D-Optimal Design in the Development of Rheologically Improved In Situ Forming Ophthalmic Gel

In situ forming ophthalmic gels need to be fine tuned considering all the biopharmaceutical challenges of the front of the eye in order to increase drug residence time at the application site resulting in its improved bioavailability and efficacy. The aim of this study was to develop in situ forming ophthalmic poloxamer P407/poloxamer P188/chitosan gel fine tuned in terms of polymer content, temperature of gelation, and viscosity. Minimizing the total polymer content while retaining the advantageous rheological properties has been achieved by means of D-optimal statistical design. The optimal in situ forming gel was selected based on minimal polymer content (P407, P188, and chitosan concentration of 14.2%, 1.7%, and 0.25% w/w, respectively), favorable rheological characteristics, and in vitro resistance to tear dilution. The optimal in situ forming gel was proved to be robust against entrapment of active pharmaceutical ingredients making it a suitable platform for ophthalmic delivery of active pharmaceutical ingredients with diverse physicochemical properties.     

Effects of high fat diet,ovariectomy, and physical activity on leptin receptor expression in rat brain and white fat tissue

Aim

To evaluate in a rat animal model whether ovariectomy, high fat diet (HFD), and physical activity in the form of running affect leptin receptor (Ob-R) distribution in the brain and white fat tissue compared to sham (Sh) surgery, standard diet (StD), and sedentary conditions.

Methods

The study included 48 female laboratory Wistar rats (4 weeks old). Following eight weeks of feeding with standard or HFD, rats were subjected to either OVX or Sh surgery. After surgery, all animals continued StD or HFD for the next 10 weeks. During these 10 weeks, ovariectomy and Sh groups were subjected to physical activity or sedentary conditions. Free-floating immunohistochemistry and Western blot methods were carried out to detect Ob-R in the brain and adipose tissue.

Results

StD-ovariectomy-sedentary group had a greater number of Ob-R positive neurons in lateral hypothalamic nuclei than StD-Sh-sedentary group. There was no difference in Ob-R positive neurons in arcuatus nuclei between all groups. Ob-R distribution in the barrel cortex was higher in HFD group than in StD group. Ob-R presence in perirenal and subcutaneous fat was decreased in StD-ovariectomy group.

Conclusion

HFD and ovariectomy increased Ob-R distribution in lateral hypothalamic nuclei, but there was no effect on arcuatus nuclei. Our results are first to suggest that HFD, ovariectomy, and physical activity affect Ob-R distribution in the barrel cortex, which might be correlated with the role of Ob-R in election of food in rats.Obesity is one of the leading health issues worldwide, associated with an increased risk of morbidity and mortality (1). In 1997, the World Health Organization (WHO) formally recognized obesity as a global epidemic (2). Increase in body fat stores and obesity is caused by an imbalance between energy intake and energy expenditure (3,4). Since childhood obesity is a predictor of an increased death rate, the “obesity epidemic” may reverse the current declining rate of death from cardiovascular diseases (5). Factors that contribute to obesity can be environmental (6), social (7), behavioral (8), psychological (9), and genetic (10,11).Women generally have more body fat than men (12). Nevertheless similar odds ratios were recorded in women and men for the association of abdominal obesity with acute myocardial infarction (13). Weight gain is common after menopause, indicating an association between hormones and fat stores (14). A large scale observational study found that both the body mass index and the level of physical activity were independent predictors of mortality and that a higher level of physical activity did not eliminate the risk associated with adiposity. At the same time, women who were both lean and physically active had the lowest mortality (15). In animal studies menopause can be induced by ovariectomy (OVX) (16).Obesity can also be called a disorder of appetite and it is controlled by complex homeostatic mechanisms involving the hypothalamus and brainstem (17). Many gut peptides like cholecystokinin, ghrelin, glucagon-like peptide-1 (GLP-1), and -2 and peptide YY (PYY) act on the brain to control eating behavior (18). There are two different system for controlling feeding behavior: short-term and long-term (19). Short-term regulation involves neural signals from the GI tract and its hormones, like insulin, glucagon, and ghrelin (20). A hormone that functions mainly within long-term regulation is leptin (16 kD), a hormonal product of the obesity (ob) gen, primarily secreted by adipocytes (21) and released in the brain. It generates a feeling of satisfaction and acts like an appetite-suppressing agent. Circulating leptin levels are lower in ovariectomized rats (22).Food intake is regulated via neural circuits located in the hypothalamus (23). Leptin acts via its leptin or Ob receptors (Ob-R) and is primarily expressed in hypothalamic neurons (19) especially in arcuate, ventromedial, and dorsomedial nuclei (24). Leptin is transported across the blood-brain barrier (BBB) by a saturable transporter (25). Ob-R is also detected in nonhypothalamic areas in the mice and in human brain neocortex, cerebellum, entorinal cortex, amygdale, and rostral medulla (26). Adipocytes, endothelial cells, and macrophages also have leptin receptor at its surface, which suggests autocrine and paracrine action for leptin in human adipose tissue (27). Association between the expression of Ob-R in target tissues and physiological and hormonal controlled processes is still unclear. Leptin receptors mRNA is found in each of the major components of the CNS “feeding” circuitry – the brainstem, hypothalamus, and is distributed reward centers (Allan brain) (28). Therefore, the aim of the current study was to evaluate whether HFD affects Ob-R distribution compared with StD specifically in the barrel field and piriform cortex compared to standard feeding centers in the hypothalamus. We supposed that the combination of OVX and HFD is interesting for further research on selected brain regions, which might be alleviated by physical activity. We also supposed that changes in Ob-R level in white fat tissue would correlate with the changes in brain regions.     

