Immediate and delayed leukocyte apoptosis in two models of peritonitis.

Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity: (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apoptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD1lb expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.

Prenatal exposure to the dopamine agonist SKF-38393 disrupts the timing of the initial response of the suprachiasmatic nucleus to light.

The abuse of social drugs such as cocaine during pregnancy represents enormous risks to the offspring. Recent studies showed that drugs administered to the pregnant rat can activate cell populations in the fetal brain, possibly altering the timing of key neuronal developmental events. The current study examined the ontogeny of light-responsiveness of the neonatal rat suprachiasmatic nucleus using c-FOS protein in SCN nuclei as a marker. The effect of acute administration of the dopamine D1 agonist, SKF-38393, on the development of light responsiveness was also examined. Pregnant dams received either SKF-38393 (10 mg/kg) or vehicle 7 h after dawn on gestational day 20. Litters were then assigned to one of seven experimental time points from 4 h after subjective dark onset on the day of birth (P0-CT16) at 4-h intervals until CT16 on the day after birth (P1-CT16). Half of the pups in each litter were exposed to a 200 lux/2 h light pulse and the other half remained in darkness. Three time points (P1-CT0, P1-CT8 and P1-CT16) were used to examine the prenatal drug effects on light-responsiveness. Light exposure at the time of subjective lights on, the day after birth (P1-CT0), resulted in a significant increase in c-FOS-positive cells. The number of positive cells recorded in the SCN after a light pulse at P1-CT0 and P1-CT8 was significantly less in SKF-38393 pretreated pups compared to vehicle treated animals. The exposure to dopaminergic stimulation during gestation may have altered the timing of development of afferent connections to the fetal SCN, resulting in alteration of the initial response of the circadian timing system to light.

Effect of stimulation of endogenous melatonin secretion during constant light exposure on 6-sulphatoxymelatonin rhythmicity in rats.

When light is presented unexpectedly at night to rats, melatonin production and secretion is acutely inhibited and the time of onset of production on the subsequent night is altered. In a series of experiments, we examined the effects of 6-12 hr light (200 lux) at night on melatonin metabolite excretion (6-sulphatoxymelatonin, aMT.6S). During the light exposure, we administered isoproterenol to stimulate endogenous production of melatonin by the pineal gland to determine if replacement of melatonin would block any phase shifting effects of the light. Exposure to 6 hr of light either during the first or second half of the night suppressed aMT.6S excretion during the light treatment and delayed the onset of melatonin secretion by 3.7 +/- 0.6 and 2.5 +/- 0.6 hr, respectively, compared to a change of 0.5 +/- 0.1 hr in animals maintained in darkness. Twelve hours light exposure (i.e. one night of continuous light) suppressed aMT.6S excretion completely and resulted in a delay in the onset the next night of 2.1 +/- 0.7 hr. When propranolol (10 mg/kg) was administered at 2-hr intervals during darkness, aMT.6S excretion was suppressed throughout the night, but on the subsequent release into constant darkness the onset of excretion was not delayed (0.6 +/- 0.1 hr delay). Administration of isoproterenol (10 mg/kg) to animals in constant light, at the time of expected lights off (CT12), and 5 hr later (CT17) resulted in an increase in melatonin production and aMT.6S excretion that was similar in duration and amount to the control night. The stimulation of endogenous melatonin production failed to block the phase shifting effects of the light exposure and, in fact, appeared to potentiate the delay at least on the first night (4.2 +/- 0.9 hr). The timing of the release into constant darkness following the light treatment had an unexpected effect on melatonin production on the cycle after treatment. Thus, animals exposed to 12 hr light and released into darkness at the normal time of lights off as above had a delay of about 2 hr and excreted 71 +/- 18% of the aMT.6S excreted on a control night. Animals released into darkness at the expected time of lights on failed to excrete more than 20 pmol/hr(i.e. no onset of excretion could be determined) at any time on the first subjective night after light treatment, which was no different from the excretion during the light treatment. On the next subjective night, the onset was delayed as expected and the amount of aMT.6S produced was restored. Treatment with isoproterenol at CT12 and CT17 failed to affect either the amount of aMT.6S excreted on the first subjective night after light treatment or the phase delay on the second night after treatment. The failure to produce melatonin on the first subjective night after 12 hr light exposure and release into darkness at CTO was not due to failure at the level of the pineal gland since injection ofisoproterenol at CT12 and CT17 on the first subjective night after light restored the normal amount of melatonin production. These results suggest that the absence of melatonin during light stimulation at night is not responsible for the phase delay in melatonin production and excretion on subsequent nights. The basis of the failure of the rats to commence melatonin production following 12 hr extended light exposure followed immediately by continuous darkness is not known.

