Delayed sodium pyruvate treatment improves working memory following experimental traumatic brain injury

Prior work indicates that cerebral glycolysis is impaired following traumatic brain injury (TBI) and that pyruvate treatment acutely after TBI can improve cerebral metabolism and is neuroprotective. Since extracellular levels of glucose decrease during periods of increased cognitive demand and exogenous glucose improves cognitive performance, we hypothesized that pyruvate treatment prior to testing could ameliorate cognitive deficits in rats with TBI. Based on pre-surgical spatial alternation performance in a 4-arm plus-maze, adult male rats were randomized to receive either sham injury or unilateral (left) cortical contusion injury (CCI). On days 4, 9 and 14 after surgery animals received an intraperitoneal injection of either vehicle (Sham-Veh, n = 6; CCI-Veh, n = 7) or 1000 mg/kg of sodium pyruvate (CCI-SP, n = 7). One hour after each injection rats were retested for spatial alternation performance. Animals in the CCI-SP group showed no significant working memory deficits in the spatial alternation task compared to Sham-Veh controls. The percent four/five alternation scores for CCI-Veh rats were significantly decreased from Sham-Veh scores on days 4 and 9 (p < 0.01) and from CCI-SP scores on days 4, 9 and 14 (p < 0.05). Measures of cortical contusion volume, regional cerebral metabolic rates of glucose and regional cytochrome oxidase activity at day 15 post-injury did not differ between CCI-SP and CCI-Veh groups. These results show that spatial alternation testing can reliably detect temporal deficits and recovery of working memory after TBI and that delayed pyruvate treatment can ameliorate TBI-induced cognitive impairments.     

Augmented peripheral chemoreflex in patients with heart failure and inspiratory muscle weakness

We hypothesized that heart failure patients with inspiratory muscle weakness (IMW) present greater peripheral chemoreflex responsiveness and augmented exercise ventilatory oscillation compared to patients with preserved inspiratory muscle strength. We studied 19 heart failure patients: 9 with IMW (maximal inspiratory pressure [PImax] < 70% of predicted) and 10 with preserved inspiratory muscle strength. Inspiratory muscle strength was measured via pressure transducer. Peripheral chemoreflex was evaluated by the single-breath CO₂ test. Exercise ventilatory oscillation was determined as the ratio between amplitude and mean of each oscillation during incremental exercise. Patients with IMW had greater peripheral chemoreflex response (0.11 ± 0.03 l min⁻¹ Torr⁻¹) than those with preserved inspiratory muscle strength (0.07 ± 0.03 l min⁻¹ Torr⁻¹, p = 0.02). Moreover, there was a significant and inverse correlation between PImax and peripheral chemoreflex response (r = −0.57, p = 0.01). Likewise, there was a significant and inverse correlation between PImax and ventilatory oscillations (r = −0.46, p = 0.04). Our findings indicate that IMW is linked to increased peripheral chemoreflex and augmented exercise ventilatory oscillation in patients with chronic heart failure.     

Desensitization of the bradykinin-induced rise in intracellular free calcium in cultured endothelial cells

We studied the cellular mechanism involved in the desensitization of cultured endothelial cells to bradykinin. Bradykinin (10 nmol/l) evoked a rise in the intracellular free calcium concentration ([Ca _i ²⁺ ]), measured with the fluorescent probe indo-1, from 137±30 (±SEM) to 623±101 nmol/l. Cells were desensitized to bradykinin by repetitive stimulation with the peptide over 10 min, after which they no longer responded to bradykinin. However, purinergic stimulation with ATP (10 mol/l) elicited the same increase in [Ca _i ²⁺ ] in endothelial cells desensitized to bradykinin as in cells never exposed to bradykinin. The initial peak of [Ca _i ²⁺ ] after stimulation with bradykinin or ATP was not affected by removal of extracellular calcium ions, indicating mobilization of Ca²⁺ from intracellular stores. Since GTP-binding proteins (G-proteins) are probably involved in the receptor-mediated stimulation of endothelial cells, we also tested the effects of sodium fluoride (NaF), a reported direct stimulator of G-proteins, on endothelial [Ca _i ²⁺ ]. NaF (5 mmol/l) increased [Ca _i ²⁺ ] to 412±88 nmol/l in control cells and was equally effective in cells desensitized to bradykinin. We conclude that the homologous desensitization to bradykinin does not occur at the level of intracellular signal transduction but at the level of membrane receptors.     