Single acute stress-induced progesterone and ovariectomy alter cardiomyocyte contractile function in female rats

Aim

To assess how ovarian-derived sex hormones (in particular progesterone) modify the effects of single acute stress on the mechanical and biochemical properties of left ventricular cardiomyocytes in the rat.

Methods

Non-ovariectomized (control, n = 8) and ovariectomized (OVX, n = 8) female rats were kept under normal conditions or were exposed to stress (control-S, n = 8 and OVX-S, n = 8). Serum progesterone levels were measured using a chemiluminescent immunoassay. Left ventricular myocardial samples were used for isometric force measurements and protein analysis. Ca²⁺-dependent active force (F_active), Ca²⁺-independent passive force (F_passive), and Ca²⁺-sensitivity of force production were determined in single, mechanically isolated, permeabilized cardiomyocytes. Stress- and ovariectomy-induced alterations in myofilament proteins (myosin-binding protein C [MyBP-C], troponin I [TnI], and titin) were analyzed by sodium dodecyl sulfate gel electrophoresis using protein and phosphoprotein stainings.

Results

Serum progesterone levels were significantly increased in stressed rats (control-S, 35.6 ± 4.8 ng/mL and OVX-S, 21.9 ± 4.0 ng/mL) compared to control (10 ± 2.9 ng/mL) and OVX (2.8 ± 0.5 ng/mL) groups. F_active was higher in the OVX groups (OVX, 25.9 ± 3.4 kN/m² and OVX-S, 26.3 ± 3.0 kN/m²) than in control groups (control, 16.4 ± 1.2 kN/m² and control-S, 14.4 ± 0.9 kN/m²). Regarding the potential molecular mechanisms, F_active correlated with MyBP-C phosphorylation, while myofilament Ca²⁺-sensitivity inversely correlated with serum progesterone levels when the mean values were plotted for all animal groups. F_passive was unaffected by any treatment.Conclusion Stress increases ovary-independent synthesis and release of progesterone, which may regulate Ca²⁺-sensitivity of force production in left ventricular cardiomyocytes. Stress and female hormones differently alter Ca²⁺-dependent cardiomyocyte contractile force production, which may have pathophysiological importance during stress conditions affecting postmenopausal women.The relation between stress, gender, and cardiovascular diseases is well established (1-4). Some of the known risk factors for cardiovascular disease such as smoking, unhealthy diet, and behavioral and psychosocial stress have deleterious effects on the cardiovascular system via activation of the sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis (5-8). Acute restraint stress is a preferred and widely used method to induce physical stress in animal models (9). Moreover, restraint and immobilization are important as models for psychological stress, which was shown to adversely affect ovarian function (10) and to play a pivotal role in the pathomechmanism of Takotsubo (stress) cardiomyopathy in postmenopausal women (11).Gender is a very important factor in the development of cardiovascular diseases. Premenopausal women have better lipid profile, endothelial function (12), and a lower risk to develop coronary artery disease and myocardial infarction (MI) than men. These advantages of female gender, however, are abolished after menopause, which is associated with increased prevalence of left ventricular (LV) hypertrophy, decreased LV ejection fraction, and LV contractility (13). One of the explanations for the distinct myocardial responses is the cardioprotective effect of female sex hormones (eg, estrogens) (14,15).Progesterone performs several actions on the heart: it exerts an antiarrhythmic effect by accelerating cardiac repolarization (16) and has a preventive role in ischemia-reperfusion injury via reducing inflammatory response (17). It has been shown to inhibit cardiomyocyte apoptosis (18), induce vasodilation, and reduce blood pressure via increasing nitric oxide (NO) levels in normotensive and hypertensive patients (19). Importantly, progesterone is produced by the both ovaries and the adrenal gland: Moreover, the adrenal progesterone content is similar or even larger than that in the ovaries (20). Adrenal progesterone production and secretion increase along with corticosterone regardless of gender and estradiol under stress conditions (21). Progesterone, being an indirect precursor of cortisol (22), increases in response to adrenocorticotrophic hormone (ACTH) stimulation (23).In the heart, there are multiple estrogen hormone receptor types (24). The expression of aromatase in the heart suggests that estrogen may be synthesized also within the cardiomyocyte to exert autocrine/paracrine actions (25). Myocyte contractility seems to be modulated by systemic estrogen levels and altered in cardiomyocytes derived from ovariectomized (OVX) rats (26). In particular, myofilament Ca²⁺-sensitivity is increased in isolated myofibrillar preparations from OVX rats, and restored to the basal levels with estrogen supplementation (27,28).Activation of the sympathetic nervous system plays a central role in the regulation of cardiomyocyte contractile function and myofilament Ca²⁺-sensitivity through beta-adrenergic receptor stimulation, activating the protein kinase A (PKA). PKA-mediated phosphorylation of Ca²⁺-handling and myofilament proteins (myosin binding protein-C [MyBP-C], troponin I [TnI], titin) were shown to alter cardiomyocyte contractile function (29,30). It has been suggested that female cardiomyocytes operate at lower levels of intracellular Ca²⁺ than those of males, particularly under inotropic conditions (31). This difference in Ca²⁺ homeostasis may be related to the fact that estrogen suppresses the L-type Ca²⁺ current (32,33) and may reduce the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) (34), which was shown to be larger in myocytes from OVX rats (35). Not only cardiomyocyte contraction, but relaxation may also be affected by estrogen via altered Ca²⁺ re-uptake into the SR and modified Ca²⁺ efflux via increased sarcolemmal Na⁺/Ca²⁺ exchange (36). Interestingly, despite similar SR Ca²⁺ content in males and females (37), studies using OVX models report conflicting results concerning changes in the expression and activity of the SR Ca²⁺-ATPase and its regulator protein phospholamban (38-41). Much less is known about the possible effect of progesterone on cardiomyocyte contractile function. We hypothesized that progesterone affected force production of single isolated cardiomyocytes. Therefore, in the present study we aimed to investigate how sex hormones (particularly progesterone) and single acute restraint stress altered cardiomyocyte contractile function and to identify the consequent posttranslational myofilament protein modifications in OVX rats.     

