Labile Period Research Articles

Abstract 6: Frequency, Risk Factors, Causes, and Outcomes of Early Neurological Deterioration in Hyperacute Cerebral Ischemia

Background: Early neurological deterioration (END) is a feared complication of acute cerebral ischemia, but studies have provided widely varying estimates of END frequency, have often not systematically investigated END mechanism, and have not characterized END frequency among hyperacute patients presenting within the first 2h of onset, during the period of greatest neurologic deficit lability. Methods: We analyzed subjects with a final diagnosis of acute cerebral ischemia in the NIH Field Administration of Stroke Therapy-Magnesium Phase 3 multicenter clinical trial. END was defined as worsening by ≥ 4 NIHSS points up to Day 4. Causes of worsening were adjudicated by a central expert panel reviewing detailed medical records and imaging studies. Results: Among 1245 acute cerebral ischemia patients transported by EMS to 55 receiving stroke centers, time from last known well to ED arrival was median 59 mins (IQR 80-46). Overall, 211 (16.9%) experienced END by Day 4, including 17.4% within the first 6 hours of ED arrival, 32.2% between 6-24h, and 50.3% between 24h-Day 4. Patient features associated with END included older age (73.1 vs 70.5, p=0.01), worse initial deficits (NIHSS 13.1 vs 8.4, p<0.0001), history of hypertension, diabetes, higher serum glucose, BUN, and creatinine. Arrival imaging findings associated with END included more advanced acute ischemic change (ASPECTS 7.8 vs 8.8, p<0.0001) and presence of prior infarction. Leading causes of END were: extension of initial cerebral infarct (contributing in 50.6% of END patients), cerebral edema (25.3%), hemorrhagic transformation (23.0%), and infection (8.4%). END was associated with substantially reduced functional independence (mRS 0-2) at 90 days, 9.1% vs 67.9%, p<0.0001, and increased 90-day mortality, 42.1% vs 6.5%, p<0.0001). Conclusions: END occurs in 1 in 6 hyperacute cerebral ischemia patients, and is associated with dramatic reductions in functional independence and increases in mortality by 3 months. The leading causes of END are ischemia progression, cerebral edema, and hemorrhagic transformation, which collectively contribute to 4 in 5 of all END events, and are important treatment targets to improve ischemic stroke outcome.

Asenapine in the treatment of trichotillomania with comorbid bipolar disorder: A case report

Trichotillomania has been found to be associated with mood disorders, particularly bipolar disorder. Trichotillomania has shared similarities with bipolar disorder by virtue of phenomenology, co-morbidity, and psychopharmacologic observations. In the past, trichotillomania with comorbid bipolar disorder was treated with lithium and sodium valproate. There has been little, if any, literature on using asenapine to augment treatment in patients with trichotillomania with comorbid bipolar disorder. A patient presented with hair-pulling episodes for a year, resulting in bald scalp patches. She had no mood symptoms prior to this. She developed low mood, anhedonia, poor sleep and poor appetite subsequently as she could not stop pulling her hair. She was started on escitalopram 10 mg daily for he depressive symptoms. Three years later, she developed hypomanic symptoms such as irritability and spending sprees. Her hair pulling behaviour worsened at this time. At this point, a diagnosis of bipolar disorder type 2 was considered and she was started on lithium 300 mg daily. Her escitalopram was discontinued. As her mood was still labile 10 months later, asenapine was added to augment lithium in the treatment of the bipolar disorder. With asenapine, her hair pulling frequency started to decrease rapidly. Asenapine was increased to 10 mg daily and her hair pulling ceased. Her mood also stabilized and she no longer had erratic periods of mood lability. In conclusion, asenapine augmentation of lithium has potential to be used in patients who have trichotillomania with comorbid bipolar disorder due to its unique receptor profile.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Reconsolidation-Extinction Interactions in Fear Memory Attenuation: The Role of Inter-Trial Interval Variability.

Fear extinction typically results in the formation of a new inhibitory memory that suppresses the original conditioned response. Evidence also suggests that extinction training during a retrieval-induced labile period results in integration of the extinction memory into the original fear memory, rendering the fear memory less susceptible to reinstatement. Here we investigated the parameters by which the retrieval-extinction paradigm was most effective in memory updating. Specifically, we manipulated the inter-trial intervals (ITIs) between conditional stimulus (CS) presentations during extinction, examining how having interval lengths with different degrees of variability affected the strength of memory updating. We showed that randomizing the ITI of CS presentations during extinction led to less return of fear via reinstatement than extinction with a fixed ITI. Subjects who received variable ITIs during extinction also showed higher freezing during the ITI, indicating that the randomization of CS presentations led to a higher general reactivity during extinction, which may be one potential mechanism for memory updating.

Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels

The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

Hyperalgesia as a Pain Memory

Although pain conditioning does not involve central pathways for pain perception and memory, can it — like fear conditioning (NEJM JW Psychiatry Feb 26 2014) — be extinguished during a labile period when a conditioned stimulus is re-presented? Researchers, some with industry funding, conducted studies on hyperalgesia in mice. Injection of capsaicin into a paw produced hyperalgesia (measured by paw withdrawal from touch) …

Exposure to a fearful context during periods of memory plasticity impairs extinction via hyperactivation of frontal-amygdalar circuits.

An issue of increasing theoretical and translational importance is to understand the conditions under which learned fear can be suppressed, or even eliminated. Basic research has pointed to extinction, in which an organism is exposed to a fearful stimulus (such as a context) in the absence of an expected aversive outcome (such as a shock). This extinction process results in the suppression of fear responses, but is generally thought to leave the original fearful memory intact. Here, we investigate the effects of extinction during periods of memory lability on behavioral responses and on expression of the immediate-early gene c-Fos within fear conditioning and extinction circuits. Our results show that long-term extinction is impaired when it occurs during time periods during which the memory should be most vulnerable to disruption (soon after conditioning or retrieval). These behavioral effects are correlated with hyperactivation of medial prefrontal cortex and amygdala subregions associated with fear expression rather than fear extinction. These findings demonstrate that behavioral experiences during periods of heightened fear prevent extinction and prolong the conditioned fear response.

Gonadal sex differentiation in the Senegalese sole (Solea senegalensis) and first data on the experimental manipulation of its sex ratios

Like other flatfishes, the Senegalese sole exhibits sexual growth dimorphism in favor of females and thus all-female stocks would be desirable. However, reproduction-associated problems persist and basic aspects of its biology such as sex determination and differentiation are still unknown. In this study, we histologically examined the process of gonadal sex differentiation in the Senegalese sole and tested the effects of treatment with sex steroids and an aromatase inhibitor on sex ratios. Sex differentiation in females started before 98days post fertilization (dpf), when fish had an average total length (TL) of ~33mm. In contrast, males began sex differentiation by 127dpf with a mean TL of ~44mm. At this time, an incipient growth advantage in favor of females was already observed. All fish were sexually differentiated by ~48mm TL (~170dpf). Treatment between 98 and 169 dpf with the synthetic androgen 17α-methyldihydrotestosterone (10mg/kg) or the aromatase inhibitor fadrozole (100mg/kg) significantly increased the proportion of males up to ~95% while the natural estrogen, estradiol-17β (10mg/kg), increased the proportion of females by about 50% but reduced growth with respect to the control values. Together, these results show that the Senegalese sole is a differentiated gonochoristic species in which gonads start to form within the first three months and sex differentiation is completed after animals reach their fifth month of age, with females differentiating earlier than males. Results also indicate that the labile period starts at <98dpf and lasts until ~125dpf. These results show that sex control can be achieved in the Senegalese sole, an important step towards the establishment of all-female stocks to improve the currently compromised production of this economically important species.

Effect of commercial grade endosulfan on growth and reproduction of the fighting fish Betta splendens

To study the effects of endosulfan on survival, growth and reproduction of the obligate air-breathing male heterogametic fighting fish Betta splendens, posthatchlings of the fighting fish were discretely immersed for 3 h/day during the labile period on the 2nd, 5th, and 8th day posthatching (dph) at selected concentrations of commercial grade endosulfan ranging from 175 to 1400 ng/L. The immersions at 1,400 ng/L led to 21% mortality, among the 79% of surviving fry, 80% developed into females. The endosulfan reduced the air-breathing frequency of 5- and 8-day old hatchlings, and the reduction in the frequency persisted even after a depuration period of 172 days. In the ovary of the treated females, reduced number of vitellogenic oocytes with increased vacuolar cavities was observed. In the testis of the treated males, the reduced number of spermatogonia with increased vacuolar cavities was observed. The treated male induced the female to spawn a fewer eggs, which were subsequently incubated in his smaller bubble nest. The control females attained puberty on the 138th dph and spawned 120 eggs once in every 15 days, the females, which were previously treated at 1400 ng/L, postponed puberty to the 179th dph and spawned 70 eggs once in every 32 days. During the 240-day experiment, endosulfan is found to reduce significantly the cumulative progeny production from 760 to 144, reducing significantly to 19% of the control.