Influence of B2 receptor antagonists on bradykinin-induced vasodilation and edema formation in isolated rabbit hindlimbs

In search of new possibilities to prevent acute inflammatory vascular reaction, we examined the effect of two selective B₂ receptor antagonists, CP 0127 ([Bissuccimidohexane (L-Cys⁶)^–1] and HOE 140 (D-Arg[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]BK), on changes in perfusion pressure and on edema formation caused by bradykinin (BK) in the isolated perfused rabbit hindlimbs. CP 0127 and HOE 140 were added to the perfusion fluid 2 min prior to the first BK-administration (5 × 10^–9 mol/l). A second BK-stimulation was performed after 30 minutes. The antagonists were tested in groups of 6 experiments each at concentrations of 10^–6 mol/l, 5 × 10^–9 mol/l and 10^–10 mol/l. CP 0127 was also tested in a concentration of 10^–8 mol/l. The application of BK resulted in an acute decrease of the mean arterial pressure and in a continual edema formation, reflected by an increase of organ weight (controls,n=6). Pretreatment with CP 0127 as well as with HOE 140 attenuated dose-dependently the BK-induced vasodilation (p < 0.005) and edema formation. The current results indicate that CP 0127 and HOE 140 are able to reduce BK-induced effects on vascular tone and edema formation.     

Blood CADPS2ΔExon3 expression is associated with intelligence and memory in healthy adults

Ca²⁺-dependent activator protein for secretion 2 (CADPS2), a secretory granule associate protein, mediates monoamine transmission and neurotrophin release. Both monoamines and neurotrophins play a crucial role in cognition, learning and memory. An aberrant splice variant of CADPS2, CADPS2ΔExon3, was reported to be associated with autism. Therefore, we examined the possible association between the expression of CADPS2/CADPS2ΔExon3 in peripheral blood and brain functions such as intelligence and memory. Quantitative polymerase chain reaction analysis was performed in 271 healthy adults (age range 20-74 years, mean ± SD 43.3 ± 15.3). Data on intelligence quotient (IQ) and memory were obtained by using full versions of the Wechsler Adult Intelligence Scale-Revised (WAIS-R), and the Wechsler Memory Scale-Revised (WMS-R), respectively. CADPS2 expression levels were not significantly associated with any scores/sub-scores of these scales. However, CADPS2ΔExon3 expression was significantly associated with lower IQ (p = 0.022; effect size: η_p² = 0.031), particularly verbal IQ of WAIS-R (p = 0.019; η_p² = 0.032), lower verbal memory (p = 0.026; η_p² = 0.026) and delayed recall (p = 0.042; η_p² = 0.021) of WMS-R. Our results suggest that CADPS2ΔExon3 affects intelligence and memory in the non-clinical population.     

Differential cerebrovascular CO2 reactivity in anterior and posterior cerebral circulations

The potential differences in cerebrovascular responses between the anterior and posterior circulations to changes in CO₂ are unclear in humans. Using transcranial Doppler ultrasound, we compared the CO₂ reactivity of the (1) BA and PCA and (2) MCA and PCA during hyperoxic rebreathing in supine position. The reactivity in the BA and PCA was similar in both absolute (1.27 ± 0.5 and 1.27 ± 0.6 cm/s/Torr; P = 0.992) and relative (3.98 ± 1.3 and 3.66 ± 1.5%/Torr CO₂; P = 0.581) measures, suggesting that the PCA is an adequate surrogate measure of reactivity for the BA. The MCA reactivity was greater than the PCA in absolute (2.09 ± 0.7 and 1.22 ± 0.5 cm/s/Torr CO₂; P < 0.001), but not relative measures (3.25 ± 1.0 and 3.56 ± 1.6%/Torr CO₂; P = 0.629). Our findings (a) confirm regional differences in the absolute reactivity in the human brain and (b) suggest that in cerebrovascular studies investigating functions mediated by posterior brain structures (e.g., control of breathing), the posterior vasculature should also be insonated.     

