Cerebral palsy can result from third-trimester hypoxic-ischaemic injury to the brain’s striatum. Mild or moderate hypothermia may offer neuroprotection. Mild hypothermia (3˚C) for 3 h post-insult delayed striatal injury, but was not neuroprotective. Moderate hypothermia (5–6˚C) for 3–6 h post-insult has yielded conflicting evidence on striatal protection. This study aimed to use stereological methods to determine whether mild or moderate hypothermia for 6 h post-insult is neuroprotective.

Hypoxia-ischaemia was induced in anaesthetised, male, postnatal day (PN) 7 Sprague-Dawley rats by right common carotid artery ligation, followed 2 h later by exposure to 8% oxygen for 1.5 h. Normothermic pups (n = 16) were maintained at a rectal temperature of 35˚C, while hypothermic pups were cooled to a rectal temperature of either 33˚C (mild, n = 8) or 29˚C (moderate, n = 8), for 6 h. Animals were sacrificed on PN 14 and 40 μm serial sections were cut through the entire right striatum. The total number of striatal medium-spiny neurons was determined stereologically using the optical disector/Cavalieri technique. Results were compared statistically using the Mann-Whitney U test.

There was no significant difference in the total number of medium-spiny neurons in the right striatum after mild hypothermia versus normothermia (1 024 000 ± 154 000 vs 1 107 000 ± 179 000; mean ± SEM, p = 0.7, two-tailed test). There was also no significant difference in the total number of medium-spiny neurons in the right striatum after moderate hypothermia vs normothermia (1 744 000 ± 173 000 vs 1 432 000 ± 229 000; p = 0.7, two-tailed test).

These results suggest that neither mild nor moderate hypothermia is neuroprotective for striatal medium-spiny neurons after hypoxic-ischaemic injury during the third-trimester equivalent. Thus hypothermia alone may not be useful in the clinical setting.

Male and female Wistar rats used for this study were offspring of female rats maintained on a control low-soy diet (containing 112 μg/g isoflavanoid) prior to conception through to weaning. After weaning, the juvenile rats were fed the same low-soy diet into adulthood. Six adult male rats were transferred to the experimental high-soy diet (465 μg/g isoflavanoid); the remaining male rats were continued on the low-soy control diet (n = 8). On days 3, 6, 12 and 25 following the commencement of the high-soy diet, the male rats were housed overnight individually with pro-estrous female rats. Female rats were housed separately until parturition. Litter size and sex ratios were recorded. After the final mating, the male rats were killed and the epididymides removed. Sperm counts were performed on the initial segment, caput, corpus and cauda of one epididymis from each rat.

At 25 days there were fewer sperm in the initial segment (p <0.05), corpus (p <0.05) and cauda (p <0.01) epididymides of experimental rats, as compared with control rats. The litter sizes for the treatment groups showed an exposure-dependent response. The litter sizes of the day 3 (p <0.01) and day 6 (p <0.05) high-soy groups were significantly lower than the low-soy group, while litter sizes of the day 12 and 25 groups were not significantly different from low-soy-fed animals. The sex ratios of the litters from both groups were not significantly different.

In conclusion, short-term exposure to high phytoestrogen levels reduces male fertility. The mechanisms involved in these changes are being investigated.

Investigations of developmental alcohol exposure in animal models have demonstrated central nervous system dysfunctions representative of fetal alcohol syndrome or fetal alcohol effects. The cerebellar Purkinje cells (Pcells) have been studied extensively in the rat, and a temporal window of vulnerability to ethanol-induced death exists from postnatal day (PD) 4 to 6. The endpoint of this temporal window has been thoroughly investigated, but the time of onset has not. This study aimed to determine the earliest postnatal time point at which Pcells are vulnerable to ethanol-induced death.

On PD 0, pups from timed, pregnant Sprague-Dawley dams were randomly assigned to two groups – alcohol-exposed (AE) (4.5 g/kg/day given in two feedings via intragastric intubations), or intubation controls (IC). On PD 0–4, pups were perfusion-fixed at 8, 10 and 12 hours after the initial alcohol exposure. Previous work has shown that maximal ethanol-induced Pcell death is visualised at these time points. Cerebelli were removed, wax-embedded, and serial 5 μm thick sections cut. Sections were immunolabelled for active caspase-3, an enzyme considered to be a marker of the initiation of irreversible cell death via apoptosis. The total number of caspase-3-positive Pcells in the whole cerebellar vermis were counted using the physical dissector/fractionator method.

