The Role of Botulinum Neurotoxin A in the Conservative Treatment of Fractures: An Experimental Study on Rats.

This paper explores the role of botulinum neurotoxin in aiding fracture recovery through temporary muscle paralysis. Specifically, it investigates the effects of botulinum neurotoxin-induced paralysis of the sternocleidomastoid muscle on clavicle fractures in rats. The research aims to assess safety, effectiveness, and the impact on fracture healing. Healthy male Albino Wistar rats were divided into four groups: clavicle fracture, botulinum neurotoxin injection, both, and control. Surgeries were conducted under anaesthesia, and postoperatively, animals were monitored for 28 days. Euthanasia and radiological assessment followed, examining fracture healing and muscle changes, while tissues were histopathologically evaluated. The modified Lane-Sandhu scoring system was used for the radiographic evaluation of clavicle fractures, and the results varied from complete healing to nonunion. Histopathological examination at 28 days postfracture showed fibrous tissue, mesenchymal cells, and primary callus formation in all groups. Despite varied callus compositions, botulinum neurotoxin administration did not affect clavicle healing, as evidenced by similar scores to the control group. Several studies have explored botulinum neurotoxin applications in fracture recovery. Research suggests its potential to enhance functional recovery in certain types of fractures. Theoretical benefits include managing muscle spasticity, aiding reduction techniques, and preventing nonunion. However, botulinum neurotoxin's transient effect and nonuniversal applications should be considered. The present study found that botulinum toxin had no clear superiority in healing compared to controls, while histological evaluation showed potential adverse effects on muscle tissue. Further research is essential to understand its risk-benefit balance and long-term effects.

Metabolic Phenotyping Study of Mouse Brain Following Microbiome Disruption by C.difficile Colonization.

Clostridioides difficile infection (CDI) is responsible for an increasing number of cases of post-antibiotic diarrhea worldwide, which has high severity and mortality among hospitalized elderly patients. The disruption of gut microbiota due to antibacterial medication facilitates the intestinal colonization of C. difficile. In the present study, a murine model was used to investigate the potential effects of antibiotic administration and subsequent colonization by C. difficile, as well as the effects of three different 10-day treatments (metronidazole, probiotics, and fecal microbiota transplantation), on the brain metabolome for the first time. Four different metabolomic-based methods (targeted HILIC-MS/MS, untargeted RP-LC-HRMS/MS, targeted GC-MS/MS, and untargeted GC-MS) were applied, resulting in the identification of 217 unique metabolites in the brain extracts, mainly glycerophospholipids, glycerolipids, amino acids, carbohydrates, and fatty acids. Univariate and multivariate statistical analysis revealed that CDI, as well as the subsequent treatments, altered significantly several brain metabolites, probably due to gut dysbiosis, and affected the brain through the gut-brain axis. Notably, none of the therapeutic approaches completely restored the brain metabolic profile to the original, healthy, and non-infected phenotype, even after 10 days of treatment.

Hepatic Metabolic Profiling of Lifelong Exercise Training Rats.

Regular physical exercise has been investigated as a primary preventive measure of several chronic diseases and premature death. Moreover, it has been shown to synchronize responses across multiple organs. In particular, hepatic tissue has proven to be a descriptive matrix to monitor the effect of physical activity. In this study, we performed an untargeted metabolomics-based analysis of hepatic tissue extracts from rats that have undergone either lifelong or chronic exercise training. For this purpose, 56 hepatic samples were collected and were analyzed by UHPLC-TOF-MS in negative ionization mode. This approach involved untargeted metabolite detection on hepatic tissue extracts accompanied by an in-house retention time/accurate mass library enabling confident metabolite identification. Unsupervised (PCA) and supervised (OPLS-DA) multivariate analysis showed significant metabolic perturbation on a panel of 28 metabolites, including amino acids, vitamins, nucleotides, and sugars. The training regime employed in this study resulted in a probable acceleration of the bioenergetic processes (glycolysis, glycogen metabolism), promoted catabolism of purines, and supplied biosynthetic precursors via the pentose phosphate pathway and pentose and glucuronate interconversions. Overall, the applied methodology was able to discriminate the different training schedules based on the rat liver metabolome.

The Action of Botulinum Toxin A on the Sternocleidomastoid Muscle: An Experimental Study on Rats.