Acute effects of vitamin B6 and fixed combinations of vitamin B1, B6, B12 on nociceptive activity evoked in the rat thalamus: Dose-response relationship and combinations with morphine and paracetamol

Summary Nociceptive activity was elicited in neurones of the thalamus by supramaximal electrical stimulation of afferent C fibres in the sural nerve of rats under urethane anesthesia. The fixed combination of vitamin B₁, B₆, B₁₂ (Neurobion^®) as well as of vitamin B₆ administered by i.p. injection dose-dependently reduced the evoked nociceptive activity. The ED₅₀ of Neurobion^® is 4.6 ml/kg (at 100 min after injection) and that of vitamin B₆ is 189mg/kg (at 90 min after injection). The minimum effective doses of Neurobion^® and vitamin B₆ are 0.5 ml/kg and 40 mg/kg, respectively. When Neurobion^® or vitamin B₆ were given at their minimum effective doses, and the minimum effective doses of morphine (0.025 mg/kg) or paracetamol (5 mg/kg) were injected i.v. 80 min later, i.e., when the maximum effect of higher doses of Neurobion^® or vitamin B₆was about to develop, no supraadditive effect developed. It is concluded that the antinociceptive effect caused by a single injection of Neurobion^® is largely due to vitamin B₆. Vitamin B₁₂ may contribute to this effect, whereas vitamin B₁ alone exhibited only a slight effect on nociception. Moreover, it appears that Neurobion^® produces its antinociceptive effect after a single injection and after repeated administration during several days by different mechanisms so that the effect of analgesic agents is not enhanced following a single injection of Neurobion^® but may be enhanced after repeated administration of the compound.     

Further antinociceptive effects of myricitrin in chemical models of overt nociception in mice

The present work explored the antinociceptive effects of the flavonoid myricitrin in models of overt nociception triggered by intraplantar injection of chemical algogens into the hind paw of mice. The nociception induced by bradykinin (3 nmol/paw i.pl.) was abolished by prior treatment with myricitrin (10–100 mg/kg, i.p.) with ID₅₀ of 12.4 (8.5–18.1) mg/kg. In sharp contrast, myricitrin failed to affect the nociception elicited by prostaglandin E₂ (3 nmol/paw i.pl.). Cinnamaldehyde (10 nmol/paw i.pl.)-induced nociception was reduced by myricitrin (100 mg/kg, i.p.) and camphor (7.6 mg/kg, s.c.) in 43 ± 10% and 57 ± 8%, respectively. Myricitrin (30–100 mg/kg, i.p.) and amiloride (100 mg/kg, i.p.) inhibited nociceptive responses induced by acidified saline (pH 5/paw i.pl.), with ID₅₀ of 22.0 (16.1–30.0) mg/kg and inhibition of 71 ± 6% and 64 ± 5%, respectively. Moreover, myricitrin (10–30 mg/kg, i.p.) and ruthenium red (3 mg/kg, i.p.) significantly reduced the nociception induced by menthol (1.2 μmol/paw i.pl.) with the mean ID₅₀ of 2.4 (1.5–3.7) mg/kg and inhibition of 95 ± 3% and 51 ± 7%, respectively. In addition, myricitrin administration (30 and 100 mg/kg, i.p.) markedly reduced menthol-induced mechanical allodynia. However, myricitrin (100 mg/kg, i.p.) prevented (only in time of 60 min) cold allodynia induced by menthol. Collectively, the present results extend prior data and show that myricitrin promotes potent antinociception, an action that is likely mediated by an inhibition of the activation of nociceptors by bradykinin and TRPs agonist (i.e. cinnamaldehyde, acidified saline and menthol), probably via inhibition of PKC pathways. Thus, myricitrin could constitute an attractive molecule of interest for the development of new analgesic drugs.     