The mean total number of labelled Pcells was significantly greater (p <0.05) in ethanol-treated animals compared with controls for all days studied eg, PD 1 at 10 h: AE = 2853.50 ± 1510.55 (mean ± SD); IC = 390.50 ± 238.60 (n = 6 /group/time point).

This study shows that the window of vulnerability for Pcells to ethanol exposure during development is longer than previously thought. This has implications for binge alcohol consumption in the third trimester of human pregnancy.

We have previously shown that epicatechin gallate (ECG) significantly improves wound healing and scar formation. In this study we have extended these observations by determining the effect of ECG on new blood vessel formation (angiogenesis), using the endothelial cell marker CD31. An increase in cyclo-oxygenase (COX) and its product prostaglandin E2 have been shown to increase the expression of vascular endothelial growth factor (VEGF). Therefore COX activity, COX isoforms and VEGF levels were also measured following ECG administration.

Full thickness incisional wounds were initiated in male Sprague-Dawley rats (250 ± 25 g). Animals were treated with either 50 μl saline (controls) or 0.8 mg/ml ECG, intradermally for 7 days. Wounds were harvested at days 1, 3, 7, 14 and 21 days post-wounding, from 6–8 rats per group, per time point. Unwounded skin (day 0) served as a control.

Blind immunohistochemical labelling with an antibody to CD31, showed a significant increase in the number of blood vessels on days 1 (p <0.01) and 3 (p <0.05) post-wounding in the ECG-treated group compared with controls. Western blot analysis (using Biorad, Quantity One image analysis software) showed a significant increase in the protein levels of VEGF on days 1 (p <0.01) and 3 (p <0.05). COX activity reached a peak at day 3 and thereafter declined. COX protein levels mirrored this activity with COX-2 being the predominant isoform. COX-2 levels were significantly elevated on days 1 (p <0.05) and 3 (p <0.01) compared with the equivalent controls.

The significant increase in COX-2 and resultant increase in the expression of VEGF may be one of the contributing factors responsible for the accelerated angiogenesis and improved scarring in ECG-treated rats.

Leptin is an adipose-derived hormone that regulates body fat levels by decreasing appetite. Leptin acts in the hypothalamus through signal transduction mechanisms involving the activation of STAT3 proteins through phosphorylation. Despite increased serum leptin concentrations during pregnancy, maternal food intake increases, leading to deposition of fat. We have previously demonstrated that intracerebroventricular leptin administration is unable to suppress food intake during pregnancy, as it does in non-pregnant rats. Hence, pregnancy appears to be a state of hypothalamic leptin resistance. To examine leptin signal transduction during pregnancy, STAT3 phosphorylation was measured following intracerebroventricular administration of leptin to non-pregnant (diestrous) and pregnant rats. Our hypothesis was that leptin-induced phosphorylation of STAT3 is impaired during pregnancy.

Following a 16-hour fast, leptin (4 μg) or vehicle was injected into the left lateral ventricle of diestrous (n = 16) and day 14 pregnant rats (n = 16). Rats were killed 30 minutes later and brains were removed. Hypothalamic nuclei were microdissected from frozen coronal brain sections using a punch technique. Tissue was then processed for Western blot analysis. Membranes were probed with a phosphorylated STAT3-specific antibody, then stripped and reprobed with a STAT3-specific antibody. Immunoreactivity was quantified by densitometry.

In the arcuate nucleus, leptin treatment increased phosphorylated STAT3 (P-STAT3) levels in diestrous rats, but not in pregnant rats. In the ventromedial nucleus there were lower levels of STAT3 in pregnant rats compared with diestrous rats. Leptin treatment also led to a smaller percentage increase in P-STAT3 in pregnant rats. These results indicate that during pregnancy there is a decrease in activation of the STAT3 signalling pathway, thus supporting the hypothesis that during pregnancy leptin signal transduction is impaired. This impairment appears to result from a decrease in the potential for STAT3 to become phosphorylated and/or a decrease in the amount of STAT3, depending on the specific hypothalamic nucleus.

Attention-deficit/hyperactivity disorder (ADHD) is a common childhood disorder characterised by increased locomotor activity and cognitive impairment. These symptoms can be successfully treated with the psychostimulants methylphenidate and D-amphetamine.