Hormonal sex reversal in Atlantic cod, Gadus morhua

Pre-harvest sexual maturation, a major constraint to aquaculture operations, commonly occurs in both sexes of Atlantic cod (Gadus morhua). Production of all-female triploids would alleviate this problem because they are sterile. The objective of this project was to develop effective endocrine manipulations to produce monosex stocks of cod, as the first step towards producing all-female triploids, using dietary 17β-estradiol (E2) and 17α-methyldihydrotestosterone (MDHT) fed during the labile period of gonadal differentiation. In the first experiment, triplicate treatments of E2 at 5, 10 and 20mg/kg feed and MDHT at 0.67, 2 and 6mg/kg feed were fed to fish during the growth interval from 17±2 to 43±4mm standard length (SL). The only treatment which affected sex ratio was the highest MDHT dose, with a significant reduction in proportion of females compared to controls. Based on these results and a thorough histological examination of gonadal development, a follow-up experiment used duplicate treatments of E2 at 20, 40, 80 and 120mg/kg and MDHT at 3, 6, 12 and 18mg/kg fed to fish during the growth interval from 7.8±1.2 to 45±2mm SL. This resulted in a dose-dependent shift in sex ratio in both estrogen and androgen treated groups, with no females observed in the groups fed the two highest doses of MDHT and a significantly reduced male ratio in E2 groups at the two highest doses. These results indicate that successful sex reversal can be achieved through dietary hormonal manipulation when the treatment starts at around 8mm SL.

Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: Lysosomal responses

In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

Seasonality in cell and tissue-level biomarkers in Mytilus galloprovincialis: relevance for long-term pollution monitoring

Mussels Mytilus galloprovincialis were collected monthly in 2 localities in the Northern Iberian Peninsula, Oia (Galicia) and Mundaka (Basque Coast), from April 2005 to May 2006, to inves- tigate whether seasonal variability in biomarkers follows the same pattern at both geographical areas. The following battery of biomarkers was analysed: lysosomal membrane stability and struc- tural changes, intracellular neutral lipid accumulation, cell type composition in the digestive gland epithelium, structural changes of digestive alveoli and digestive gland histopathology. Additionally, gonad index was measured as a supporting parameter. Overall, results demonstrate that most stud- ied biomarkers showed similar seasonal variability pattern in both localities. Lysosomal membrane stability (labilisation period (LP)) and most histopathological conditions showed similar seasonal vari- ability patterns in both localities, although values of LP and prevalences of some parasites were higher in Mundaka. Data provide baseline values to be used as reference values for the biomonitor- ing programmes carried out in coastal areas from Galicia to the Basque Coast. It is concluded that for monitoring programmes based on biomarkers, samples must be taken at least twice per year (spring and autumn) to avoid the effect on biomarkers of the late stages of each reproductive cycle.

An initial transient is followed by steady‐state regulation of blood pressure during hibernation bouts in the Syrian hamster

Hamster blood pressure and heart rate decrease during hibernation to conserve energy, but it is unclear how blood pressure changes over the course of a hibernation bout. We tested the hypothesis that blood pressure falls and then slightly recovers to a stable, regulated state during a hibernation bout. A blood pressure telemetry transmitter was implanted in a Syrian hamster (Mesocricetus auratus) for continuous recordings. Each hamster was placed in a 14:10 Light:Dark cycle at 23°C for 3 weeks and then transferred to a 8:16 Light:Dark cycle at 6°C. While euthermic, the hamster maintained mean arterial pressure at 87 ± 3 mmHg and heart rate at 352 ± 16 bpm at ambient temperatures of 23°C as well as at 6°C. Each hamster displayed multiple 3–5 day hibernation bouts. Each bout showed a two stage pattern: an initial, labile period (&lt; 1 day) where mean pressure transiently decreased (for some bouts as low as 20 mmHg) and a steady period (2–4 days) where mean pressure was maintained at 46 ± 4 mmHg. Spontaneous baroreflex sensitivity, normalized to baseline heart rate, was −2.7 ± 1.5 and −6.7 ± 2.5 (during euthermia and hibernation, respectively). Data support the hypothesis that following a transient, mean arterial pressure is stable during a bout of hibernation. (Support: R01 HL091763)

Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density – Vv – and labilisation period –LP) and combined (lysosomal response index – LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists.