Pregnancy decreases GABAergic inhibition of the hypothalamic paraventricular nucleus

Depressor responses to peripheral or central infusion of Angiotensin II type 1 (AT₁) receptor antagonists (AT₁X) are greater in pregnant (P) compared to nonpregnant (NP) animals. AT₁ and ionotropic excitatory amino acid (EAA) receptors contribute to pressor responses to GABA_A receptor blockade with bicuculline (Bic) in the paraventricular nucleus (PVN) of male rats. Therefore, we hypothesized that GABAergic inhibition is decreased and AT₁ receptors play a greater excitatory role in the PVN of P versus NP rats. Unilateral microinjection of Bic was performed before (Bic₁), after AT₁X (Bic₂), and after AT₁X + EAA blockade (kynurenate, Kyn) (Bic₃) in the PVN. Increases in mean arterial pressure (MAP: NP = 20 ± 2; P = 12 ± 2 mmHg), heart rate (HR: NP = 57 ± 6; P = 19 ± 6 beats/min) and renal sympathetic nerve activity (RSNA: NP = 70 ± 9; P = 33 ± 7%) due to Bic (Bic₁) were attenuated in P rats. Responses to AT₁X and Kyn alone were insignificant in both groups. In NP rats, AT₁X attenuated (+ 12 ± 4 mmHg), and AT₁X + Kyn further decreased the pressor response to Bic in the PVN (+ 6 ± 2 mmHg). In P rats AT₁X reduced the pressor response to Bic (+ 5 ± 1 mm Hg), and Kyn had no additional effect (+ 3 ± 1 mmHg). Effects of PVN Bic to alter the autospectra of RSNA were suppressed by prior AT₁X and Kyn in both groups. Thus, tonic GABAergic inhibition is decreased and the contribution of AT₁ receptors in the PVN may be greater in P rats.     

Early treadmill training promotes motor function after hemorrhagic stroke in rats

Rehabilitation after a stroke is very important because it has beneficial effects on brain function, including the promotion of plasticity. However, an optimal time window for rehabilitation interventions after hemorrhagic stroke has not been clearly defined. The aim of this study was to determine whether early exercise training initiated 24 h after an intracerebral hemorrhage (ICH) might enhance neurologic recovery more than exercise initiated 1 week after ICH without hematoma expansion and edema volume increase. We subjected adult male Sprague–Dawley rats to experimental ICH by the intrastriatal administration of bacterial collagenase. The rats were randomly divided into the following 2 groups: early training group (treadmill exercise started 24 h post-ICH; n = 18) and late training group (treadmill exercise started 1-week post-ICH; n = 18). Two weeks after surgery we performed neurologic tests (rota-rod, modified limb-placing, and adhesive-dot removal tests), and measured hematoma volumes and brain water content. In the late training group, compared with the pre-ICH performance on the rota-rod test (98.3 ± 69.4 s), the animals had significantly worse performance after the post-ICH rehabilitation (40.5 ± 52.6 s; p < 0.01, paired t-test). In the early training group however, the motor performance after the post-ICH rehabilitation (56.4 ± 73.5 s) was not significantly different from the baseline pre-ICH performance (79.8 ± 33.9 s; p = 0.24). There were no significant differences between the two groups with respect to the other neurologic tests. Early exercise did not increase hematoma size or brain water content. Early treadmill training could be performed safely, and enhanced motor recovery in a rat model of ICH. Further studies are required to translate the results into clinical significance.     

Fitness alters fluid regulatory but not behavioural responses to hypohydrated exercise