A commonly used animal model of ADHD is the spontaneously hypertensive rat (SHR), which shows comparable behaviours. In order to identify cellular differences underlying the increased locomotor activation in ADHD, in vivo intracellular records were made from spiny projection neurons in urethane-anaesthetised rats, from the SHR, their genetic control, the Wistar-Kyoto (WKY) and the normal Wistar (WI) strains. Action potential amplitude, threshold and duration, afterhyperpolarisation amplitude, input resistance and membrane potentials in the UP and DOWN states were measured in 33 neurons (n = 11 per strain). There was a significant difference in the action potential threshold (one-way ANOVA; p <0.05). The SHR threshold (-59.9 ± 7.4 mV, mean ± SD) was significantly hyperpolarised in comparison with that of the WKY (-49.4 ± 5.0 mV) and WI (-52.5 ± 7.7 mV) strains. There were no significant differences in other measured parameters.

The difference in spiny projection neuron action potential threshold could reflect differences in membrane conductances active at the time of action potential generation. Many of the currents present are modulated by dopamine neurotransmission, which may be impaired in both ADHD and the SHR. Preliminary results from the SHR strain (n = 7) indicate that intraperitoneal administration of D-amphetamine (0.5 mg/kg) has at least a short-term depolarising effect on the spiny projection neuron action potential threshold level (pre-D-amphetamine -52.5 ± 3.8 mV; post-D-amphetamine -48.9 ± 6.8 mV; p <0.05 one-tailed paired t test). These results could provide evidence for the cellular action of psychostimulants used in the treatment of ADHD.

The effect of therapy using a cervico-mandibular support collar (CMSC) to manage obstructive sleep apnoea (OSA) was compared with standard therapy, nasal continuous positive airway pressure (nCPAP). The pathogenesis of OSA includes repetitive periods of partial or complete upper airway obstruction during sleep and symptoms such as daytime sleepiness and decreased vigilance. OSA may be influenced by the relative positions of the cranium, mandible and cervical spine. The CMSC was thus designed to reduce the risk of obstruction by preventing both the downward movement of the mandible and neck flexion during sleep.

Ten adult subjects with mild–moderate OSA (apnoea-hypopnoea index (AHI) 10–60/h) completed the study. Subjects received treatment with CMSC or nCPAP each for one month in random order. An overnight polysomnogram, with the device in situ, and symptom questionnaires were completed at the end of each treatment period. Head posture was measured from cephalograms taken with and without the CMSC. The study was analysed on an intention-to-treat basis. Treatment success was defined as AHI ≤10/h and symptom resolution.

The CMSC maintained the head in the natural head position and prevented downward movement of the mandible during sleep. However, treatment success with the CMSC was achieved in only 2/10 subjects, partial success in 2/10, and in six subjects there was no therapeutic benefit. AHI (mean ± SD) at baseline was 29 ± 13/h and with CMSC, 27 ± 17/h. In contrast, treatment success was achieved in 7/10 subjects using nCPAP (10 ± 8/h). Subjective reporting by subjects, though not significantly different, indicated a trend towards improvement in daytime symptoms and better compliance with the CMSC compared with nCPAP.

The success of the CMSC in maintaining the natural postural relationships of the cranium, mandible and cervical spine was insufficient to maintain airway patency. It is concluded that factors in addition to posture influence airway patency in OSA subjects during sleep.

International guidelines recommend a range of confirmatory tests for the diagnosis of asthma. These are based largely on identifying variable airflow obstruction and responses to bronchodilators or a trial of steroid. More recently, attention has focussed on assessing airway inflammation using exhaled nitric oxide (eNO) measurements and the analysis of induced sputum, but systematic comparisons to confirm their diagnostic role have not been made.

We investigated 47 consecutive patients who were referred by their general practitioner with symptoms suggestive of asthma. The diagnosis or exclusion of asthma was confirmed according to current internationally agreed gold standards following comprehensive investigation. Sensitivities and specificities for a diagnosis of asthma were derived for peak flow measurements, spirometry, and changes in these parameters following a trial of oral steroid. Comparisons were made against measurements of eNO and sputum cell counts.

