Effect of bismuth subgallate (a hemostatic agent) on bone repair; a histologic, radiographic and histomorphometric study in rats.

Bismuth subgallate (BS) is a hemostatic agent used for soft tissue surgery in otorhinolaryngology and dermatology. Its effect on bone repair has not been studied. The present study undertook a quantitative and qualitative evaluation of post-extraction bone healing in the presence of BS. Under intraperitoneal anesthesia, forty male Wistar rats, 80+/-5g body weight, underwent the extraction of both lower first molars. BS was placed in the right post-extraction socket (group E) and the contralateral socket served as control (group C). The animals were killed in groups immediately, 7, 14 and 30 days post-extraction. The mandibles were resected, radiographed and processed for embedding in paraffin. The mesial socket was sectioned along the bucco-lingual axis and stained with hematoxylin-eosin. Total tissue volume and trabecular bone volume of the apical third of the sockets were determined histomorphometrically. At 14 and 30 days post-extraction, group E exhibited bone tissue that resembled that of group C. Histomorphometric analyses showed no statistically significant differences between groups C and E. Bismuth subgallate did not interfere with post-extraction bone healing. Further studies will analyze the effect of this hemostatic agent on bone repair in aniticoagulated rats.

Two new highly polymorphic markers in the 3' UTR region of the PLA2G7 gene.

We described two new highly polymorphic markers located 31 bp downstream of the last nucleotide of exon 12 in the 3' UTR region of the gene PLA2G7: 1344 +31TG(n) AG(m). Eight and 14 alleles were observed for the AG and TG repeats, respectively. These two markers have the highest heterozygosity until now reported for PLA2G7 gene.

Homocysteine, traditional risk factors and impaired renal function in coronary artery disease.

BACKGROUND: To establish whether the frequent finding of a moderate-intermediate increase in plasma total homocysteine (tHcy) causes coronary artery disease (CAD), the authors evaluated the number of coexisting major traditional risk factors, as well as the major tHcy determinants, in patients with the same degree of CAD but different tHcy levels. MATERIALS AND METHODS: The authors studied 180 patients with CAD, who were divided into three groups according to tHcy levels: 60 patients with normal tHcy, 60 patients with moderate (15-30 micromol L(-1)) and 60 patients with intermediate hyperhomocysteinaemia (30-100 micromol L(-1)). The patient groups were matched for gender, age and number of affected coronary vessels. All patients were checked for the presence of traditional risk factors for CAD (i.e. hypertension, diabetes, hyperlipidaemia, smoking habit, familial history, obesity), as well as determinants of tHcy levels. The population was subdivided into those having, or not, a substantial burden of traditional risk factors (i.e. < 4 and > or = 4, respectively). RESULTS: There was a significant trend towards a reduced number of subjects within the group with > or = 4 risk factors across increasing tHcy levels (51.7%, 37.8%, 26%, for normal, moderate, intermediate tHcy, respectively, chi2 for linear-trend = 0.006). Folate and vitamin B12 concentrations, estimated glomerular filtration rate (GFR), MTHFR 677C > T polymorphism were the major determinants of tHcy in this population. CONCLUSIONS: In patients with the same degree of CAD, those with hyperhomocysteinaemia had a reduced burden of traditional risk factors as compared with those with normal tHcy levels. Hyperhomocysteinaemia was significantly associated with an emerging non-traditional risk factor such as lower GFR.

Interaction between metabolic syndrome and PON1 polymorphisms as a determinant of the risk of coronary artery disease.

The enzyme serum paraoxonase plays an important role in antioxidant defences and prevention of atherosclerosis. Metabolic syndrome (MS) is a clinical condition associated with increased oxidant stress and cardiovascular mortality. Two common polymorphisms of serum paraoxonase, PON1 Leu(55)Met and Gln(192)Arg, have been postulated to modulate the cardiovascular risk. We studied 915 subjects with angiographic documentation: 642 subjects with coronary atherosclerosis and 273 with normal coronary arteries. Two hundred and twenty-four subjects met the diagnostic criteria of MS. We found a significant interaction between MS and both the PON1 polymorphisms in determining the risk of coronary artery disease (P<0.05 by likelihood-ratio test). The 55Leu and the 192Arg alleles, associated with reduced protection against lipid peroxidation, were associated with coronary artery disease only in the MS subgroup. Subjects with MS and both 55Leu and 192Arg alleles had significantly increased risk (OR=9.38 with 95% CI=3.02-29.13 after adjustment by multiple logistic regression) as compared to subjects without MS and with 55Met/Met-192Gln/Gln genotype. No increased risk was found for subjects with MS and the 55Met/Met-192Gln/Gln genotype. This study highlights a potential example of genetic (paraoxonase polymorphisms)-clinical (MS) interaction influencing cardiovascular risk.