Dental and Skeletal Age in Patients with Palatally Displaced Canines

ObjectiveTo determine potential associations between dental and skeletal maturation and palatally displaced canines (PDC) considering gender and chronological age.Material and methodsThis study included pretreatment panoramic and lateral cephalometric radiographs of 43 subjects with PDCs and 203 randomly selected orthodontic subjects with normally erupted canines. Both groups were non syndromic patients. Chronological age of subjects was rounded and noted in years with decimal points and compared with chronological age according to Demirjian''s dental age assessment. Skeletal maturation was determined by cervical vertebrae changes on cephalometric radiographs.ResultsFemale subjects with PDC were more affected by left side canine displacement than males (p=0.027) with five times higher odds ratio (OR = 4.9; 95% CIL=1.2-19.7). A comparison of chronologic and skeletal age indicated that PDC subjects were skeletally younger than unaffected groups with statistically significant differences at the age of 10, 12 and 13. (p=0.05).ConclusionYoung subjects with PDCs showed skeletal maturation delay compared to control group, indicating that skeletal maturation assessment could be one of unexplored predicting factors of a PDC, especially at the age between 10 and 13 years in both genders. Subjects with PDC showed intensive growth spurt after the age of 12 years in females, and after the age of 13 in males. Dental maturation delay showed no statistical significance in PDC prediction.     

Existence of spare alpha 1-adrenoreceptors, but not alpha 2-adrenoreceptors, for respective vasopressor effects of cirazoline and B-HT 933 in the pithed rat

The existence of a receptor reserve (spare receptors) was investigated for postsynaptic vascular alpha1- and alpha2-adrenoceptors in the pithed rat by evaluating the effects of progressive inactivation of alpha1- and alpha2-adrenoceptor pools by the irreversible antagonist phenoxybenzamine on the pressor responses of cirazoline and B-HT 933. Dose-response curves for the alpha1-adrenoceptor-mediated vasoconstrictor effects of cirazoline were shifted in a rightward direction with no depression of the maximum response by lower does of phenoxybenzamine (0.1-0.2 mg/kg, i.v.). Progressively higher doses of phenoxybenzamine (greater than 1 mg/kg, i.v.) produced further rightward shifts in the dose-response curves of cirazoline, but also depressed the maximum response. In contrast, all doses of phenoxybenzamine that inhibited the alpha2-adrenoceptor-mediated pressor effects of B-HT 933 produced a reduction in the maximum response. These results are highly suggestive of the existence of a receptor reserve in the pithed rat for the postsynaptic vascular alpha1-adrenoceptor-mediated effects of cirazoline, but not for the postsynaptic vascular alpha2-adrenoceptor-mediated effects of B-HT 933. Confirmation of the existence of a receptor reserve for only the postsynaptic vascular alpha1-adrenoceptor-mediated effects of cirazoline came from further analysis of the antagonism, by phenoxybenzamine, of cirazoline and B-HT 933 dose-response curves. The maximum pressor response that could be elicited by the alpha1-adrenoceptor agonist cirazoline was a hyperbolic function on the size of the alpha1-adrenoceptor pool, the latter being progressively decreased by phenoxybenzamine treatment. Such a hyperbolic relationship is indicative of a receptor reserve. In marked contrast, the maximum pressor response that could be evoked by the alpha2-adrenoceptor agonist B-HT 933 was a linear function of the size of the intact alpha2-adrenoceptor pool, characteristic of a lack of spare receptors. Further analysis showed that the occupancy-response relationship is fivefold more favorable for the alpha1-adrenoceptor-mediated pressor effects of cirazoline than for the alpha2-adrenoceptor-mediated pressor effects of B-HT 933, indicating that any given maximum pressor response in the pithed rat may be obtained with one-fifth as many alpha1-adrenoceptors being activated by cirazoline than alpha2-adrenoceptors being stimulated by B-HT 933. (ABSTRACT TRUNCATED AT 250 WORDS)     

Epidemiological and clinical aspects of congenital heart disease in children in Tuzla Canton,Bosnia-Herzegovina

