhibited clinical and histologie signs of EAE. The mean ± SD of incubation time, clinical score (0–4) and histologic score (0–3) for this group were 12.8 ± 1.0 days, 2.7 ± 0.6, and 2.3 ± 0.7, respectively. Restraint stress from day 8 significantly suppressed EAE: the mean ± SD of incubation time, clinical score, and histologic score for this group were 17.2 ± 2.2 days (p < 0.001), 1.8 ± 1.3 (p < 0.05), and 1.5 ± 0.9 (p < 0.02), respectively. Restraint stress from day 1 did not modify EAE. The findings suggest that stressful factors may exert an influence on the clinical course of MS.     

Drug/nutrition interaction in the developing brain: Dipyrone enhances spreading depression in rats

The abuse of pharmaceutical drugs and the inadequate ingestion of nutrients constitute external factors that can alter brain development, both individually and in combination. We used cortical spreading depression (CSD) as a neurophysiological parameter to investigate the combined effects of the antipyretic/analgesic/anti-inflammatory drug dipyrone and malnutrition (M) in the developing rat brain. Suckling malnourished rats (M; n = 69) and well nourished controls (W; n = 76) received dipyrone (300 mg/kg/day) or saline per gavage for 7 consecutive days during the 2nd, 3rd, or 4th postnatal week. At 35–45 days, CSD was recorded at 2 points in the parietal region. In both groups, dipyrone increased CSD propagation velocities compared to respective saline controls (P < 0.05). This effect was intensified when dipyrone application during the 4th postnatal week intensified the increase compared to the 2nd and 3rd weeks. In saline-treated groups, the velocities (mean ± s.d., in mm/min) were 3.70 ± 0.11, 3.77 ± 0.16, and 3.78 ± 0.13 (W) and 4.13 ± 0.10, 4.16 ± 0.10, and 4.14 ± 0.09 (M), for animals treated in the 2nd, 3rd and 4th postnatal weeks. In dipyrone-treated groups, the respective values were 3.99 ± 0.14, 4.03 ± 0.16, and 4.30 ± 0.19 (W) and 4.47 ± 0.17, 4.70 ± 0.31, and 5.01 ± 0.28 (M). Results support the hypothesis that dipyrone has a CSD-facilitating effect, which is more intense at a late brain developmental stage and is facilitated by malnutrition. This may help explain the developmental brain excitability changes that are associated with pharmacological and nutritional factors.     

Angiotensin II-induced hypertension differentially affects estrogen and progestin receptors in central autonomic regulatory areas of female rats

Estrogen receptor (ER) activation in central autonomic nuclei modulates arterial blood pressure (ABP) and counteracts the deleterious effect of hypertension. We tested the hypothesis that hypertension, in turn, influences the expression and trafficking of gonadal steroid receptors in central cardiovascular circuits. Thus, we examined whether ER- and progestin receptor (PR)-immunoreactivity (ir) are altered in medullary and hypothalamic autonomic areas of cycling rats following chronic infusion of the hypertensive agent, angiotensin II (AngII). After 1 week AngII-infusion, systolic ABP was elevated from 103 ± 4 to 172 ± 8 mmHg (p < 0.05; N = 8/group) and all rats were in diestrus (low estrogen). In AngII-infused rats the number of PR-immunoreactive nuclei was reduced (− 72%) compared to saline-infused controls also in diestrus (p < 0.05). Furthermore, the intensity of ERα-ir increased selectively in nuclei (16%) and cytoplasm (21%) of cells in the commissural nucleus of the solitary tract (cNTS; p < 0.05) while neither the number nor intensity of ERβ-labeled cells changed (p > 0.05). Following chronic AngII-infusion, electron microscopy showed a higher cytoplasmic-to-nuclear ratio of ERα-labeling selectively in tyrosine hydroxylase (TH)-labeled neurons in the cNTS. Furthermore, AngII-infusion increased ERα-ir in the cytosol of TH- and non-TH neuronal perikarya and increased the amount of ERα-ir associated with endoplasmic reticulum only in TH-containing perikarya. The data suggest that hypertension modulates the expression and subcellular distribution of ERα and PR in central autonomic regions involved in blood pressure control. Considering that ERα counteracts the central and peripheral effects of AngII, these receptor changes may underlie adaptive responses that protect females from the deleterious effects of hypertension.     