Dehydration is typical during prolonged exercise. Because training stimulates numerous adaptations, some involving fluid regulation, it is conceivable that training involves adaptations to dehydration. This study tested the hypothesis that trained individuals have altered fluid regulatory, but not behavioural or perceptual responses to exercise when hypohydrated. Six trained (V.O_{2 peak}: 65 ± 8 mL kg^− 1 min^− 1) and six untrained (V.O_{2 peak}: 45 ± 4 mL kg⁻¹ min⁻¹) males cycled for 40 min at 70%V.O_{2 peak}, once whilst euhydrated (EUH) and once whilst hypohydrated by ~ 2% body mass (HYPO), before a 40-min performance trial with euhydration (in EUH) or ad libitum drinking (in HYPO), in temperate conditions (24.3 °C, 50% rh). Baseline hydration was achieved by complete or partial rehydration from exercise + heat stress on the previous evening. Body mass was reduced (− 1.8 ± 0.1%) and plasma osmolality was increased (5 ± 1 mosmol kg^− 1) similarly between fitness groups in HYPO compared to EUH (P < 0.05). During exercise, plasma [AVP] rose more in HYPO than EUH; the elevation was greater in the Untrained (4.1 ± 1.7 vs. 2.0 ± 0.8 pmol L^− 1, P < 0.01) than Trained (1.4 ± 0.6 vs. 1.1 ± 0.5 pmol L^− 1, P < 0.01; P = 0.02). Increases in plasma [AVP] relative to osmolality were higher in Untrained than Trained (0.47 ± 0.06 vs. 0.025 ± 0.05 pmol mosmol^− 1, P = 0.03). Fitness groups had equivalent thirst ratings during fixed exercise but Trained were thirstier than Untrained when self regulating in HYPO (4.0 ± 1.5 vs. 2.7 ± 1.2; P = 0.05); thus Trained tended to consume more fluid (1.20 ± 0.16 vs. 0.88 ± 0.16 L; P = 0.19), but maintained similar hypohydration consistent with their greater sweat rate during HYPO. In conclusion, aerobic fitness attenuates the neuroendocrine ([AVP]) response to hypohydrated exercise, but not perceptual (thirst) or behavioural (ad libitum drinking) responses.     

The concentrations of serum,plasma and platelet BDNF are all increased by treadmill VO2max performance in healthy college men

The most current human-based studies in which brain-derived neurotrophic factor (BDNF) levels in the peripheral blood system are analyzed use it as an indicator that represents BDNF levels in the CNS. However, whether circulating BDNF (serum and plasma) is positively or inversely associated with cardiorespiratory fitness levels (VO_2max) is still controversial, and no study has done to investigate exercise effects on the concentration of BDNF stored in circulating platelets which, in fact, store a large amount of circulating BDNF. Thus, the purpose of this study was to determine the relation between VO_2max and all circulating BDNF levels (serum, plasma and platelets) in college male students (N = 18; age, 19 ± 1 years; height, 173.22 ± 7.65 cm; weight, 78.25 ± 14.25 kg; body fat percent, 13.82 ± 5.68%). Dual X-ray energy absorptiometry whole body scan was used to measure their body composition. After the overnight fast, all participants were performed VO_2max test, and their blood was collected at rest and immediately after the exercise. Our data resulted in significant increases in platelet counts and serum, plasma and platelet BDNF levels immediately after the exercise (p < 0.01). VO_2max had a significant negative correlation with serum BDNF, plasma BDNF and platelet BDNF at rest (p < 0.05) but a significant positive correlation with serum, plasma BDNF, and platelet BDNF immediately after the exercise (p < 0.01). However, our data show no correlation between VO_2max and platelet count both at rest and immediately after the exercise. In conclusion, this is the first study showing that basal BDNF levels are inversely correlated with cardiorespiratory fitness levels but that the inverse correlations turn into positive correlations with all circulating BDNF levels immediately after the exercise. Moreover, it is the first time to provide evidence that platelet BDNF levels are also positively affected by the exercise. However, future studies will be needed to investigate what tissues provide BDNF into the circulating system and to elucidate the role of circulating BDNF.     

Differential hippocampal pharmacokinetics of phenobarbital and carbamazepine in repetitive seizures induced by 3-mercaptopropionic acid

