Mohamed A. Fahim

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity.

Both spinal hemisection (SH) at C2 and tetrodotoxin (TTX) phrenic nerve blockade result in diaphragm muscle paralysis and inactivity of the phrenic axon terminals. However, phrenic motoneuron somata are inactive with SH but remain active with TTX phrenic nerve blockade. Neuromuscular transmission failure with repeated activation decreases following SH and increases following TTX phrenic nerve blockade, suggesting that matching (or mismatching) of somal and synaptic inactivities of phrenic motoneurons differentially regulates synaptic vesicle pools at diaphragm neuromuscular junctions. At individual type-identified rat diaphragm presynaptic terminals, the size of the releasable pool of synaptic vesicles was analyzed by fluorescence confocal microscopy of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64) uptake and synaptic vesicle density at active zones was determined using transmission electron microscopy. After 14 days of SH and TTX-induced diaphragm muscle inactivity, neuromuscular junction size was not different at type I or IIa fibers, but increased at type IIx and/or IIb fibers (by 51% in SH and 35% in TTX) compared with control. With SH, synaptic vesicle pool size and density increased at presynaptic terminals innervating type I or IIa fibers (17 and 63%, respectively; P<0.001) and type IIx and/or IIb fibers (41 and 31%, respectively; P<0.001) when compared with controls. Following TTX, synaptic vesicle pool size and density decreased by 64 and 17%, respectively, at presynaptic terminals innervating type I or IIa fibers, and by 50 and 36%, respectively, at type IIx and/or IIb fibers (P<0.001, for all comparisons). Thus, matching motoneuron soma and axon terminal inactivity (SH) increases the size and density of releasable synaptic vesicle pools at adult rat diaphragm neuromuscular junctions. Mismatching motoneuron soma and axon terminal inactivities (TTX) results in converse presynaptic adaptations. Inactivity-induced neuromuscular plasticity reflects specific adaptations in the size and density of synaptic vesicle pools that depend on motoneuron soma rather than axon terminal (or muscle fiber) inactivity.

Evaluation of the direct systemic and cardiopulmonary effects of diesel particles in spontaneously hypertensive rats.

Recent data suggest that ultrafine pollutant particles (diameter <0.1microm) may pass from the lung into the systemic circulation. However, the systemic and cardiorespiratory effects of translocated particles are not well known. In this study, we determined the direct acute (24h) effect of the systemic administration of 0.01mg/kg and 0.02mg/kg diesel exhaust particles (DEP) on systolic blood pressure, heart rate, and both systemic and pulmonary inflammation in spontaneously hypertensive rats (SHR). Compared to the blood pressure in control group, rats exposed to DEP exhibited a dose-dependent increase in systolic blood pressure, at 0.01mg/kg (P<0.05) and 0.02mg/kg (P<0.01). Likewise, the heart rate was also dose-dependently increased at 0.01mg/kg (P:NS) and 0.02mg/kg (P<0.01) compared to control SHR. DEP exposure (0.02mg/kg) significantly elevated the number of leukocytes in blood (P<0.05), interleukin-6 (IL-6, P<0.005), tumor necrosis factor alpha (P<0.05) and leukotriene B4 (LTB4, P<0.005) concentrations in plasma. Moreover, in SHR given 0.02mg/kg, the number of platelet was significantly reduced (P<0.05), whereas the tail bleeding time was prolonged (P<0.05). Pulmonary inflammations were confirmed by the presence of a significant increase in the number of macrophages (0.02mg/kg) and neutrophils (0.01 and 0.02mg/kg) and protein contents (0.02mg/kg) in bronchoalveolar lavage (BAL) compared to saline-treated SHR. Also, IL-6 (0.01mg/kg; P<0.05 and 0.02mg/kg; P<0.01), LTB4 (0.02mg/kg; P<0.05) concentrations in BAL and the superoxide dismutase activity (0.02mg/kg; P=0.01) were significantly elevated compared to control group. We conclude that, in SHR, the presence of DEP in the systemic circulation leads not only to cardiac and systemic changes, but also triggers pulmonary inflammatory reaction involving IL-6, LTB4 and oxidative stress.