Asthma was diagnosed in 17 patients (36%), with the remaining 30 patients having alternative diagnoses (chronic rhinosinusitis (13, 28%), extended post-viral respiratory syndrome (8, 17%), gastro-oesophageal reflux disease (6, 13%), eosinophilic bronchitis (2, 4%), chronic obstructive pulmonary disease (1, 2%)).

Mean eNO levels were significantly higher in the asthmatic compared with the non-asthmatic patients (21.2 ppb (95% CI 14.6, 27.8) and 6.7 ppb (95% CI 4.9, 8.4) respectively, p <0.001). The sensitivity for each of the conventional tests (morning–evening peak flow variation, spirometry measurement and response to oral steroid) was low (0 to 35%), but much higher for eNO (71–88%) and sputum eosinophils (86%). Although specificity was higher for the conventional tests, it was still greater than 80% for both eNO and eosinophils. Overall, eNO and eosinophils had significantly greater predictive values compared with the conventional tests.

Currently recommended tests to confirm asthma have relatively low diagnostic accuracy. In contrast, eNO measurements and induced sputum analysis offer superior outcomes, with the advantage that the eNO test is quick and easy to perform.

Amyloid-β is a neurotoxic peptide involved in the pathophysiology of Alzheimer’s disease. Less certain is the involvement of secreted-amyloid precursor protein (sAPP), a neurotrophic, memory-enhancing fragment cleaved from the same precursor protein as amyloid-β. Long-term potentiation (LTP), an activity-dependent enhancement of synaptic transmission, is a model used to study memory mechanisms. The present study investigates the involvement of sAPP in LTP, generated in the rat hippocampus in vivo.

Adult male rats anaesthetised with urethane were implanted bilaterally with a recording electrode and cannula in the dentate gyrus, and a stimulating electrode in the perforant path. In the first experiment, after baseline recordings of evoked synaptic potentials, either an antibody targeted to sAPP, a control antibody with no target sequence, or saline was injected into the dentate gyrus. LTP was induced by high-frequency stimulation (HFS; 50 trains), and responses were monitored for 3.5 h. The anti-APP group showed a sustained and significant reduction in LTP of the synaptic potentials (26 ± 2%, mean ± SEM, n = 5, p <0.0001; paired t test) compared with the combined control group (57 ± 9%, n = 7). In the second experiment, after baseline recordings, sAPP (100 nM) was injected into the dentate gyrus. LTP was induced 10 min later by a reduced number of HFS trains (20) to permit observation of LTP enhancement. Shortly after induction, there was a significantly increased initial LTP in the sAPP group (53 ± 4%, n = 5, p <0.001) compared with the saline group (32 ± 5%, n = 5), but this effect was not persistent.

Taking these results together, the endogenous release of sAPP in the hippocampus may contribute to the induction of LTP, and therefore a loss of sAPP may contribute to the memory deficits in Alzheimer’s disease.

A number of checkpoints operate during the cell cycle to ensure accurate replication and segregation of chromosomes. The mitotic spindle checkpoint is activated and arrests cells in prometaphase if a single chromosome is incorrectly attached to the spindle microtubules. If the spindle checkpoint is non-functional then cells may ultimately become aneuploid and possibly tumourigenic. A variety of proteins have been implicated in the spindle checkpoint but a full understanding of how this pathway functions is incomplete. The aim of this investigation was to identify further components of the spindle checkpoint.

A yeast two-hybrid study was carried out to screen for new protein interactions with the mouse checkpoint protein Bub1b. Proteins previously known to interact with Bub1b were identified (Bub3 and Cdc20) as well as a small number of new interactors. One of these, a protein phosphatase 2A (PP2A) regulatory sub-unit was identified multiple times and this interaction was further investigated.

PP2A is a hetero-trimeric enzyme comprising structural, catalytic and regulatory sub-units. The regulatory sub-unit determines substrate specificity and is encoded by a large number of alternative genes in several gene families. The gamma regulatory sub-unit of the B56 family was identified in this study, and subsequently several other genes from this family (B56 beta, delta and epsilon) as well as three B56gamma alternative splice variants were cloned and shown also to interact with Bub1b.

Subcellular localisation of the proteins has been determined using transient expression of green fluorescent protein fusions, as well as the effect of over-expression on mitotic checkpoint function. The Bub1b site of protein interaction with these phosphatase regulatory sub-units has been localised to within a region of 12 amino acids.