The last fifty years of neonatal surgical management.

Neonatal surgical mortality has steadily fallen over the last five decades. Improved survival does not appear to be related to the introduction of new operative procedures. Most of the basic procedures were developed by 1960. Eight developments appear to be responsible: (1) The growth of pediatric surgery resulted in widespread availability of neonatal surgeons and dissemination of knowledge about newborn surgical emergencies. (2) The parallel growth of pediatric anesthesia, beginning in 1946, provided specialized intraoperative management of the neonate. (3) Understanding neonatal physiology is the key to successful management; major advances occurred between 1950 and 1970. (4) New inventions revolutionized patient care; the transistor (1947) made it possible for medical devices to sense, amplify and control physiologic responses and opened the communication and computer age. (5) Neonatal mechanical ventilation had a prohibitive mortality and was seldom utilized; the development of CPAP and a continuous flow ventilator in the 1970s allowed safe ventilatory support. (6) Total parenteral nutrition (1968) prevented starvation that frequently affected infants with major anomalies. (7) The effective treatment of infection began with the clinical use of penicillin (1941); antibiotics have reduced mortality but infants suffering from the septic syndrome have a prohibitive mortality; cytokine, proinflammatory agent research, and the development of anti-inflammatory and blocking agents in the 1980s have not affected mortality. (8) The establishment of newborn intensive care units (1960) provided an environment, equipment, and staff for effective physiologic management.

Controlled-release melatonin implants delay puberty in rats without altering melatonin rhythmicity.

There is increasing evidence that continuous availability of melatonin via implants can produce the same physiological changes in animals as timed administration of the hormone. The mechanisms underlying this apparent contradiction are not known. In an attempt to gain further understanding of the way continuous melatonin administration affects reproductive activity, the effects of melatonin implants on gonadal development and melatonin production were investigated in rats treated neonatally with testosterone. Five-day-old male rats maintained on a 12L:12D photoperiod were injected with 1 mg testosterone propionate to induce photo-responsiveness and implanted at 21 days of age with novel melatonin implants designed to raise the daytime blood melatonin concentration into the nighttime range, i.e., from less than 60 pM in the controls during the day to 380 +/- 33 pM in the implanted rats. Following 21 days treatment, seminal vesicle and ventral prostate weights of implanted rats were significantly less than the controls (27.0 +/- 1.9 vs. 18.5 +/- 1.5 mg/ 100 g BW (P = 0.003) and 33.8 +/- 2.1 vs. 26.7 +/- 2.2 mg/100 g BW (P = 0.02), respectively). To determine the effect of the implants upon melatonin production, urine was collected at hourly intervals during the last four days of the experiment and the hourly 6-sulphatoxymelatonin (aMT.6S) excretion rate was determined. Rats bearing melatonin implants maintained a rhythm of aMT.6S excretion in 12L:12D, which was indistinguishable from that in the control animals except for a raised daytime excretion of the metabolite. Following one cycle of urinary aMT.6S measurements in the light/dark cycle, the animals were released into constant darkness, with the implants still in place or after their removal four hours before darkness to evaluate the characteristics of the melatonin rhythm in the absence of masking effects of the light/dark cycle. The melatonin rhythm persisted in both control and implanted rats and no differences in the onset, offset, or amplitude could be determined. The results of this study indicate that, like many other mammals, for laboratory rats controlled continuous release of melatonin can mimic the effects of short daylength or timed melatonin administration. Despite the reproductive consequences of continuous melatonin delivery, the timing of endogenous melatonin production is unaffected.