Dynamics of recovery of morphometrical variables and pQCT-derived cortical bone properties after a short-term protein restriction in maturing rats.

Severe protein restriction during the post-weaning period in the rat markedly reduces femoral bone mass and produces a number of alterations in the shaft biomechanical properties. Body weight and femur length show an immediate and complete catch-up during nutritional rehabilitation. The aim of the present investigation was to assess whether the accelerated bone growth that occurs during protein rehabilitation is accompanied by recovery of cortical bone properties. The dynamics of the recovery of both material and geometric properties were thus evaluated on the femoral diaphyses in 45-day old female rats after a 10-day period of dietary protein restriction by peripheral quantitative computed tomography (pQCT). Protein starvation led to marked reduction of both body weight and femoral length (37% and 14% at day 10, respectively) which showed a complete catch-up after 30 d of protein refeeding. Protein restriction was associated with the interruption of the natural increase in cortical area (CtCSA), volumetric cortical bone mineral content (vCtBMC) and volumetric cortical bone mineral density (vCtBMD) which were 19.7, 25.8, and 14%, respectively, in malnourished than in control rats at the end of the protein starvation period. These parameters recovered completely during protein refeeding. Treatment also reduced by 30% both rectangular (xCSMI) and polar (pCSMI) moments of inertia. Although an improvement of these architectural indicators occurred with time, an approximately 20% deficit was still present at the end of the observation period (70 d), as was the bone strength index (BSI). It is concluded that protein restriction affected the adaptation of diaphyseal design which should reduce the mechanical competence of the femoral diaphysis because of an inadequate architectural distribution of cortical bone, and that the alteration did not show complete catch-up during the studied period.

Clinical, biochemical and molecular findings in a series of families with hereditary hyperferritinaemia-cataract syndrome.

Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant disease caused by mutations in the iron responsive element (IRE) of the l-ferritin gene. Despite the elucidation of the genetic basis, the overall clinical spectrum of HHCS has been less well studied as, to date, only individual case reports have been described. Therefore, we studied a total of 62 patients in 14 unrelated families, with nine different mutations. No relevant symptoms other than visual impairment were found to be associated with the syndrome. A marked phenotypic variability was observed, particularly with regard to ocular involvement (i.e. age range at which cataract was diagnosed in 16 subjects with the C39T: 6-40 years). Similarly, serum ferritin levels varied substantially also within subjects sharing the same mutation (i.e. range for the A40G: 700-2412 microg/l). We followed an HHCS newborn in whom well-defined lens opacities were not detectable either at birth or at 1 year. The lens ferritin content was analysed in two subjects who underwent cataract surgery at different ages, with different cataract morphology. Values were similar and about 1500-fold higher than in controls. These observations suggest that: (i) in HHCS the cataract is not necessarily congenital; (ii) in addition to the IRE genotype, other genetic or environmental factors may modulate the phenotype, especially the severity of the cataract.

Effects of human recombinant erythropoietin on differentiation and distribution of erythroid progenitor cells on murine medullary and splenic erythropoiesis during hypoxia and post-hypoxia.