Cross-sensitivity between isoflurane and diazepam: evidence from a bidirectional tolerance study in mice

We examined in mice the effect of chronic diazepam treatment on the sensitivity to isoflurane, and that of repeated isoflurane exposure on the sensitivity to diazepam. Mice were divided into four groups: group 1, treated with diazepam, 10 mg/kg i.p. twice daily; group 2, vehicle-treated controls; group 3, exposed to 3% isoflurane for 25 min twice daily; and group 4, untreated controls. After 14 days the effect of the treatment was assessed. Twenty-four hours after the last 10 mg/kg diazepam treatment, groups 1 and 2 received diazepam, 5 mg/kg i.p., and were subjected to the horizontal wire test (HWT). All control mice but only 10% of the diazepam-treated mice failed the HWT. Groups 1 and 2 were then exposed to increasing concentrations of isoflurane. Diazepam-treated mice (group 1) lost the HWT at 0.7±0.7%, compared with 0.6±0.1% in controls (group 2) (P<0.001); the ED₅₀ was 0.75% vs. 0.65%. Group 1 mice lost the righting reflex at 0.94±0.07% isoflurane vs. 0.87±0.06% in group 2 (P<0.01); the ED₅₀ was 0.93% vs. 0.82%. Recovery time was 175±161 s in group 1 vs. 343±275 s in group 2 (P<0.02). Twenty-four hours after the last of the repeated exposures to isoflurane, we examined the responses of groups 3 and 4 to increasing concentrations of isoflurane. Mice in group 3 lost the righting reflex at 1.0±0.06% isoflurane vs. 0.9±0.04% in controls (group 4) (P<0.001); the ED₅₀ was 0.96% vs. 0.85%. Recovery time was 113±124 s vs. 208±126 s in groups 3 and 4 (P<0.09). Diazepam, 3 mg/kg i.p. administered to groups 3 and 4, caused loss of the HWT reflex in 33% of group 3 mice and in 82% of controls (group 4) (P<0.001). It appears that prolonged exposure to both diazepam and isoflurane caused reduced sensitivity to each drug separately, as well as to the other drug. This finding may strengthen the theory that inhalational anesthetics may act via the same mechanism as the benzodiazepines.     

Long-term effects of intracerebroventricular insulin microinjection on renal sodium handling and arterial blood pressure in rats

The role of the central nervous system (CNS) in the control of hydrosaline homeostasis has been strikingly demonstrated by several studies. Our laboratory recently showed that centrally administered insulin produced a dose-related increase in the urinary output of sodium, which was abolished by bilateral renal denervation, nitric oxide synthase inhibition and cerebroventricular streptozotocin administration in rats. Recent studies have shown that hyperinsulinemia induces subtle derangements of intracellular insulin–insulin receptor trafficking and insulin metabolism, which are associated with an impairment of insulin signaling. The long-term effect of high insulin levels on the periventricular region could alter insulin signaling, which in turn, may modify the central natriuretic and cardiovascular effects of this peptide. In order to evaluate this hypothesis, we investigated the effects of 7-day i.c.v. insulin administration on tubular handling and blood pressure in conscious, unrestrained rats and their controls, randomly assigned to one of two separate groups: (a) i.c.v. 0.15 M NaCl-injected (n = 7) and (b) i.c.v. 126.0 ng insulin-injected rats (n = 7). In the current study, there were no significant differences between the blood pressure, daily tap water intake and serum sodium, potassium, lithium and creatinine levels in control i.c.v. 0.15 M NaCl-injected rats, compared with the insulin-treated group. Conversely, there was a significant decrease in the daily solid rat chow intake (Co: 16.4 ± 3.5 g vs. Ins: 10.3 ± 2.6 g, P = 0.003) in 7-day long-term insulin-treated rats, compared with the control group. We confirmed that centrally administered insulin produced a substantial increase in the urinary output of Na⁺, Li⁺ and K⁺, and that the response was significantly enhanced in long-term i.c.v. insulin pre-treated animals, when compared with controls (fractional sodium excretion (FE_Na) from basal: 0.047 ± 0.18% to Ins-treated: 0.111 ± 0.035%, P = 0.001). Additionally, we demonstrated that insulin-induced natriuresis occurred by increasing fractional proximal (FEP_Na) from basal (16.8 ± 2.6% to Ins-treated: 26.7 ± 2.8%, P = 0.001) and post-proximal sodium excretion (FEPP_Na) from basal (0.37 ± 0.03% to Ins-treated: 0.42 ± 0.05%, P = 0.043), despite a decreased Na⁺ filtered load and rat food intake. The current data suggest that centrally injected insulin maintain its CNS ability to amplify neuronal hypotensive and natriuretic pathways that counteract the known peripheral antinatriuretic effects of insulin.     

