Subramanian Dhanasekaran

Contrasting actions of diesel exhaust particles on the pulmonary and cardiovascular systems and the effects of thymoquinone

BACKGROUND AND PURPOSE Acute exposure to particulate air pollution has been linked to acute cardiopulmonary events, but the underlying mechanisms are uncertain.

Pulmonary exposure to diesel exhaust particles promotes cerebral microvessel thrombosis: Protective effect of a cysteine prodrug l-2-oxothiazolidine-4-carboxylic acid

Inhaled particulate matter is associated with increased cerebro- and cardiovascular events. However, the systemic mechanisms underlying these effects remain unclear. In the present study, we investigated the mechanisms underlying the relationship between airway and systemic inflammation and pial cerebral venular thrombosis, 24h after intratracheal (i.t.) instillation of diesel exhaust particles (DEP; 15 or 30 microg/mouse) or saline (control). Doses of 15 and 30 microg/mouse induced a dose-dependent macrophage and neutrophil influx into the bronchoalveolar lavage (BAL) fluid with elevation of total proteins and Trolox equivalent antioxidant capacity (TEAC), but without IL-6 release. Similarly, in plasma, IL-6 concentrations did not increase but the TEAC was significantly and dose-dependently decreased. The number of platelets and the tail bleeding time were both significantly reduced after exposure to DEP (30 microg). Interestingly, the same dose showed platelet proaggregatory effect in mouse pial cerebral venules. Pretreatment with the cysteine prodrug l-2-oxothiazolidine-4-carboxylic acid (OTC, 80 mg/kg) 24 and 1h before i.t. DEP (30 microg), abolished the DEP-induced macrophage and neutrophil influx, and the increase of TEAC in BAL. Lung histopathology confirmed the protective effect of OTC on DEP-induced lung inflammation. OTC also reversed the decrease of TEAC concentrations in plasma, the shortening of the bleeding time, and the thrombotic effect of DEP in pial cerebral venules. We conclude that pulmonary exposure to DEP cause oxidative stress responsible, at least partially, for the pulmonary and systemic inflammation and thrombotic events in the pial cerebral microvessels of mice. OTC pretreatment abrogated these effects through its ability to balance oxidant-antioxidant status.

Time-course effects of systemically administered diesel exhaust particles in rats

Nanosized fraction of particulate air pollution has been reported to translocate from the airways into the bloodstream and act on different organs. However, the direct effect of these translocated particles is not well understood. In this study, we determined the time-course (6 h, 18 h, 48 h and 168 h) effects of the systemic administration of 0.02 mg/kg diesel exhaust particles (DEP) on systolic blood pressure (SBP), systemic inflammation, oxidative status, and morphological alterations in lungs, heart, liver and kidneys in Wistar rats. SBP was significantly decreased at 6 h (P < 0.05) but no significant effects have been observed at later time points. The leukocyte numbers were increased at 6 h (P < 0.05) and 18 h (P < 0.05). However, the platelet numbers were significantly decreased (P < 0.05) 6 h following the systemic administration of DEP. The IL-6 concentrations in plasma was increased at 6 h (P < 0.05) and 18 h (P < 0.05). Similarly, superoxide dismutase activity was significantly increased at 6 (P = 0.01) and 18 h (P < 0.05) following DEP exposure. The direct addition of DEP (0.1-1 microg/ml) to untreated rat blood significantly induced in vitro platelet aggregation in a dose-dependent fashion. The activation of intravascular coagulation was confirmed by a dose-dependent shortening of activated partial thromboplastin time and the prothrombin time following in vitro exposure to DEP (0.25-1 microg/ml). Histological analysis revealed the presence of DEP in the lungs, heart, liver and kidneys. However, the morphological changes were only observed in the lungs, where the presence of infiltration of inflammatory cells was observed as early as 6 h, increased at 18 h, and decreased in intensity at 48 h and at 168 h. We conclude that the direct systemic administration of DEP caused acute effect on SBP (6 h) and systemic inflammation and oxidative stress mainly at 6 h and 18 h. Despite the presence of DEP in lungs, heart, liver and kidneys, the histopathological changes were only seen in the lung which suggests that, at the dose and time-points investigated, DEP cause inflammation and have a predilection for pulmonary tissue.

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.

Anisotropic propagation in the small intestine.

