Erythropoietin effects on cryopreserved/transplanted cat ovarian tissue: A comparison of two incubation methods.

Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.

Exploring the therapeutic potential of an antinociceptive and anti-inflammatory peptide from wasp venom.

Animal venoms are rich sources of neuroactive compounds, including anti-inflammatory, antiepileptic, and antinociceptive molecules. Our study identified a protonectin peptide from the wasp Parachartergus fraternus' venom using mass spectrometry and cDNA library construction. Using this peptide as a template, we designed a new peptide, protonectin-F, which exhibited higher antinociceptive activity and less motor impairment compared to protonectin. In drug interaction experiments with naloxone and AM251, Protonectin-F's activity was decreased by opioid and cannabinoid antagonism, two critical antinociception pathways. Further experiments revealed that this effect is most likely not induced by direct action on receptors but by activation of the descending pain control pathway. We noted that protonectin-F induced less tolerance in mice after repeated administration than morphine. Protonectin-F was also able to decrease TNF-α production in vitro and modulate the inflammatory response, which can further contribute to its antinociceptive activity. These findings suggest that protonectin-F may be a potential molecule for developing drugs to treat pain disorders with fewer adverse effects. Our results reinforce the biotechnological importance of animal venom for developing new molecules of clinical interest.

Effects of erythropoietin on ischaemia-reperfusion when administered before and after ovarian tissue transplantation in mice.

RESEARCH QUESTION: Is the optimal timing for administering erythropoietin to minimize ischaemic injury in ovarian tissue transplantation before ovary removal for cryopreservation and subsequent transplantation or after transplantation? DESIGN: Thirty Swiss mice (nu/nu) were divided into three groups: treatment control group (n = 10); erythropoietin before harvesting group (EPO-BH) (n = 10) and erythropoietin after transplantation group (EPO-AT) (n = 10). Animals underwent bilateral ovariohysterectomy and their hemiovaries were cryopreserved by slow freezing. At the same time, previously cryopreserved hemiovaries were transplanted subcutaneously in the dorsal region. Erythropoietin (250 IU/kg) and sterile 0.9% saline solution were administered every 12/12 h over 5 consecutive days in the EPO-AT and EPO-BH groups, respectively. RESULTS: Administration of erythropoietin in the EPO-AT group improved the viability of ovarian follicles, reducing degeneration and increasing the number of morphologically normal growing follicles at 14 days after transplantation compared with the EPO-BH group (P = 0.002). This group also showed higher percentages of proliferative follicles at 7 days after transplantation (P ≤ 0.03), increased blood vessel count (P ≤ 0.03) and greater tissue area occupied by blood vessels at days 7 and 14 after transplantation (P ≤ 0.03), compared with hormone administration before cryopreservation (EPO-BH group) and the treatment control group. Additionally, treatment with erythropoietin before or after transplantation reduced fibrotic areas at 7 days after transplantation (P = 0.004). CONCLUSION: Erythropoietin treatment after transplantation reduced ischaemic damage in transplanted ovarian tissue, increased angiogenesis, maintenance of ovarian follicle proliferation and reduced fibrosis areas in the grafted tissue.

Metabolic activity in cryopreserved and grafted ovarian tissue using high-resolution respirometry.

Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value < 0.05). Despite decreased follicular viability and mitochondrial activity, the cryopreservation followed by transplantation of ovarian tissue proved feasible for attempts to restore ovarian function.

Short-term high-fat diet induces cognitive decline, aggression, and anxiety-like behavior in adult zebrafish.

Obesity is a global health problem with high prevalence and defined by a high body mass index (BMI). Several comorbidities affecting the central nervous system (CNS) are associated with obesity (e.g., neurodegenerative diseases, cognitive deficit, and psychobehavioral disturbs). The zebrafish (Danio rerio) has been considered a suitable model organism to investigate the neurobehavioral features of various human diseases. Here, we verify the impact of a high-fat diet (HFD) on the CNS by specifically assessing the effects of short-term HFD on anxiety-like responses, aggression, social preference, and memory, which are essential behaviors for survival and reproduction. Animals were separated in three experimental groups. The standard diet group (SD) received 7.5 mg/fish of dry food, while HFD groups received 5 mg/fish dry food plus 7.5 (HFD-7.5) or 15 mg/fish (HFD-15) of chicken egg yolk daily. Dietary fat content (w/w) was approximately 6.5%, 16.9%, and 21.1%, respectively. We performed behavioral tests and morphometric analyses after two weeks of HFD. In comparison to SD animals, HFD groups showed typical obesogenic responses with increases in BMI, abdominal length, and body weight. HFD individuals also showed increased aggression and anxiety-like behaviors in the mirror-induced aggression and novel tank diving tests, respectively. Interestingly, HFD did not change the social preference behavior, mean swimming speed or spontaneous activity levels, while the HFD-15 group showed cognitive deficits in the inhibitory avoidance test. Collectively, this "proof-of-concept" study is the first report to characterize the effects of short-term HFD on different behavioral domains of zebrafish with high degree of face validity. Moreover, our data reinforce the growing utility of zebrafish to explore the neurobehavioral basis of obesity, providing clinically translatable data, complementing the existing rodent models and supporting future mechanistic studies.

Heart defibrillation: relationship between pacing threshold and defibrillation probability.

BACKGROUND: Considering the clinical importance of the ventricular fibrillation and that the most used therapy to reverse it has a critical side effect on the cardiac tissue, it is desirable to optimize defibrillation parameters to increase its efficiency. In this study, we investigated the influence of stimuli duration on the relationship between pacing threshold and defibrillation probability. RESULTS: We found out that 0.5-ms-long pulses had a lower ratio of defibrillation probability to the pacing threshold, although the higher the pulse duration the lower is the electric field intensity required to defibrillate the hearts. CONCLUSION: The appropriate choice of defibrillatory shock parameters is able to increase the efficiency of the defibrillation improving the survival chances after the occurrence of a severe arrhythmia. The relationship between pulse duration and the probability of reversal of fibrillation shows that this parameter cannot be underestimated in defibrillator design since different pulse durations have different levels of safety.

Ventricular myocyte injury by high-intensity electric field: Effect of pulse duration.

Although high-intensity electric fields (HEF) application is currently the only effective therapy available to terminate ventricular fibrillation, it may cause injury to cardiac cells. In this study we determined the relation between HEF pulse length and cardiomyocyte lethal injury. We obtained lethality curves by survival analysis, which were used to determine the value of HEF necessary to kill 50% of cells (E50) and plotted a strength-duration (SxD) curve for lethality with 10 different durations: 0.1, 0.2, 0.5, 1, 3, 5, 10, 20, 35 and 70 ms. For the same durations we also obtained an SxD curve for excitation and established an indicator for stimulatory safeness (stimulation safety factor - SSF) as the ratio between the SxD curve for lethality and one for excitation. We found that the lower the pulse duration, the higher the HEF intensity required to cell death. Contrary to expectations, the highest SSF value does not correspond to the lowest pulse duration but to the one of 0.5 ms. As defibrillation threshold has been described as duration-dependent, our results imply that the use of shorter stimulus duration - instead of the one typically used in the clinic (10 ms) - might increase defibrillation safeness.