Successful production of monosex female brook trout Salvelinus fontinalis using gynogenetic sex reversed males by a combination of methyltestosterone immersion and oral treatments

Recent advances towards the production of all-female populations of brook trout Salvelinus fontinalis have shown that methyltestosterone treatments applied after first feeding were not efficient in inducing masculinisation in neomales. A process to create all-female populations of brook trout using methyltestosterone was determined from 4 experiments. Different protocols of masculinisation by oral and/or hormonal immersion treatments were tested and the sex ratio of progeny produced from the cross of sex-reversed gynogenetic males with normal females was checked. No sexual dimorphism for growth was recorded, at least until two years of age. Masculinisation trials using oral treatments failed to produce any reversal of sex. Only the application of four weekly immersions beginning one week before hatching, combined with an oral treatment, provided 100% masculinisation. Gynogenetic sex-reversed males crossed with normal females produced 100% females, demonstrating the efficiency of this protocol in reversing the sex of brook trout. This result also highlights an earlier and longer period of sex lability, compared with most other Salmonids, and the need to initiate immersion treatments a long time before the initiation of gonadal differentiation.

Time-course study of the early lysosomal responses to pollutants in mussel digestive cells using acid phosphatase as lysosomal marker enzyme

Lysosomal biomarkers are early warning signals of the biological effects caused by environmental pollutants but the promptness of lysosomal responses to pollutants has not been investigated yet. This work is aimed to determine the response-time of digestive cell lysosomes in mussels exposed to metals and hydrocarbons. Mussels, Mytilus galloprovincialis, were exposed, under laboratory conditions to Cd and to the water-accommodated fraction of a lubricant oil. One mussel per experimental group was sacrificed and processed every hour from 0 h to 30 h. Changes in AcP activity, immunoreactivity and LMS test based on AcP histochemistry, discriminates significantly control and exposed mussels within 5 h exposure. The present results suggested that after 15–20 h exposure digestive cell loss might be accompanied by increased AcP activity (extralysosomal) without a parallel increase in the levels of immunoreactive AcP protein, especially after Cd-exposure. The reduced labilisation period of lysosomal membrane constitute a cost effective early warning signal that, however, is not necessarily correlated with the exposure time. The routine application of immunochemical techniques deserves more research efforts before its implementation although, these techniques are very valuable to understand and interpret correctly lysosomal responses to pollutants.

Activation of Hippocampal Nuclear Factor-κB by Retrieval Is Required for Memory Reconsolidation

Initially, memory is labile and requires consolidation to become stable. However, several studies support that consolidated memories can undergo a new period of lability after retrieval. The mechanistic differences of this process, termed reconsolidation, with the consolidation process are under debate, including the participation of hippocampus. Up to this point, few reports describe molecular changes and, in particular, transcription factor (TF) involvement in memory restabilization. Increasing evidence supports the participation of the TF nuclear factor-kappaB (NF-kappaB) in memory consolidation. Here, we demonstrate that the inhibition of NF-kappaB after memory reactivation impairs retention of a hippocampal-dependent inhibitory avoidance task in mice. We used two independent disruptive strategies to reach this conclusion. First, we administered intracerebroventricular or intrahippocampal sulfasalazine, an inhibitor of IKK (IkappaB kinase), the kinase that activates NF-kappaB. Second, we infused intracerebroventricular or intrahippocampal kappaB decoy, a direct inhibitor of NF-kappaB consisting of a double-stranded DNA oligonucleotide that contains the kappaB consensus sequence. When injected immediately after memory retrieval, sulfasalazine or kappaB decoy (Decoy) impaired long-term retention. In contrast, a one base mutated kappaB decoy (mDecoy) had no effect. Furthermore, we also found NF-kappaB activation in the hippocampus, with a peak 15 min after memory retrieval. This activation was earlier than that found during consolidation. Together, these results indicate that NF-kappaB is an important transcriptional regulator in memory consolidation and reconsolidation in hippocampus, although the temporal kinetics of activation differs between the two processes.