Abstract  Measuring propagation anisotropy may help in determining the tissue layers involved in the propagation of electrical impulses in the intestine. We used 240 extracellular electrograms recorded from the isolated feline duodenum. The conduction velocities of slow waves and of individual spikes were measured from their site of origin into all directions. Both slow waves and spikes propagate anisotropically in the small intestine but in different directions and to a different degree. Slow waves propagated anisotropically faster in the circumferential (1.7 ± 0.8 cm s−1) than in the axial direction (1.3 ± 0.5 cm s−1; P < 0.001). Spikes, on the other hand, propagated faster in the longitudinal direction (7.8 ± 4.5 cm s−1) than in the circumferential direction (3.3 ± 4.3 cm s−1; P < 0.001). Furthermore, the average conduction velocity of spikes (6.3 ± 4.5 cm s−1) was significantly higher than that of slow waves (1.5 ± 1.1 cm s−1; P < 0.001). The anisotropic propagation of spikes supports the argument that these propagate in the longitudinal muscle layer. The anisotropic propagation of slow waves may be the result of the interaction between the myenteric layer of interstitial cells of Cajal and their electrotonic connection to both the longitudinal and the circular muscle layer.

Sensory and autonomic nerve changes in the monosodium glutamate‐treated rat: a model of type II diabetes

Rats that had been injected with monosodium glutamate (MSG) neonatally were studied for up to 70 weeks and compared with age‐matched control rats to study changes in glucose tolerance and in sympathetic and sensory nerves. At 61 and 65 weeks of age, there were significant differences in glucose tolerance between the MSG and control groups, and the MSG group had raised fasting blood glucose. These changes were not associated with changes in the number of β‐cells in the islets of Langerhans. In addition, the diabetic MSG‐treated rats had central obesity and cataracts. Hypoalgesia to thermal stimuli was present in MSG‐treated rats as early as 6 weeks and persisted at 70 weeks. However, no differences were observed in the distribution of substance P, the neurokinin‐1 receptor or calcitonin gene‐related peptide in the dorsal horn of L3–L5 at this age (70 weeks). Diabetic MSG‐treated animals at 65 and 70 weeks of age had significantly reduced noradrenaline concentrations in the heart, tail artery and ileum, while concentrations in the adrenal gland and corpus cavernosum were significantly increased. There was also a significant increase in adrenal adrenaline, dopamine and serotonin, largely attributable to changes in weight of the adrenal gland in the MSG‐treated animals. The results indicate that MSG‐treated animals develop a form of type II diabetes by about 60 weeks of age, and that there are significant changes in amine levels in various tissues associated with these developments.

Acute respiratory and systemic toxicity of pulmonary exposure to rutile Fe-doped TiO2 nanorods

Nanomaterials are extensively used in medicines, industry and daily life, but little is known about their possible health effects. Titanium dioxide (TiO₂) nonmaterial-based photocatalysis is useful in the complete mineralization of organic pollutants in waste water and air. While the Fe-doping of TiO₂ enhances their photocatalytic activity, their potential pathophysiologic effects are unknown. Here, rutile Fe-doped (9%) pure titanium dioxide (TiO₂) nanorods were prepared and characterized. Subsequently, we assessed the acute (24 h) pulmonary and extrapulmonary effects of intratracheal (i.t.) instillation of these nanorods (1 and 5 mg/kg) in Wistar rats. In the bronchoalveolar lavage, the treatment induced a significant and dose-dependent increase of neutrophils, an increase of interleukin-6 (IL-6, at 5 mg/kg), and caused a dose-dependent-decrease of superoxide dismutase (SOD) activity. The lung sections of rats exposed to rutile Fe-TiO₂ nanorods showed infiltration of inflammatory cells in dose-dependent manner. Similarly, the heart rate, systolic blood pressure, plasma IL-6, and leukocyte and platelet numbers were increased at 5 mg/kg. The plasma SOD and reduced glutathaione activities were dose-dependently decreased after exposure to the nanorods. Histopathologically, the liver showed mild inflammatory cells infiltration of few portal tracts, but the kidneys and heart were unaffected. In plasma, the levels of lactate dehydrogenase and hepatic enzymes, i.e., alanine aminotranferease and aspartate aminotransferase were increased significantly. The in vitro exposure of human lung cancer cells NCI-H460-Luc2 and human hepatoma cells HepG2 to FeTiO₂ (6.25-100 μg/ml) dose-dependently reduced cellular viability. Also, the In vitro direct addition of these nanorods (0.1-1 μg/ml) to untreated rat blood, significantly and dose-dependently induced platelet aggregation. In conclusion, exposure to rutile Fe-TiO₂ promotes pulmonary and systemic inflammation and oxidative stress. It affects the liver, enhances thrombotic potential, heart rate and systolic blood pressure. Moreover, the rutile Fe-TiO₂ elicited direct toxicity on NCI-H460-Luc2 and HepG2 cells.