Hemopoietic cells, the extracellular matrix, growth factors and the microenvironment are involved in the regulation of hemopoiesis. Although the regulation of erythropoiesis is well understood at the cellular level in vivo and in vitro, the role of hemopoietic sites of erythroid progenitors production has not been well defined in both steady state conditions and in stress erythropoiesis. In this study we examined the qualitative erythroid differentiation and quantitative changes of the erythroid progenitors in different erythropoietic organs during erythropoiesis of stress in a hypoxia-induced polycythemia and post-hypoxic changes in a mice model. Chronic intermittent exposure to hypobaric hypoxia induced polycythemia in mice and the post-hypoxic period was characterized by total suppression of erythropoiesis. The number and distribution in hemopoietic sites of Immature Erythroid Burst (BFU-EI), Mature Erythroid Burst (BFU-EM) and Erythroid Colony Forming Units (CFU-E) was evaluated in bone marrow and spleen of hypoxic and post-hypoxic mice after removal from the chamber. The number of BFU-EI and CFU-E, was evaluated in both femoral bone marrow and spleen of ex-hypoxic polycythemic mice, at two times intervals after the end of hypoxia. We found that in both bone marrow and spleen, the kinetics of the CFU-E pool was characterized by a sharp fall from above normal to lower than normal levels. BFU-EM increased from normal to higher than normal levels. These results have been correlated with both erythropoietin (EPO) and the erythropoietic activity. The results show that EPO levels largely control both the differentiation and the amplification of the CFU-E pool and they suggest that EPO may acts as a "survival factor" at the CFU-E level and/or increase the flow of cells from BFU-E to CFU-E. After the termination of the period of hypoxia and during post-hypoxia there was a reduction in EPO production which subsequently caused a depletion of the CFU-E population, indicating that the size of the CFU-E pool is EPO-dependent. After the injection of 1U of recombinant human erythropoietin (rHuEPO) the size of that pool was increased and the pool of BFU-EI was decreased. It is noteworthy that our studies show that the spleen functions as a large reservoir of erythroid precursors for hypoxia-induced stress erythropoiesis.

Prolonged exposure to hypobaric hypoxia transiently reduces GABA(A) receptor number in mice cerebral cortex.

The central nervous system is severely affected by hypoxic conditions, which produce alterations in neural cytoarchitecture and neurotransmission, resulting in a variety of neuropathological conditions such as convulsive states, neurobehavioral impairment and motor CNS alterations. Some of the neuropathologies observed in hypobaric hypoxia, corresponding to high altitude conditions, have been correlated with a loss of balance between excitatory and inhibitory neurotransmission, produced by alterations in glutamatergic and GABAergic receptors. In the present work, we have studied the effect of chronic hypobaric hypoxia (506 hPa, 18 h/day x 21 days) applied to adult male mice on GABA(A) receptors from cerebral cortex, to determine whether hypoxic exposure may irreversibly affect central inhibitory neurotransmission. Saturation curves for [3H]GABA specifically bound to GABA(A) receptors in isolated synaptic membranes showed a 30% decrease in maximal binding capacity after hypoxic exposure (Bmax control, 4.70+/-0.19, hypoxic, 3.33+/-0.10 pmol/mg protein), with no effect on GABA binding sites affinity (Kd control: 159.3+/-13.3 nM, hypoxic: 164.2+/-15.1 nM). Decreased B(max) values were observed up to the 10th post-hypoxic day, returning to control values by the 15th post-hypoxic day. Pharmacological properties of GABA(A) receptor were also affected by hypoxic exposure, with a 45 to 51% increase in the maximal effect by positive allosteric modulators (pentobarbital and 5alpha-pregnan-3alpha-ol-20-one). We conclude that long-term hypoxia produces a significant but reversible reduction on GABA binding to GABA(A) receptor sites in cerebral cortex, which may reflect an adaptive response to this sustained pathophysiological state.

Bone growth in nonorganic nutritional dwarfing rats.