Previous evidence has shown that chronic 3-mercaptopropionic acid (MP) administration induced brain P-glycoprotein (P-gp) overexpression altering target site accumulation of phenytoin. The aim of the present work was to assess the involvement of P-glycoprotein in carbamazepine and phenobarbital hippocampal pharmacokinetics in an experimental model of epilepsy, induced by repetitive MP administration. Seizures were induced in Wistar rats by injection of MP (45 mg kg⁻¹, i.p.) during 10 days. Control rats (C) were injected with saline solution. In order to monitor extracellular brain antiepileptic levels, a concentric probe was inserted into the hippocampus. Animals were administered with carbamazepine (10 mg kg⁻¹, i.v.) or phenobarbital (20 mg kg⁻¹, i.v.) 30 min after intraperitoneal administration of vehicle or nimodipine (2 mg kg⁻¹), a well known P-glycoprotein inhibitor. No differences were found in hippocampal concentrations of carbamazepine comparing all groups. In vehicle pre-treated rats, hippocampal phenobarbital concentrations were lower in MP (maximal concentration, C_max: 6.0 ± 0.6 μg ml⁻¹, p < 0.05) than in C animals (C_max: 9.4 ± 0.9 μg ml⁻¹). Control rats pre-treated with nimodipine showed similar results (C_max: 10.7 ± 0.6 μg ml⁻¹) than those pre-treated with vehicle. Nimodipine pre-treatment in MP rats enhanced hippocampal phenobarbital concentrations (C_max: 10.2 ± 1.0 μg ml⁻¹, p < 0.05) as compared with vehicle pre-treatment.     

Activation of the central cholinergic pathway increases post-exercise tail heat loss in rats

The aim of this study was to evaluate the effects of stimulation of the central cholinergic pathway on the regulation of post-exercise tail heat loss in rats. Either 2.0 μL of 25 × 10⁻³ M physostigmine (Phy) or 0.15 M NaCl solution (Sal) were injected into the right lateral cerebral ventricle of both resting (n = 8) and post-exercising rats (n = 6; 24 m min⁻¹; 25 min; 5% inclination). Tail temperature (T_tail) was measured using a thermistor taped to the tail, and intraperitoneal temperature, an index of core temperature (T_c), was recorded using a telemetry sensor implanted into the peritoneal cavity. In resting rats, Phy induced an increase in both T_tail (26.8 ± 0.3 °C Phy versus 25.2 ± 0.6 °C Sal; P < 0.05) and in heat loss index (0.26 ± 0.03 Phy versus 0.14 ± 0.05 Sal; P < 0.05; 30 min after injection), and a decrease in T_c compared to the Sal injection group (36.6 ± 0.2 °C Phy versus 37.0 ± 0.2 °C Sal; P < 0.05). In post-exercising rats, Phy injection attenuated the decrease in both T_tail (28.3 ± 0.8 °C Phy versus 26.4 ± 0.6 °C Sal; P < 0.05) and heat loss index (0.37 ± 0.07 Phy versus 0.19 ± 0.02 Sal; P < 0.05) without altering T_c. We conclude that activation of the central cholinergic pathway increases post-exercise tail heat loss in rats.     

The phosphorescence oxygen analyzer as a screening tool for disorders with impaired lymphocyte bioenergetics

This study aimed to show the feasibility of using the phosphorescence oxygen analyzer to screen for clinical disorders with impaired cellular bioenergetics. [O₂] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, O₂ consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics. Cyanide inhibited O₂ consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. The rate of respiration (mean ± SD, in μM O₂ per min per 10⁷ cells, set as the negative of the slope of [O₂] vs. t) for adults was 2.1 ± 0.8 (n = 18), for children 2.0 ± 0.9 (n = 20), and for newborns (umbilical cord samples) 0.8 ± 0.4 (n = 18), p < 0.0001. For an 8-year-old patient with reduced NADH dehydrogenase and pyruvate dehydrogenase activities in the muscle, the rate was 0.7 ± 0.2 (n = 3) μM O₂ per min per 10⁷ cells. For a 3-month-old patient with hepatocerebral mitochondrial DNA depletion syndrome (MDS) with confirmed mutations in the MPV17 gene, the rate was 0.6 μM O₂ per min per 10⁷ cells. For an18 month-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.5 μM O₂ per min per 10⁷ cells. For a 6-year-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.6 μM O₂ per min per 10⁷ cells. For 1-week-old patient with congenital lactic acidemia and hypotonia (confirmed mutations in DLD gene), the rate was 1.5 μM O₂ per min per 10⁷ cells. For three siblings (9-year-old male, 8-year-old male and 2-month-old female) with congenital progressive myopathy, the rates were 0.9, 0.6 and 1.2 μM O₂ per min per 10⁷ cells, respectively. Four patients with congenital lactic acidemia (with inadequate work-up) were also studied; their rates were 0.2, 1.5, 0.3 and 1.7 μM O₂ per min per 10⁷ cells. This novel approach permits non-invasive, preliminary assessment of cellular bioenergetics. Potential applications and limitations of this technique are discussed.     