WINDOWS OF EMBRYONIC SEXUAL LABILITY IN TWO LIZARD SPECIES WITH ENVIRONMENTAL SEX DETERMINATION

Temperature-dependent sex determination (TSD) occurs in all major reptile lineages, but the selective forces and physiological mechanisms that link sex to incubation temperature may differ among and within those groups. Different models for TSD evolution make different predictions about when offspring sex will respond to environmental cues. Although TSD has evolved in several lizard lineages, there is less detailed information on these taxa than in turtles and crocodilians with TSD. We incubated eggs of an agamid lizard (Amphibolurus muricatus) and a scincid lizard (Bassiana duperreyi), two species with TSD. Rather than manipulate incubation temperature to identify periods of sexual lability (as in most previous studies of this topic), we topically applied the aromatase inhibitor fadrozole to eggs at a variety of times through the incubation period. Fadrozole application sex-reversed the resultant hatchlings if applied from the time of oviposition until at least 60% of the way through incubation. In all of the TSD lizard species studied so far, offspring sex is determined either while the eggs are held inside the mother's body or soon after oviposition, providing substantial maternal control over incubation temperatures at this critical period. Hence, the hypothesis that TSD evolves because it enables offspring sex to be matched to conditions that are unpredictable at the time of laying is less likely to apply to squamates than to turtles, sphenodontians, and (especially) crocodiles, in which the period of sexual lability is delayed until long after oviposition.

Molecular mechanisms of memory reconsolidation

Memory reconsolidation has been argued to be a distinct process that serves to maintain, strengthen or modify memories. Specifically, the retrieval of a previously consolidated memory has been hypothesized to induce an additional activity-dependent labile period during which the memory can be modified. Understanding the molecular mechanisms of reconsolidation could provide crucial insights into the dynamic aspects of normal mnemonic function and psychiatric disorders that are characterized by exceptionally strong and salient emotional memories.

Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using mussels as sentinel organisms

With the aim of assessing the biological effects of pollution along three gradients of pollution in the NW Mediterranean Sea, a biomonitoring survey was implemented using a battery of biomarkers (lysosomal membrane stability, lysosomal structural changes, metallothionein (MT) induction and peroxisome proliferation) in mussels over a period of two years as part of the EU-funded BEEP project. Mussels from the most impacted zones (Fos, Genova and Barcelona harbours) showed enlarged lysosomes accompanied by reduced labilisation period of lysosomal membranes, indicating disturbed health. MT levels did not reveal significant differences between stations and were significantly correlated with gonad index, suggesting that they were influenced by gamete development. Peroxisomal acyl-CoA oxidase (AOX) activity was significantly inhibited in polluted stations possibly due to interactions among mixtures of pollutants. In conclusion, the application of a battery of effect and exposure biomarkers provided relevant data for the assessment of biological effects of environmental pollution along the NW Mediterranean Sea.

Male Coho Salmon (Oncorhynchus kisutch) Exposed to a Time-Course of Urban Sewage Effluent Exhibit a Sporadic Low Incidence of Sex Reversal and Intersex

Abstract Developing coho salmon alevin (Oncorhynchus kisutch) were exposed to concentrations of 1 to 10% municipal wastewater effluent (MWWE) during the labile period of sexual differentiation. MWWE was collected at regular time intervals from a municipal wastewater treatment plant during a 4-week period that coincided with the latter part of the spawning season of coho salmon. The salmon were exposed, from just prior to hatch until 28 d post hatch, to MWWE collected at 3 a.m., 8 a.m., 3 p.m. or 8 p.m. Subsequent gonadal development was assessed by histology and compared to genetic sex as determined by Y-chromosomal DNA markers. The MWWE (analyzed using GC-HRMS) had relatively high concentrations of steroidal estrogens and other endocrine active substances at some time points during the exposure period. A low incidence of intersex and sex reversal was noted in the coho salmon exposed to MWWE collected at only two time points; 3 a.m. (1% MWWE) and 8 p.m. (10% MWWE), and ≤19% of the male fish in these exposure groups were affected. The coho salmon utilized were of wild genetic backgrounds and thus the sporadic nature of the response observed may be due to a significant genetic influence on the ability to respond to exogenous hormones at this early stage of development.