Natural lipophilic inhibitors of mitochondrial complex I are candidate toxins for sporadic neurodegenerative tau pathologies

Annonacin, a natural lipophilic inhibitor of mitochondrial complex I has been implicated in the etiology of a sporadic neurodegenerative tauopathy in Guadeloupe. We therefore studied further compounds representing the broad biochemical spectrum of complex I inhibitors to which humans are potentially exposed.We determined their lipophilicity, their effect on complex I activity in submitochondrial particles, and their effect on cellular ATP levels, neuronal cell death and somatodendritic redistribution of phosphorylated tau protein (AD2 antibody against pS396/pS404-tau) in primary cultures of fetal rat striatum.The 24 compounds tested were lipophilic (logP range 0.9–8.5; exception: MPP⁺ logP = − 1.35) and potent complex I inhibitors (IC₅₀ range 0.9 nM–2.6 mM). They all decreased ATP levels (EC₅₀ range 1.9 nM–54.2 μM), induced neuronal cell death (EC₅₀ range 1.1 nM–54.5 μM) and caused the redistribution of AD2⁺ tau from axons to the cell body (EC₅ range 0.6 nM–33.3 μM). The potency of the compounds to inhibit complex I correlated with their potency to induce tau redistribution (r = 0.80, p < 0.001).In conclusion, we propose that the widely distributed lipophilic complex I inhibitors studied here might be implicated in the induction of tauopathies with global prevalence.     

Defining the mechanisms that underlie cortical hyperexcitability in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis [ALS] is a rapidly progressive neurodegenerative disorder of motor neurons, heralded by the development of cortical hyperexcitability. Reduction of short interval intracortical inhibition [SICI] in ALS, a feature linked to the development of cortical hyperexcitability, may be mediated by degeneration of inhibitory circuits or alternatively activation of high threshold excitatory circuits. As such, determining the mechanisms of SICI reduction in ALS has clear diagnostic and therapeutic significance. Consequently, the present study utilized a novel threshold tracking paired-pulse paradigm to determine whether SICI reduction in ALS represented reduced inhibition or excessive excitation. Using a 90 mm circular coil, SICI was assessed at three different conditioning stimulus intensities: 40%, 70% and 90% of resting motor threshold [RMT]. Motor evoked potential responses were recorded over the abductor pollicis brevis muscle. Short interval intracortical inhibition was uniformly reduced across all three levels of conditioning intensities in ALS [40% RMT, ALS −0.6 ± 0.7%, controls 2.0 ± 0.6%, P < 0.01; 70% RMT, ALS 0.6 ± 2.7%, controls 12.8 ± 2%, P < 0.001; 90% RMT, ALS −15.9 ± 1.3%, controls 2.2 ± 4.1%, P < 0.01]. In addition, the resting motor threshold was reduced, while the motor evoked potential amplitude was increased in ALS patients, in keeping with cortical hyperexcitability. These findings establish that SICI reduction in ALS represents degeneration of inhibitory cortical circuits, combined with excessive excitation of high threshold excitatory pathways. Neuroprotective strategies aimed at preserving the integrity of intracortical inhibitory circuits, in addition to antagonizing excitatory cortical circuits, may provide novel therapeutic targets in ALS.