Slow wave propagation and plasticity of interstitial cells of Cajal in the small intestine of diabetic rats

The number of myenteric interstitial cells of Cajal (ICC‐MY), responsible for the generation and propagation of the slow wave in the small intestine, has been shown to decrease in diabetes, suggesting impairment of slow‐wave (SW) propagation and related motility. To date, however, this expected decrease in SW propagation has neither been recorded nor analysed. Eleven rats were treated with streptozotocin and housed in pairs with 11 age‐matched control animals. After 3 or 7 months, segments of duodenum, jejunum and ileum were isolated and divided into two parts. One part was processed for immediate freezing, cryosectioning and immunoprobing using anti‐c‐Kit antibody to quantify ICC‐MY. The second part was superfused in a tissue bath, and SW propagation was recorded with 121 extracellular electrodes. In addition, a cellular automaton was developed to study the effects of increasing the number of inactive cells on overall propagation. The number of ICC‐MY was significantly reduced after 3 months of diabetes, but rebounded to control levels after 7 months of diabetes. Slow‐wave frequencies, velocities and extracellular amplitudes were unchanged at any stage of diabetes. The cellular automaton showed that SW velocity was not linearly related to the number of inactive cells. The depletion of ICC‐MY is not as severe as is often assumed and in fact may rebound after some time. In addition, at least in the streptozotocin model, the initial reduction in ICC‐MY is not enough to affect SW propagation. Diabetic intestinal dysfunction may therefore be more affected by impairments of other systems, such as the enteric system or the muscle cells.

Two‐dimensional high‐resolution motility mapping in the isolated feline duodenum: methodology and initial results

Several types of electrical events occur in the small intestine but their spatial and temporal contributions to overall motility are not clear. In order to quantify local motility in greater detail, a new technique of recording and analysing movements at multiple sites was developed. Use was made of isolated segments of feline duodenum superfused in a tissue bath. Multiple marker dots (20–75) were placed on the serosal surface by applying fine spots of candle soot in rectangular arrays (1–2 mm dot separation). A digital video camera was used to record spontaneous movements of the dots for periods of 10–30 min. After each experiment, 4–6 periods (10–60 s each) of video frames were transferred to a computer (25 fps, 720 × 576 pixels) and the movements of the dots was tracked every 40 ms using custom‐made software. Initial results (eight experiments) show that spontaneous motility is remarkably variable, both in space and time. Three types of movement could be discerned: (i) periodic, rolling or pendular movements, with a frequency of approximately 15 min–1 occurring predominantly in the longitudinal direction; (ii) twitches, wherein a subset of dots were suddenly displaced longitudinally; and (iii) drifts of most of the dots in a circular or oblique direction. All three types of movement occurred throughout every recording session although their relative magnitudes differed greatly from moment to moment. Occasionally, it was possible to detect propagated ‘contractions’ with an apparent velocity of 10 mm s–1. Immobilizing the preparation at one point by inserting a needle through the middle of the array of markers had a negligible effect on the displacements, whereas application of verapamil (10–5  mol L–1) reduced or abolished motility. In summary, we present a new technique to map in detail two‐dimensional motility at the surface of the intestine. Initial results seem to suggest that motility at the serosal surface is not uniform and highly anisotropic.

The Effect of Sildenafil on Cisplatin Nephrotoxicity in Rats

Sildenafil, the first drug for erectile dysfunction, has cardiopulmonary protective actions. A recent study has reported that sildenafil given intraperitoneally (i.p.) attenuated cisplatin (CP)-induced nephrotoxicity. Here, we evaluated whether sildenafil, given by two different routes and at two different doses, can attenuate CP-induced nephrotoxicity and would also affect renal haemodynamics in CP-treated rats. Six groups of rats were treated with saline (controls), CP [5 mg/kg, intraperitoneally (i.p.) once], sildenafil (0.4 mg/kg/day, i.p. for 5 days), sildenafil (0.4 mg/kg/day i.p. for 5 days) plus CP (5 mg/kg, i.p., once), sildenafil [10 mg/kg/day, subcutaneous (s.c.) for 5 days] or sildenafil (10 mg/kg/day, s.c. for 5 days) plus CP (5 mg/kg, i.p. once). Five days after the end of the treatments, urine was collected from all rats, which were then anaesthetized for blood pressure and renal blood flow monitoring. This was followed by intravenous (i.v.) injection of norepinephrine for the measurement of renal vasoconstrictor responses. Thereafter, blood and kidneys were collected for measurement of several biochemical, functional and structural parameters. CP reduced body-weight and renal blood flow but did not affect norepinephrine-induced renal vasoconstriction. It increased the plasma concentrations of urea and creatinine, and reduced creatinine clearance. CP caused extensive renal tubular necrosis, increased urine volume and N-acetyl-β-D-glucosaminidase activity. When sildenafil (0.4 mg/kg/day, i.p. for 5 days) was combined with cisplatin, there was a dramatic improvement in renal histopathology, reduction in N-acetyl-β-D-glucosaminidase and increase in renal blood flow. However, sildenafil (10 mg/kg/day, s.c. for 5 days) did not affect CP nephrotoxicity, suggesting the importance of dose and route selection of sildenafil as a nephroprotectant.