Oleanolic Acid: A Novel Cardioprotective Agent That Blunts Hyperglycemia-Induced Contractile Dysfunction

Diabetes constitutes a major health challenge. Since cardiovascular complications are common in diabetic patients this will further increase the overall burden of disease. Furthermore, stress-induced hyperglycemia in non-diabetic patients with acute myocardial infarction is associated with higher in-hospital mortality. Previous studies implicate oxidative stress, excessive flux through the hexosamine biosynthetic pathway (HBP) and a dysfunctional ubiquitin-proteasome system (UPS) as potential mediators of this process. Since oleanolic acid (OA; a clove extract) possesses antioxidant properties, we hypothesized that it attenuates acute and chronic hyperglycemia-mediated pathophysiologic molecular events (oxidative stress, apoptosis, HBP, UPS) and thereby improves contractile function in response to ischemia-reperfusion. We employed several experimental systems: 1) H9c2 cardiac myoblasts were exposed to 33 mM glucose for 48 hr vs. controls (5 mM glucose); and subsequently treated with two OA doses (20 and 50 µM) for 6 and 24 hr, respectively; 2) Isolated rat hearts were perfused ex vivo with Krebs-Henseleit buffer containing 33 mM glucose vs. controls (11 mM glucose) for 60 min, followed by 20 min global ischemia and 60 min reperfusion ± OA treatment; 3) In vivo coronary ligations were performed on streptozotocin treated rats ± OA administration during reperfusion; and 4) Effects of long-term OA treatment (2 weeks) on heart function was assessed in streptozotocin-treated rats. Our data demonstrate that OA treatment blunted high glucose-induced oxidative stress and apoptosis in heart cells. OA therapy also resulted in cardioprotection, i.e. for ex vivo and in vivo rat hearts exposed to ischemia-reperfusion under hyperglycemic conditions. In parallel, we found decreased oxidative stress, apoptosis, HBP flux and proteasomal activity following ischemia-reperfusion. Long-term OA treatment also improved heart function in streptozotocin-diabetic rats. These findings are promising since it may eventually result in novel therapeutic interventions to treat acute hyperglycemia (in non-diabetic patients) and diabetic patients with associated cardiovascular complications.

Exacerbation of thrombotic events by diesel exhaust particle in mouse model of hypertension

Several epidemiological studies have shown that acute exposure to particulate air pollution is associated with increases in cardiovascular morbidity and mortality, and that these effects are especially exacerbated among individuals with pre-existing compromised cardiovascular function such as hypertension. This study was undertaken to determine the cardiovascular effect of diesel exhaust on TO mice made hypertensive by implanting osmotic minipump infusing angiotensin II or vehicle (control). On day 13, the animals were intratracheally instilled with either DEP (15 μg/mouse) or saline. 24 h later, pulmonary exposure to DEP had significantly decreased the systolic blood pressure (SBP) in hypertensive (HT) mice (P<0.01), but not in normotensive (NT) mice. The number of leukocytes and red blood cells, and the plasma interleukin 6 concentration in plasma, however, were not affected in any of the animals. The PaO₂ was decreased, and PaCO₂ increased in DEP-treated HT mice compared to NT mice treated with DEP (P<0.05). The number of circulating platelets was significantly increased in DEP-treated HT versus saline-treated HT and DEP-treated NT mice. Moreover, in NT mice, DEP exposure induced a prothrombotic effect in pial arterioles compared with saline-treated NT mice (P<0.05). Interestingly, in DEP-treated HT mice, the prothrombotic events were significantly aggravated compared with saline-treated HT and DEP-treated NT mice. The direct addition of DEP (0.1-1 μg/ml) to untreated mouse blood significantly induced in vitro platelet aggregation in a dose-dependent fashion, and these effects were more pronounced in blood of HT mice. In vitro exposure to DEP (0.25-1 μg/ml) led to activated intravascular coagulation, an effect that was confirmed by a shortening of both the activated partial thromboplastin time (aPTT) and the prothrombin time (PT). The effect of DEP on aPTT was potentiated in the plasma of HT mice. It can be concluded that the thrombotic events caused by DEP are exacerbated by hypertension in mice. Our findings, therefore, provide a possible plausible explanation for the cardiovascular morbidity and mortality accompanying urban air pollution.