Since no data are available to characterize mandibular growth in nonorganic nutritional dwarfing (ND), the purpose of the present study was to describe the effects of a diet on mandible and femur growth in a nutritional dwarfish animal model. Male Wistar rats were divided into two groups of 10 animals each: Control (C) and Experimental (E80: diet-restricted group). C rats were fed a standard diet ad libitum. E80 rats received 80% of the amount of standard diet eaten by group C. Food intake and body weight (Wt) and length (Lt) were recorded periodically. Growth data (Wt and Lt) were expressed as a Z-score of weight-for-length (WLZ) ratio, an index of body size. Five animals of each group were selected at random at 4 and 8 weeks and sacrificed. Additionally at t = 0, 5 animals were sacrificed for baseline measurements. Mandibular growth was estimated directly on the right mandible by measuring ten dimensions. Femur growth was estimated from Wt and Lt measurements of the bone. Mandibular weight, area, length and height were negatively affected by dietary restriction during the first 4 weeks of the experimental period. Mandibular growth ceased after this point. Dimensions corresponding to the alveolar unit did not change with time. However, all other dimensions were negatively influenced but not to the same extent. Femur rather than mandibular weight was severely affected. Therefore, the negative effects of the nutritional stress that occurs after weaning would be stronger for the femur, than for the mandible. Femur length was also negatively affected by suboptimal nutrition. In summary, the results of the present study showed that mandible and femur growth respond differently to mild chronic food restriction. These observations could be explained in terms of the different critical bone growth periods and of the time at which nutritional stress was imposed.

Bone growth in nonorganic nutritional dwarfing rats

Since no data are available to characterize mandibular growth in nonorganic nutritional dwarfing (ND), the purpose of the present study was to describe the effects of a diet on mandible and femur growth in a nutritional dwarfish animal model. Male Wistar rats were divided into two groups of 10 animals each: Control (C) and Experimental (E80: diet-restricted group). C rats were fed a standard diet ad libitum. E80 rats received 80

of the amount of standard diet eaten by group C. Food intake and body weight (Wt) and length (Lt) were recorded periodically. Growth data (Wt and Lt) were expressed as a Z-score of weight-for-length (WLZ) ratio, an index of body size. Five animals of each group were selected at random at 4 and 8 weeks and sacrificed. Additionally at t = 0, 5 animals were sacrificed for baseline measurements. Mandibular growth was estimated directly on the right mandible by measuring ten dimensions. Femur growth was estimated from Wt and Lt measurements of the bone. Mandibular weight, area, length and height were negatively affected by dietary restriction during the first 4 weeks of the experimental period. Mandibular growth ceased after this point. Dimensions corresponding to the alveolar unit did not change with time. However, all other dimensions were negatively influenced but not to the same extent. Femur rather than mandibular weight was severely affected. Therefore, the negative effects of the nutritional stress that occurs after weaning would be stronger for the femur, than for the mandible. Femur length was also negatively affected by suboptimal nutrition. In summary, the results of the present study showed that mandible and femur growth respond differently to mild chronic food restriction. These observations could be explained in terms of the different critical bone growth periods and of the time at which nutritional stress was imposed.

Bone growth in nonorganic nutritional dwarfing rats.

Since no data are available to characterize mandibular growth in nonorganic nutritional dwarfing (ND), the purpose of the present study was to describe the effects of a diet on mandible and femur growth in a nutritional dwarfish animal model. Male Wistar rats were divided into two groups of 10 animals each: Control (C) and Experimental (E80: diet-restricted group). C rats were fed a standard diet ad libitum. E80 rats received 80

of the amount of standard diet eaten by group C. Food intake and body weight (Wt) and length (Lt) were recorded periodically. Growth data (Wt and Lt) were expressed as a Z-score of weight-for-length (WLZ) ratio, an index of body size. Five animals of each group were selected at random at 4 and 8 weeks and sacrificed. Additionally at t = 0, 5 animals were sacrificed for baseline measurements. Mandibular growth was estimated directly on the right mandible by measuring ten dimensions. Femur growth was estimated from Wt and Lt measurements of the bone. Mandibular weight, area, length and height were negatively affected by dietary restriction during the first 4 weeks of the experimental period. Mandibular growth ceased after this point. Dimensions corresponding to the alveolar unit did not change with time. However, all other dimensions were negatively influenced but not to the same extent. Femur rather than mandibular weight was severely affected. Therefore, the negative effects of the nutritional stress that occurs after weaning would be stronger for the femur, than for the mandible. Femur length was also negatively affected by suboptimal nutrition. In summary, the results of the present study showed that mandible and femur growth respond differently to mild chronic food restriction. These observations could be explained in terms of the different critical bone growth periods and of the time at which nutritional stress was imposed.

Catch-up in mandibular growth after short-term dietary protein restriction in rats during the post-weaning period.