In this study, we aim to investigate the effective dose of botulinum neurotoxin A that results in paralysis of the sternocleidomastoid muscle for a minimum duration of 28 days in Wistar rats. This research is the first in a series of studies to investigate the value of botulinum toxin A in the healing of clavicle fractures through the temporary paralysis of the sternocleidomastoid. A surgical incision was made under general anaesthesia, and botulinum neurotoxin A in respective doses of 4 and 6 international units (IU) or normal saline in equivalent volumes were injected directly into the exposed muscle. Electromyography was conducted on days 0, 7, and 28 following substance administration to determine the extent of muscle paralysis. Electromyography on day 0 showed no paralysis in either group. Animals injected with neurotoxin all exhibited paralysis on days 7 and 28 that was weaker in the group injected with the smaller dose of 4 IU. One death occurred in the group injected with the higher dose (6 IU), whereas in the control group, no paralysis was seen. Botulinum neurotoxin A in a dose of 6 IU resulted in complete paralysis of the sternocleidomastoid in rats for a minimum of 28 days. A dose of 4 IU resulted in less potent paralysis but was safer in our research. Botulinum neurotoxin is a substance utilised in cosmetics and therapeutics for many years, yet research shows that its use can be expanded to target a wider range of pathologies. In this series of studies, we aim to explore the neurotoxin's applications on the treatment of clavicle fractures. To investigate this, we need to first establish the duration of its action on the sternocleidomastoid muscle.

The addition of crocin in the freezing medium extender improves post-thaw semen quality.

Semen cryopreservation is arguably the most important method or technique contributing to the advancement of modern animal production. However, the quality of sperm after thawing is still highly variable. The addition of antioxidant compounds to the freezing medium has been used customarily to counteract the harmful effects of Reactive Oxygen Species (ROS) that are produced during the freeze/thaw process. Crocin, a potent antioxidant, improves the fertilizing capacity of spermatozoa. In this study, we evaluated the potential of crocin (0, 0.5 and 1 mM) as an extender additive to diminish the damaging effects of cryopreservation on bovine spermatozoa. Post-thaw semen quality was assessed by means of motility, viability and lipid peroxidation (LPO). We further investigated the effect of crocin supplementation upon freezing on sperm quality parameters during their incubation at 37°C for up to 2 hr. Overall, the data assessment indicates that crocin facilitated a general improvement of the quality of freeze/thawed spermatozoa, under the present experimental conditions. Crocin (1 mM) maintained a higher percentage of alive spermatozoa with intact acrosome with rapid and progressive motility, compared to the control extender. Moreover, the spermatozoa cryopreserved in the presence of crocin exhibited higher values in CASA kinematic parameters (VCL, VSL, VAP, ALH) immediately after thawing. Furthermore, the positive effect of crocin on motility parameters was also sustained over a period of 2 hr incubation at 37°C. This effect of crocin may be attributed to the observed inhibition of LPO during the incubation period. Thus, the results indicate that the addition of crocin (especially at a final concentration of 1 mM) in the freezing extender medium may benefit the preservation of the quality parameters of spermatozoa that are compromised by the freeze/thaw heat shock and the stress during handling for IVF or artificial insemination.

Effect of crocin on antioxidant gene expression, fibrinolytic parameters, redox status and blood biochemistry in nicotinamide-streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes is regarded as an epidemiological threat for the twenty-first century. Phytochemicals with known pharmaceutical properties have gained interest in the field of alleviating secondary complications of diseases. Such a substance is crocin, a basic constituent of saffron (Crocus sativus). The present study aimed at examining the beneficial effects of per os crocin administration on the antioxidant status, blood biochemical profile, hepatic gene expression and plasminogen activator inhibitor-1 activity (PAI-1) in the liver, kidney and plasma (an important marker of pre-diabetic status and major factor of thrombosis in diabetes) of healthy rats, as well as of rats with nicotinamide-streptozotocin-induced diabetes. RESULTS: Diabetes disrupted the oxidation-antioxidation balance, while crocin improved the antioxidant state in the liver by significantly affecting SOD1 gene expression and/or by restoring SOD and total antioxidant capacity (TAC) levels. In the kidney, crocin improved hydrogen peroxide decomposing activity and TAC. In blood, hepatic transaminases ALT and AST decreased significantly, while there was a trend of decrease regarding blood urea nitrogen (BUN) levels. The expression of PAI-1 gene was affected in the liver by the dose of 50 mg kg-1. CONCLUSIONS: Crocin treatment contributed in restoring some parameters after diabetes induction, primarily by affecting significantly hepatic transaminases ALT and AST, SOD1 and PAI-1 gene expression and nephric H2O2 decomposing activity. In conclusion, crocin did contribute to the alleviation of some complications of diabetes.