Serotonin agonists mimic the phase shifting effects of light on the melatonin rhythm in rats.

The effect of serotonin agonists on the rhythmic excretion of the melatonin metabolite 6-sulphatoxymelatonin was examined in rats. The animals were maintained in 12L:12D and administered saline, quipazine (10 mg/kg), (+/-)-2-propylamino-8-hydroxy-1,2,3,4-tetrahydronaphthalene hydrobromide (8-OH-DPAT, 5 mg/kg) or buspirone (10 mg/kg), 4 h after dark (ZT16). All three drugs caused an acute, transient suppression of 6-sulphatoxymelatonin excretion and a significant delay (P < 0.01) in the onset of the nocturnal rise on the following night of 2.1 +/- 0.6, 1.4 +/- 0.7 and 1.5 +/- 0.3 h respectively while saline administration had no effect (0.4 +/- 0.2 h delay, P > 0.01). To examine the effects of the time of day of agonist administration, groups of rats were treated with quipazine (10 mg/kg) or 8-OH-DPAT (5 mg/kg) 18, 24 or 30 h after the initiation of continuous darkness (CT6, CT12 or CT18) and monitored for a further two nights. Quipazine but not 8-OH-DPAT injection at CT6 resulted in a small but significant delay in the onset of 6-sulphatoxymelatonin excretion on the following night (1.0 +/- 0.2 h and 0.3 +/- 0.2 h) while treatment with both agonists at CT12 failed to affect the onset of excretion (0.8 +/- 0.2 and 0.1 +/- 0.2 h). When quipazine (10 mg/kg) was administered at CT18, 6-sulphatoxymelatonin excretion was acutely suppressed for the rest of the night and there was a large significant delay in the onset of 6-sulphatoxymelatonin excretion (1.2 +/- 0.2 h) while a smaller delay was observed following 8-OH-DPAT administration (0.8 +/- 0.2 h). The acute suppression of 6-sulphatoxymelatonin excretion and subsequent phase delay following quipazine treatment at CT18 was also evident at doses of 1 mg/kg (1.6 +/- 0.4 h) and 3 mg/kg (1.5 +/- 0.6 h). These results show that peripheral administration of serotonin agonists active at 5HT1a/5HT7 receptors mimic the dual effects of light on melatonin production in the rat and raise the possibility that serotonin pathways are more important in mediating the effects of retinally perceived light in the rat than previously believed.

Cardinal clinical signs in the differentiation of heart murmurs in children.

OBJECTIVE: To determine the diagnostic accuracy of clinical assessment of heart murmurs in children and specific clinical features that are predictive of cardiac disease. DESIGN: Concurrent case series with pretest-posttest assessment. SETTING: Pediatric cardiology outpatient clinic. PARTICIPANTS: Five full-time academic pediatric cardiologists. MEASURES: For each of 222 consecutive patients who were seen for first-time evaluation of a heart murmur, the clinical findings and diagnostic impressions were recorded after clinical assessment. The results of electrocardiograms and echocardiograms were then reviewed, and changes in diagnostic impressions were recorded and compared with the original impressions. RESULTS: The prevalence of cardiac disease was 33%. Clinical assessment differentiated those patients with pathologic murmurs with a sensitivity of 92%, specificity of 94%, positive predictive value of 88%, and negative predictive value of 96%. If diagnostic uncertainty was considered an indication for echocardiography, then sensitivity and specificity increased to 97% and 98%, respectively. Missed disease included only trivial or minor lesions. Clinical features that were independently predictive of the presence of disease included murmurs that were pansystolic (odds ratio [OR], 54.0), grade 3 or more in intensity (OR, 4.84), heard best at the left upper sternal border (OR, 4.24) and harsh in quality (OR, 2.37), and the presence of an abnormal second heart sound (OR, 4.09) and an early or midsystolic click (OR, 8.35). CONCLUSIONS: Clinical assessment by a pediatric cardiologist is sufficient to distinguish pathologic from innocent heart murmurs. A genetic approach by using specific clinical features that are independently associated with disease may have some practical utility to noncardiologists.