Afterhyperpolarization induced by the activation of nicotinic acetylcholine receptors in pelvic ganglion neurons of male rats

The electrophysiological mechanism underlying afterhyperpolarization induced by the activation of the nicotinic acetylcholine receptor (nAChR) in male rat major pelvic ganglion neurons (MPG) was investigated using a gramicidin-perforated patch clamp and microscopic fluorescence measurement system. Acetylcholine (ACh) induced fast depolarization through the activation of nAChR, followed by a sustained hyperpolarization after the removal of ACh in a dose-dependent manner (10 μM to 1 mM). ACh increased both intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺ concentrations ([Na⁺]_i) in MPG neurons. The recovery of [Na⁺]_i after the removal of ACh was markedly delayed by ouabain (100 μM), an inhibitor of Na⁺/K⁺ ATPase. Pretreatment with ouabain blocked ACh-induced hyperpolarization by 67.2 ± 5.4% (n = 7). ACh-induced hyperpolarization was partially attenuated by either the chelation of [Ca²⁺]_i with BAPTA/AM (20 μM) or the blockade of small-conductance Ca²⁺-activated K⁺ channels by apamin (500 nM). Taken together, the activation of nAChR increases [Na⁺]_i and [Ca²⁺]_i, which activates Na⁺/K⁺ ATPase and Ca²⁺-activated K⁺ channels, respectively. Consequently, hyperpolarization occurs after the activation of nAChR in the autonomic pelvic ganglia.     

Major role of GABAA-receptor mediated tonic inhibition in propofol suppression of supraoptic magnocellular neurons

Using slice patch clamp recording, we examined the effects of general anesthetic propofol (2,6-diisoprophlphenol) on dual modality of GABA_A inhibition in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs): conventional quantal synaptic transmission (IPSCs, I_phasic) and persistent tonic form of inhibitory current (I_tonic). Propofol (10 μM) enhanced I_tonic as shown by an inward shift in I_holding (16.46 ± 2.93 pA, n = 27) and RMS increase (from 3.37 ± 0.21 pA to 4.68 ± 0.33 pA, n = 27) in SON MNCs. Propofol also prolonged the decay time of IPSCs with decreased IPSCs frequency but no significant changes in IPSCs amplitude. Overall, propofol (1–10 μM) caused much smaller increase in mean I_phasic than mean I_tonic at all tested concentrations. In consistent with the enhancement of GABA_A currents, propofol attenuated ongoing firing activities of SON MNCs by ∼65% of control. Selective inhibition of I_phasic by a GABA_A antagonist, gabazine (1 μM), failed to block the propofol suppression of the firing activities, while inhibition of I_tonic and I_phasic by bicuculline (20 μM) efficiently blocked the propofol-induced neurodepression in SON MNCs. Taken together, our results showed that propofol facilitated I_tonic with marginal increase in mean I_phasic, and this could be a mechanism reducing the intrinsic SON MNCs excitability during propofol anesthesia.     