Olive oil with high polyphenolic content induces both beneficial and harmful alterations on rat redox status depending on the tissue.

Olive oil (OO) possesses a predominant role in the diet of Mediterranean countries. According to a health claim approved by the European Food Safety Authority, OO protects against oxidative stress­induced lipid peroxidation in human blood, when it contains at least 5 mg of hydroxytyrosol and its derivatives per 20 g. However, studies regarding the effects of a total OO biophenols on redox status in vivo are scarce and either observational and do not provide a holistic picture of their action in tissues. Following a series of in vitro screening tests an OO containing biophenols at 800 mg/kg of OO was administered for 14 days to male Wistar rats at a dose corresponding to 20 g OO/per day to humans. Our results showed that OO reinforced the antioxidant profile of blood, brain, muscle and small intestine, it induced oxidative stress in spleen, pancreas, liver and heart, whereas no distinct effects were observed in lung, colon and kidney. The seemingly negative effects of OO follow the recently formulated idea in toxicology, namely the real life exposure scenario. This study reports that OO, although considered a nutritional source rich in antioxidants, it exerts a tissues specific action when administered in vivo.

Antioxidant effect of a polyphenol-rich grape pomace extract on motility, viability and lipid peroxidation of thawed bovine spermatozoa.

BACKGROUND: Grape extracts of the Greek species Vitis vinifera possess potent antioxidant properties in vitro. The freeze/thaw process and the preparation of semen during assisted reproductive techniques can adversely affect the functional integrity of spermatozoa. The objective was to assess the effect of three different concentrations (1 µg ml(-1), 2 µg ml(-1) and 5 µg ml(-1)) of a polyphenol-rich grape pomace extract on motility, viability, acrosomal and lipid peroxidation status of thawed bovine spermatozoa after 2 and 4 hrs of incubation. RESULTS: The results indicate that the percentage of "Rapid" spermatozoa remained significantly increased (p <0.05) in the presence of 5 µg ml(-1) of the extract, compared to the control after 2 hrs of incubation. Additionally, the incubation of spermatozoa with 2 µg ml(-1) and 5 µg ml(-1) of the extract for 2 hrs resulted in a significantly better maintenance of viable spermatozoa with intact acrosome (p <0.05). The other parameters did not show statistically significant changes. Moreover, the presence of 2 µg ml(-1) and 5 µg ml(-1) of the extract kept the levels of Malondialdehyde (MDA) production in significantly lower level, compared to the other groups, after 2 hrs and 4 hrs of incubation (p <0.05). Particularly, a dose-dependent effect was noticed after 2 hrs of incubation. CONCLUSIONS: Our results suggest that the grape pomace extract exerts a powerful antioxidant role, by suppressing lipid peroxidation, and provides protection in terms of motility and acrosomal integrity, which are correlated with in vivo fertility. The optimal extract concentration is 5 µg ml(-1).

CCl4 induces tissue-type plasminogen activator in rat brain; protective effects of oregano, rosemary or vitamin E.

The high metabolic rate and relatively low antioxidant defenses of the lipid-rich brain tissue render it highly susceptible to reactive oxygen species (ROS) and oxidative stress, whereas the implication of ROS in the pathogenesis of several diseases in the central nervous system is well-established. The plasminogen activator (PA) system is a key modulator of extracellular proteolysis, extracellular matrix remodeling and neuronal cell signaling and has been implicated in the pathogenesis of these diseases. This study evaluates the role of tissue-type PA (t-PA) in oxidative stress and the protective role of dietary antioxidants in the rat brain. We used the CCl4 experimental model of ROS-induced lipid peroxidation and evaluated the antioxidant effect of oregano, rosemary or vitamin E. CCl4-treated Wistar rats exhibited elevated brain t-PA activity, which was decreased upon long-term administration of oregano, rosemary or vitamin E. PA inhibitor-1 (PAI-1) activity was also slightly elevated by CCl4, but this increase was not affected by the antioxidants. We hypothesize that the CCl4-induced t-PA activity indicates extracellular proteolytic activity that may be linked to neuronal cell death and brain damage. Vitamin E or antioxidants present in oregano or rosemary are effective in inhibiting t-PA elevation and can be considered as a potential protection against neuronal damage.

Crocetin administration ameliorates endotoxin-induced disseminated intravascular coagulation in rabbits.