Reduced potassium currents in old rat CA1 hippocampal neurons

Potassium currents are an important factor in repolarizing the membrane potential and determining the level of neuronal excitability. We compared potassium currents in CA1 hippocampal neurons dissociated from young (2–3 months old) and old (26–30 months old) Sprague–Dawley rats. Whole‐cell patch‐clamp techniques were used to measure the delayed rectifier (sustained) and the A‐type (transient) potassium currents. The delayed rectifier current was smaller in old (548 ± 57 pA) than in young (1193 ± 171 pA) neurons. In the absence of extracellular calcium, the delayed rectifier current was also smaller in old (427 ± 41 pA) than in young (946 ± 144 pA) neurons. The cell membrane capacitance was unchanged in old (13.3 ± 1.2 pF) compared to young (13.6 ± 1.2 pF). Therefore, the reduction in the delayed rectifier current was not due to a change in membrane surface area. Moreover, activation and inactivation of the delayed rectifier current were unchanged in old compared to young neurons. The slope of the current‐voltage relation, however, was smaller in old (B = 5.03) than in young (B = 9.62) neurons. Similarly, the A‐current was smaller in old (100 ± 16 pA) than in young (210 ± 44 pA) neurons in the presence of extracellular calcium. This reduction of potassium currents could account for the prolongation of action potentials reported previously for old rat CA1 hippocampal neurons. The age‐related reduction in potassium current indicates plasticity in neuronal function that can impact communication in the hippocampal neural network during aging. J. Neurosci. Res. 63:176–184, 2001.

Ageing of the female pelvic floor: towards treatment a la carte of the "geripause"

The study of menopause and female ageing “geripause” is receiving much attention lately from the health care community for three main reasons [1–3]. The first is the global increase in female life expectancy as a result of improved health awareness and services with continuing reduction of adult mortality, progressive transition from high to low fertility, and recent socioeconomic affluence in most countries. This unprecedented demographic change, which started in the developed world in the 19th century and more recently in developing countries, allowed women to experience menopausal manifestations during approximately one third of their lifespan and reach the geripause. Secondly, the process of medicalization within the broader context of the dominance of health as a cultural preoccupation in recent societies and women’s motivation by personal concerns and cultural forces to take more control of the effects of menopause and/or ageing on their bodies has resulted in increasing use of female sex hormones as a replacement therapy. Finally, there is a growing public and medical concern about the serious adverse effects of estrogen/progestin replacement therapy in old postmenopausal “geripausal” women that had been recently reported in the Women’s Health Initiative Trial. This is accompanied by considerable interest in the contemporary biomedical literature, in particular, about the prevalence, detrimental effects, and management of support-related pelvic floor dysfunction (pelvic organ prolapse, urinary incontinence, and fecal incontinence) in the geripausal population. There seems to be no consensus, however, whether the exact underlying mechanism is normative ageing, falling circulating estrogen levels caused by menopausal ovarian failure, or a combination of both factors [3–9]. It is widely believed that estrogen deprivation at the climacteric is primarily responsible for support-related pelvic floor dysfunction in geripausal women. This assumption is based on the detection of estrogen receptors in the components of continence-maintaining and supportive pelvic floor structures in premenopausal women and experimental animals [3–6]. In turn, estrogen replacement had been extensively used to prevent or restore the decline in pelvic floor support and/or deterioration of urinary and fecal control after the menopause but without critical analysis of the long-term cure rates or evidence-based improvement in clinical outcome after treatment in most studies [7]. Paradoxically, a recent meta-analysis and a Int Urogynecol J (2008) 19:455–458 DOI 10.1007/s00192-008-0576-0

AGING INCREASES CALCIUM INFLUX AT MOTOR NERVE TERMINAL

To determine whether increased transmitter release from soleus nerve terminals of old C57BL/6J mice is caused by an altered Ca2+ regulation, the time course of post‐tetanic potentiation of miniature endplate potential (MEPP) frequency was used as an indicator of the kinetics of Ca2+ metabolism in young (10 months) and old (24 months) mice. Post‐tetanic potentiation properties were studied in either (1) 0.2 mM Ca2+, 5.0 mM Mg2+ Krebs; or (2) Ca2+‐free/EGTA Krebs to eliminate Ca2+ influx, and thereby isolated Ca2+ buffering. In the 0.2 mM Ca2+ Krebs, the time constants of decay of augmentation (TA) and potentiation (TP) were longer in old (TA = 10.3 ± 1.0 sec, TP = 195.3 ± 5.4 sec) than in young (TA = 7.0 ± 0.7 sec, TP = 78.8 ± 6.6 sec) nerve terminals. Evoked transmitter release was measured in 0.4 mM Ca2+, 2.75 mM Mg2+ Krebs. Quantal content of the endplate potential was positively correlated with TA (r = 0.95) and with TP (r = 0.98). In the Ca2+‐free/EGTA Krebs, there was no difference in post‐tetanic potentiation properties between young and old terminals. These results suggest that Ca2+ influx into the soleus nerve terminal increases with aging. This may explain, at least in part, the increased quantal content observed at old terminals.