Effect of NMDA receptor blockade on melatonin and activity rhythm responses to a light pulse in rats.

The possible role of the excitatory amino acids as mediators of the acute suppression and subsequent delay by light of pineal melatonin production was studied in rats using the NMDA receptor antagonist MK-801. Saline or MK-801 in doses up to 3 mg/kg (IP), was administered 15 min before a 15-min light pulse (200 lx), 4 h after dark onset, and the excretion of 6-sulphatoxymelatonin (aMT.6S) determined. Under these conditions saline injected/light exposed animals exhibited an acute, total but transient suppression of urinary aMT.6S excretion and a delay in the onset of aMT.6S the following night of 1.5 +/- 0.2 h. MK-801 failed to block either the acute or phase delaying effect of light (onset delayed by 2.2 +/- 0.4 h). Pretreatment with MK-801 (3 mg/kg) failed to block the effects of shorter, less intense light pulses 15 min before the pulse (e.g., 1 min/2 lx; onset delayed by 2.0 +/- 0.4 h following saline, 1.5 +/- 0.1 h following MK-801) or 60 min before a short duration low intensity pulse. In other experiments MK-801 (1 and 3 mg/kg) failed to affect aMT.6S excretion when injected in the dark at the time of lights out or 4 h after dark onset. NMDA (10 and 30 mg/kg) injection at the time of lights out or 4 h after darkness did not mimic the effects of a light pulse by decreasing aMT.6S excretion or causing a delay in the onset of excretion the following night. Finally MK-801 (3 mg/kg) injected 4 h after dark failed to block the phase delaying effects of a 15 min light pulse (200 lx) on running activity in rats. These results do not support the hypothesis that excitatory amino acids in the retino-hypothalamic tract acting on the NMDA receptor subtype and terminating in the suprachiasmatic nucleus mediate the photic influences upon rat pineal melatonin and activity rhythms.

Melatonin binding sites and their role in seasonal reproduction.

The pineal gland has a major role in the translation of scotophase duration into a hormonal signal by the hormone melatonin. Animals such as sheep, goats and deer use the seasonal variation of this signal to coordinate reproductive behaviour with the environment. Despite intensive research over more than 30 years the site(s) of action of melatonin and the resultant intracellular responses are still not clear. This review discusses recent work that has localized the site of action of melatonin in sheep using administration into the hypothalamus in vivo as well as studies on putative melatonin receptors in the pars tuberalis and brain. There is clear evidence that melatonin acting at the level of the pars tuberalis is involved in the seasonal regulation of prolactin secretion, but the evidence for involvement of the pars tuberalis in seasonal reproduction is not compelling. Localized administration of melatonin to the sheep brain revealed that areas anatomically distinct from the pars tuberalis, the ventromedial and arcuate nuclei, simulated seasonal reproductive changes in rams and ewes. Recent studies on brain melatonin binding sites in our laboratory have shown that an antagonist of tissue transglutaminase, Bacitracin, as well as substrates for the enzyme inhibit binding of melatonin to brain membranes. As a working hypothesis, we propose that pineal melatonin secretion alters seasonal reproduction by interactions with a neural transglutaminase at the synapse of neurones involved in the control of GnRH secretion. Synaptic transglutaminase is implicated in the control of the release of neurotransmitter via the synaptic vesicle associated protein, synapsin 1; activation of transglutaminase results in the covalent modification of synapsin 1 such that vesicles are not released from the cytoskeleton. Seasonal variation in the duration of melatonin secretion may result in similar variations in the duration of suppression and activation of transglutaminase. The resultant changes in transmitter release may then be responsible for the seasonal neuronal plasticity previously observed in GnRH neurones.