Effect of APOE polymorphisms on early responses to traumatic brain injury

To investigate the relationship between apolipoprotein E (APOE) polymorphisms and the severity of traumatic brain injury (TBI) in acute stage in the cohort of mainland Chinese patients. We prospectively identified admissions to the two neurosurgical departments for head injury. A total of 110 subjects with TBI (80 males and 30 females, with mean age of 43.87 years) were enrolled from December 2003 to May 2004, and demographic and clinical data were collected. Venous blood was collected from patients with TBI on admission to determine the APOE genotype polymorphisms. The APOE genotyping was performed by means of PCR-RFLP. The deterioration of patients’ condition in acute stage (<7 days after TBI) was judged by either of following criteria: decrease of GCS, increase in hematoma volume or delayed hematoma both detected by repeated CT scanning. χ²-test and logistic regression analyses were done by SPSS. The distributions of APOE genotypes and alleles matched Hardy–Weinberg law. In 110 Chinese patients, 19 subjects presented with deteriorated clinical condition after hospitalization, and seven of 17 patients with APOE ?4 (41.2%) had a deteriorated condition which was significantly different from those without APOE ?4 (12 of 93 patients, 12.9%, P = 0.01). However, neither the presence of ?2 nor of ?3 was significantly different from those absent of it (P > 0.05). Logistic regression analyses showed that APOE ?4 was a risk factor (OR = 4.836, P = 0.011, 95% CI 1.443–16.208) to predispose to clinical deterioration after adjusting for patient age, sex, smoking or not, alcohol-drinking or not, injury severity, injury mechanisms, treatments, and pattern of TBI. This finding suggests that the patients with APOE ?4 predispose to clinical deterioration in acute phase after TBI and APOE polymorphisms play a role in early responses to TBI.     

Increased cutaneous NGF and CGRP-labelled trkA-positive intra-epidermal nerve fibres in rat diabetic skin

In this study we have determined the amount of Nerve Growth Factor (NGF) and the innervation density of the glabrous hindpaw skin of diabetic rats (n = 4) and controls (n = 3). The proportion of intra-epidermal nerve fibres (IENF) expressing the high affinity NGF receptor (trkA) and calcitonin gene-related peptide (CGRP) were also determined. Four weeks after induction of diabetes by intraperitoneal streptozotocin injection skin was analyzed for: (i) NGF content using ELISA and (ii) the innervation density of peptidergic afferents that also expressed trkA using immunocytochemistry. NGF levels were approximately three-fold higher in diabetic skin compared to controls (diabetic: 134.7 ± 24.0 (SD) pg ml⁻¹, control: 42.7 ± 21.5 pg ml⁻¹, p = 0.002). As expected there was a significant reduction in IENF density in diabetic skin (2.7 ± 1.3 fibres mm⁻¹) compared to controls (6.9 ± 1.5 fibres mm⁻¹; p = 0.01). In diabetic rats there was no significant difference in the proportion of trkA-labelled IENF (diabetic 74 ± 21%; control 83 ± 15%, p = 0.6), but significantly more trkA-positive IENF were also labelled by CGRP antibodies in diabetic skin compared to controls (diabetic 89 ± 22%; control 38 ± 2%, p = 0.03). These data suggest that in diabetes the upregulation of cutaneous NGF may ‘over-troph’ the surviving axons, increasing CGRP labelling, which may be important in the aetiology of painful diabetic neuropathy.     

Regulatory role of 1, 25-dihydroxyvitamin D3 and vitamin D receptor gene variants on intracellular granzyme A expression in pulmonary tuberculosis

Vitamin D receptor (VDR) genotypes have been shown to be associated with differential susceptibility or resistance to tuberculosis. The influence of FokI, BsmI, ApaI and TaqI variants of VDR gene on 1, 25(OH)₂ D₃ modulated granzyme A expression of cytotoxic lymphocytes induced by culture filtrate antigen (CFA) of Mycobacterium tuberculosis was studied in 40 pulmonary tuberculosis (PTB) patients and 49 normal healthy subjects (NHS) by flow cytometry. In both the study groups, addition of 1, 25(OH)₂ D₃ (10^− 7M) significantly reduced the percentage of granzyme A positive cells in both unstimulated (NHS, p < 0.0001; PTB, p = 0.02) and stimulated culture conditions (CFA, NHS, p < 0.0001; PTB, p = 0.0001) which correlated positively with the IFN-γ levels (unstimulated, p = 0.01; CFA stimulated, p = 0.004) in NHS. The ApaI aa genotype and bbaaTT extended genotype were associated with a significantly decreased percentage of granzyme A positive cells in NHS (p < 0.05). Our results suggest that 1, 25(OH)₂ D₃ suppresses granzyme A probably by down-regulating Th1 cytokine response. Moreover, the VDR gene variants might regulate cytotoxic T-cell response via 1, 25(OH)₂ D₃ mediated suppression of granzyme A expression in tuberculosis.