Alpha Tocopherol Protects Against Immunosuppressive and Immunotoxic Effects of Lead

Chronic exposure to lead (Pb) is associated with multi-organ toxicity. The precise mechanism(s) involved, however, remains incompletely defined. The present study was undertaken to analyze the effect of Pb on the immune system and determine the ability of f tocopherol (AT) to reverse Pb-induced immunotoxicity. Groups of TO Mice (6 per group) were treated ip for 2 weeks with saline alone, Pb acetate alone, Pb plus AT, or with AT alone. Spleens were then analyzed for (i) cellular composition by flow cytometry, (ii) cellular response to B and T cell mitogens and (iii) production of nitric oxide (NO). Pb treatment resulted in a significant state of splenomegaly associated mainly with an influx of CD11b + myeloid cells. Surprisingly, however, these cells exhibited no upregulation in expression of activation markers and did not produce NO. The lymphocyte mitogenic responses were inhibited by S 70% in Pb-treated group. Concurrent treatment with Pb and AT resulted in almost a complete reversal of Pb-induced splenic cellular influx. Despite this, however, mitogenic responses in Pb+AT treated group were approximately 50% of those observed in normal (saline-treated) controls. We conclude that (1) chronic treatment with Pb acetate induces a state of splenomegaly and decreased proliferation in response to mitogenic stimuli and (2) co-treatment with AT largely reversed the cellular influx but this was associated with only a partial improvement of the mitogenic responses. These results highlight the role of AT as a potentially effective antioxidant in the immune system.

Local cerebral hyperthermia induces spontaneous thrombosis and arteriolar constriction in the pia mater of the mouse

The effect of local cerebral hyperthermia on responses of pial microvessels of the mouse was investigated. A set protocol was followed, involving the performance of a craniotomy on anaesthetized animals and using intravital microscope-television closed circuitry. Controlled hyperthermic exposure was applied regionally by heating the brain surface with irrigating artificial cerebrospinal fluid. Microvascular responses such as changes in diameter, thrombosis and embolism were monitored and video-taped observations were further viewed and analysed. When both brain surface and core body temperatures were kept at 37° C, no changes in pial microvessels were noted. With core body temperature kept at 37° C and at a brain surface temperature of 43.1° C, passing emboli and arteriolar constriction were observed. A few minutes later, visible thrombosis was prevalent. Further spontaneous thrombo-embolic activity continued and at the end of a 50-min hyperthermic exposure, arterioles attained a constriction of 37%. Thrombus formation was sometimes massive enough to occlude fully the microvessel. The protocol followed in this study can be adopted to other small animal species and for a variety of experimental procedures involving hyperthermia and the pial microcirculation.

Withania coagulans Fruit Extract Reduces Oxidative Stress and Inflammation in Kidneys of Streptozotocin-Induced Diabetic Rats

The present study was carried out to investigate the changes in oxidative and inflammatory status in streptozotocin-induced diabetic rats kidneys and serum following treatment with Withania coagulans, a popular herb of ethnomedicinal significance. The key markers of oxidative stress and inflammation such as inflammatory cytokines (IL-1β, IL-6, and TNF-α) and immunoregulatory cytokines (IL-4 and IFN-γ) were increased in kidneys along with significant hyperglycemia. However, treatment of four-month diabetic rats with Withania coagulans (10 mg/kg) for 3 weeks significantly attenuated hyperglycemia and reduced the levels of proinflammatory cytokines in kidneys. In addition, Withania coagulans treatment restored the glutathione levels and inhibited lipid peroxidation along with marked reduction in kidney hypertrophy. The present study demonstrates that Withania coagulans corrects hyperglycemia and maintained antioxidant status and reduced the proinflammatory markers in kidneys, which may subsequently reduce the development and progression of renal injury in diabetes. The results of the present study are encouraging for its potential use to delay the onset and progression of diabetic renal complications. However, the translation of therapeutic efficacy in humans requires further studies.