Catch-up growth has been defined as growth with a velocity above the statistical limits of normality for age during a defined period of time which follows a period of impaired growth. Since no data are available on catch-up in mandibular growth, the present study was designed to estimate the dynamics of the mandibular size after short-term dietary protein restriction in rats during the post-weaning period. Weanling male rats, 22 d of age, were divided into two groups, control (C) and experimental (E). E rats were fed a protein-free diet during the first 10 d; from this time on, they were placed on a 20% protein diet, as were C rats during the entire experimental period, which lasted 70 d. Five rats from both groups were randomly selected every 10 d and sacrificed. Mandibular growth was estimated directly on the right mandible by measuring several dimensions (mandibular area, base length, mandibular height, mandibular length, alveolar length and incisor alveolar process length). Alveolar and incisor alveolar process lengths did not change with age or dietary protein. All other dimensions increased with age and were thus negatively affected by protein restriction. After growth restriction ceased, the rate of increase of all affected dimensions was above normal values and deficits were swiftly eliminated. Since age-independent dimensions compose roughly the anterior portion of the mandible, this portion of the bone was not affected by protein restriction. It was, thus, the posterior part of the mandible which stopped growth during the nutritional insult and showed catch-up during nutritional rehabilitation. In summary, the rat mandible has a high potential for catch-up during the post-weaning period, showing the ability to achieve complete catch-up in about 30 d.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80% suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells.

A simple in vivo bioassay suitable for routine testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80

suspension of heterologous (rat) red cells were used as assay animals and splenic 59Fe uptake as expression of the response to rHu-EPO. The assay took three days and the following schedule is proposed: 1) intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo bioassay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone.

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone

Erythropoietin assay in mice made polycythemic by transfusion of heterologous red cells

A simple in vivo bioassay suitable testing of quality control of recombinant human erythropoietin (rHu-EPO) analogues was developed. Mice made polycythemic by intraperitoneal injection of 1.2 ml of a 80 per cent suspension of heterologous (rat) red cells were used as assay animals and splenic 59 Fe uptke as expression of the response to rHu-EPO. The assay took three days and the following schedule is propose: 1)intraperitoneal injection of 1.2 ml of washed packed red cells obtained from donor rats, 2) subcutaneous injection of test material 4-5 h after transfusion, 3) intravenous administration of 59 Fe tracer 48 h later, and 4) determination of splenic isotope uptake 6 h after injection. This method for the in vivo biossay of rHu-EPO analogues is an economical and reliable alternative to the existing bioassays of the hormone(AU)

Plasma disappearance of exogenous erythropoietin in mice under different experimental conditions.

Erythropoietin (EPO) is a glycoprotein hormone produced primarily in the kidneys and to a lesser extent in the liver that regulates red cell production. Most of the studies conducted in experimental animals to assess the role of EPO in the regulation of erythropoiesis were performed in mouse models. However, little is known about the in vivo metabolism of the hormone in this species. The present study was thus undertaken to measure the plasma tl/2 of radiolabeled recombinant human EPO (rh-EPO) in normal mice as well as in mice with altered erythrocyte production rates (EPR), plasma EPO (pEPO) titer, marrow responsiveness, red cell volume, or liver function. Adult CF-1 mice of both sexes were used throughout. For the EPO life-span studies, 30 mice in each experiment were intravenously injected with 600,000 cpm of 125l-rh-EPO and bled by cardiac puncture in groups of five every hour for 6 h. Trichloroacetic acid (TCA) was added to each plasma sample and the radioactivity in the precipitate measured in a gamma-counter. EPO, pEPO, marrow responsiveness, or red cell volume were altered by either injections of rh-EPO, 5-fluorouracil, or phenylhydrazine, or by bleeding, or red cell transfusion. Liver function was altered by CI4C administration. In the normal groups of mice, the estimated tl/2 was 182.75+/-14.4 (SEM) min. The estimated tl/2 of the other experimental groups was not significantly different from normal. These results, therefore, strongly suggest that the clearance rate of EPO in mice is not subjected to physiologic regulation and that pEPO titer can be really taken as the reflection of the EPO production rate, at least in the experimental conditions reported here.