Disseminated intravascular coagulation (DIC), a life-threatening secondary complication in several diseases, is characterized by large amounts of thrombin that lead to fibrin deposition and microthrombus formation throughout the microcirculation. Recent in-vitro studies suggest that crocin, crocetin or safranal, carotenoid constituents of the spice Crocus sativus L. (saffron), have antithrombotic properties, especially anti-Xa activity. The present study was designed to evaluate the effect of crocetin on thrombosis procedure using a rabbit model of bacterial endotoxin-induced DIC. Crocetin administration (3 mg/kg), 30 min before the beginning of endotoxin infusion, improved DIC-related haemostatic indices such as platelet blood counts (P≤ 0.05), blood plasma fibrinogen and protein C concentration (P≤ 0.05). In addition, it ameliorated DIC-associated disease and fibrin deposition in the glomeruli (P≤ 0.05). These results indicate that crocetin reveals a preventive antithrombotic role in vivo and prescribe further investigation on the possibility of developing crocetin-based DIC treatment modalities.

Endosulfan-induced lipid peroxidation in rat brain and its effect on t-PA and PAI-1: ameliorating effect of vitamins C and E.

Endosulfan provokes systemic toxicity in mammals and induces reactive oxygen species (ROS) and lipid peroxidation (LPO). The brain is susceptible to LPO and several studies implicate ROS and LPO in CNS diseases. Tissue plasminogen activator (t-PA) has been accredited with plasminogen-dependent roles in the CNS, as well as plasminogen-independent functions. The aim of this study was to investigate the activities of t-PA and its inhibitor, plasminogen activator inhibitor-1 (PAI-1) in the adult rat brain, after subchronic endosulfan treatment. Furthermore, the potency of vitamins C and E to attenuate these effects was explored. Endosulfan was administered in Wistar rats either alone or with vitamin C and/or vitamin E. The induced oxidative stress was manifested by induction of LPO as determined by higher malondialdehyde levels. This was accompanied by elevation of t-PA and PAI-1 activities. Vitamins E and C, both well-known for their antioxidant properties, substantially acted in a preventive way and protected the brain from these effects.

Plasminogen activator activity in the porcine oviduct during the oestrous cycle.

The mammalian oviduct is a dynamic tissue, which lies under the influence of ovarian steroids and produces proteins that affect various stages of fertilization and post-fertilization events. In this study, expression of urokinase-type plasminogen activator (u-PA mRNA) and plasminogen activator activity (PAA) were examined in porcine oviducts by reverse transcription and polymerase chain reaction (RT-PCR) and activity assays, respectively. For this purpose, oviducts were collected from Landrace cycling sows and divided into three segments (isthmus, ampulla, infundibulum). Different concentrations of u-PA mRNA were detected in the three segments following the pattern isthmus>ampulla>infundibulum and this pattern was maintained during the oestrous cycle. On the contrary, the highest PAA was measured in the ampulla compared to the isthmus and the infundibulum and the highest ampullary PAA was detected during the first 2 days of the oestrous cycle. The different regulation of u-PA mRNA expression and PAA is probably due to the existence of PA inhibitors. Recent observations suggest that PAI-1, the main inhibitor of PAs, shows greater expression in the isthmus compared to the ampulla and the local generation of plasmin is inhibited. The latter may be related to observations that spermatozoa are quiescent in the isthmus before fertilization. This study supports the suggestion that urokinase-type plasminogen activator has a biological role within the porcine oviduct, especially at or near the time of fertilization.

Effect of plasmin on movement characteristics of ejaculated bull spermatozoa.

Proteolytic enzymes appear to have an essential role in multiple phases of mammalian fertilization. Several observations suggest that the plasminogen activator/plasmin system might also play a role in mammalian fertilization. Movement characteristics of bovine sperm incubated with different concentrations of plasmin were investigated using a computer-assisted automated semen analysis system. Sperm were incubated up to 4h in a modified Tyrode's medium (control) and 0.1, 1, 10 and 100 mU/ml of plasmin. The percentage motile sperm was significantly higher at 0 h for sperm incubated in 1, 10, and 100 mU of plasmin. Relative to sperm incubated in control medium, lateral head displacement (ALH), curvilinear velocity, beat cross frequency, path velocity and straight line velocity (VSL) of sperm treated with 100 mU of plasmin for 0 h were increased. After 2h of incubation, sperm treated with 100 mU of plasmin showed an increase in ALH, but a decrease in VSL, straightness and linearity. The effect of plasmin on most motility parameters appears to be direct since all these parameters were affected at 0 h of incubation. Our results support the notion of hyperactivation of bovine spermatozoa following incubation with different concentrations of plasmin. The present work provides additional information to further characterize motility movement of bovine sperm associated with final preparation for fertilization.