Congenital heart disease (CHD) is among the most frequent of all congenital anomalies. The purpose of this study was to present the results of an initial registration of children with CHD from January 1994 to December 1999 in Tuzla Canton, Bosnia-Herzegovina. The population studied consisted of all 39,699 live-born children in this area. Diagnosis of CHD was made by clinical findings, electrocardiography, chest X-ray, echocardiography, catheterisation or autopsy. In the 6-year period, 243 children were found to have CHD, i.e. a prevalence of 6.12 per 1000 live-born. Critical CHD was present in 58 of them, or 1.46 children per 1000 live-born. The average age at diagnosis was 1.47 years. There were 132 boys (54.3%) and 111 girls. The most frequent anomaly was ventricular septum defect with a prevalence of 2.49 per 1000 live-born, representing 40.7 % of the total anomalies. Of the total group, 46 (18.9%) had extracardiac anomalies related to syndromes. Cardiac surgery was indicated in 98 patients (40.3%) but could only be carried out in 42 (17.3%). A total of 63 (25.9%) patients died, 54 of whom within the 1st month of life. CONCLUSION: congenital heart disease is a very significant health problem in Tuzla Canton. It requires urgent measures in terms of organisation of early diagnosis and proper management.     

Loss of heterozygosity of DPC4 tumor suppressor gene in human sporadic colon cancer

We investigated the prevalence of DPC4 loss of heterozygosity in sporadic colorectal cancer. Thirty-six cases of human sporadic colon carcinoma and corresponding normal tissue samples were examined to evaluate loss of heterozygosity at the DPC4 tumor suppressor locus using variable nucleotide tandem repeat (VNTR) analysis and three polymorphic markers. From 36 analyzed samples 35 (97%) were heterozygous or informative. Loss of heterozygosity at the DPC4 locus was detected in 18 (51%) of informative tumor DNAs. The DPC4 LOH was more frequent in smaller tumors (<5 cm) than in larger ones. There was no correlation between DPC4 LOH and age or sex of patients. There was a negative correlation between DPC4 LOH and histological grade or Dukes' stage of tumors, but without statistic significance. Observed results are in agreement with the view that malignant progression is consequence of many genetic changes. It can be concluded that inactivation of the DPC4 gene plays a role in a multistep process of outgrowth and progression of colon cancer.     

Discriminative stimuli produced by clonidine in spontaneously hypertensive rats: generalization to antihypertensive drugs with different mechanisms of action

Spontaneously hypertensive rats were food deprived and trained to lever press on a fixed-ratio 10 schedule of food reinforcement in a paradigm in which responding was reinforced on one lever after an injection of clonidine (0.02 mg/kg) and on an alternate lever after an injection of saline. The spontaneously hypertensive rats learned the clonidine-saline discrimination to a criterion of correct lever selection on 10 consecutive days in an average of 39 training sessions. In subsequent tests, emission of clonidine discrimination responses was found to be both time dependent and dose dependent (ED50, 0.009 mg/kg). The antihypertensive drugs lofexidine (ED50, 0.03 mg/kg), guanabenz (ED50, 0.019 mg/kg), p-aminoclonidine (ED50, 0.23 mg/kg), methyldopa (ED50, 21.8 mg/kg), hydralazine (ED50, 0.98 mg/kg), prazosin (ED50, 0.72 mg/kg), minoxidil (ED50, 12.4 mg/kg) and pergolide (ED50, 0.021 mg/kg) were generalized to clonidine in a dose-dependent manner. Yohimbine antagonized both the antihypertensive action and the discriminative stimulus properties of clonidine in parallel without antagonizing those actions of hydralazine. Similarly, the discriminative stimulus properties and antihypertensive action of pergolide were antagonized by sulpiride in parallel without antagonizing any of those effects when produced by clonidine or hydralazine. Although the stimulus properties and response-suppressive actions of the antihypertensive or other drugs were not related, the ED50 values for generalization of the drugs to the clonidine stimulus and for their antihypertensive action were highly correlated (r = 0.99). These data suggest that clonidine produces an interoceptive discriminative stimulus that is based upon its antihypertensive action in spontaneously hypertensive rats.