Infrared analysis of bones in magnesium-deficient rats treated with vitamin K<Subscript>2</Subscript>

In this study, we compared the effects of vitamin K₂ menatetrenone on bone mechanical properties in rats fed a low-magnesium (Mg) diet. In addition, the mechanism of bone quality was examined using Fourier transform infrared imaging (FTIRI). Thirty 4-week-old male Wistar rats were divided into three groups: intact, low-Mg-control, and low-Mg-MK-4 groups. Rats in the low-Mg groups were given a diet containing 6 mg/100 g Mg (intact, 90 mg/100 g). After an 8-week-treatment, the cortical bone mineral content (CtBMC), outer perimeter, and endo perimeter of the femoral diaphysis in the low-Mg-control group were significantly higher, while the maximum load (ML) and elastic modulus (EM) were 81% and 50% of those in the intact group, respectively (respectively, P < 0.05). In the low-Mg-MK-4 group, ML and EM were significantly higher than in the low-Mg-control group (P < 0.05), with no differences in CtBMC. The mineral/matrix ratios for the periosteal and central regions in the low-Mg-control group were 162% and 120% of those in the intact group (both, P < 0.05), respectively. MK-4 significantly inhibited these increases (P < 0.05). We found that the mineral/matrix ratios for the periosteal region of the femoral diaphysis were negatively correlated with EM, suggesting that an increase in the mineral/matrix ratio may be involved in the reduction of EM and that MK-4 may improve EM by improving the mineral/matrix ratio.     

Response of serum carboxylated and undercarboxylated osteocalcin to alendronate monotherapy and combined therapy with vitamin K<Subscript>2</Subscript> in postmenopausal women

Alendronate decreases the risk of femoral neck fracture by suppressing bone turnover, and also decreases the serum total osteocalcin level. A low serum carboxylated osteocalcin level or high undercarboxylated osteocalcin level could be risk factors for femoral neck fracture. Vitamin K mediates the carboxylation of osteocalcin, but the effect of alendronate therapy with or without vitamin K₂ supplementation remains unknown. Forty-eight postmenopausal women were enrolled in a 1-year prospective randomized trial and assigned to alendronate monotherapy (5 mg/day) (group A, n = 26) or vitamin K₂ (45 mg/day) plus alendronate (5 mg/day) (group AK, n = 22). Bone mineral density was measured by dual-energy X-ray absorptiometry at 0 and 12 months; bone turnover parameters were measured at 0, 3, and 12 months. Four patients discontinued alendronate therapy, and we analyzed the remaining 44 patients (23 in group A and 21 in group AK) who completed 1 year of treatment. Alendronate decreased undercarboxylated osteocalcin; carboxylated osteocalcin was not affected. Addition of vitamin K₂ enhanced the decrease of undercarboxylated osteocalcin levels and led to a greater increase of femoral neck bone mineral density. Alendronate monotherapy does not decrease carboxylation of osteocalcin, and combination of vitamin K₂ and alendronate brings further benefits on both osteocalcin carboxylation and BMD of femoral neck in postmenopausal women with osteoporosis.     

Combination use of vitamin K<Subscript>2</Subscript> further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats

 Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K₂ treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K₂ leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K₂ with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss. Received: January 30, 2002 / Accepted: November 6, 2002 RID="*" ID="*"  Present address: Department of Health Sciences, Oita University of Nursing and Health Sciences, Oita, Japan Acknowledgments. This work was supported in part by a Special Grant for Medical Research from Ministry of Post and Telecommunications, Japan (to M.F.), a grant in aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (#13671115 to M.F.), and by a grant from the Research Society for Metabolic Bone Disease (to M.F.). We are grateful to Miss Sachiko Suzuki for technical assistance. Offprint requests to: M. Fukagawa     

Effects of vitamin K2 and calcitonin on bone resorption model induced by vitamin A in thyroparathyroidectomized rats

Vitamin K₂ is considered to have two different effects: one is to enhance bone formation, and the other is to suppress bone resorption. However, as these effects have not been observed in a single experiment, it is unclear whether bone formation can proceed during a state of accelerating bone resorption. We therefore examined the effects of vitamin K₂ and calcitonin on a vitamin A-induced bone resorption model of thyroparathyroidectomized rats using bone histomorphometry and bone metabolism markers. The seven groups of male Sprague-Dawley rats (6 weeks old) were sham operation of thyroparathyroidectomy (TPTX) (sham group), TPTX (TPTX group), treated with vitamin A (20 mg/kg per day) from 11th to 20th day after TPTX (A group), treated with vitamin A and vitamin K₂ (30 mg/kg per day) or its vehicle from 11th to 20th day after TPTX [K group or K (veh) group], and treated with vitamin A and calcitonin (10IU/kg/ per day) or its vehicle during the same period [CT group or CT (veh) group]. Serum and urine samples were taken for marker determination on days 10, 13, 16, and 19 of TPTX and at death on the 21st day after TPTX. Undecalcified sections (Villanueva bone stain) were made of the left tibiae and decalcified sections [tartrate-resistant acid phosphatase (TRAP) stain] of the right tibiae. In the undecalcified sections, secondary trabeculae were used for histomorphometry, and in the decalcified sections primary and secondary trabeculae were used. Serum Ca of the vitamin A-administered group was significantly higher than that of the TPTX group, but this change was inhibited by vitamin K₂ or calcitonin. Serum alkaline phosphatase (ALP) in the K group was significantly higher than in all the thyroparathyroidectomized groups except the K (veh) group. In the undecalcified sections, although there was no significant difference between any of the groups in bone volume, the K group showed an increase of osteoid surface and mineralizing surface. In the decalcified sections, the K group showed a decrease of TRAP-positive areas compared to the K (veh) group in primary trabeculae. There was no significant difference between the K and K (veh) groups in secondary trabeculae. Results from the CT group were compatible with bone resorption inhibition in both bone metabolism markers and bone histomorphometry. We found that vitamin K₂ enhances bone formation and suppresses bone resorption in areas with a high turnover of bone metabolism. Vitamin K₂ is therefore expected to increase bone content if it is administered over an extended period.     

Local injection of 1,25-dihydroxyvitamin D<Subscript>3</Subscript> enhanced bone formation for tooth stabilization after experimental tooth movement in rats

The present investigation evaluated the effect of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) on alveolar bone formation during tooth movement in rats. Orthodontic elastics were inserted between the maxillary first and second molars on bilateral sides in male rats. 1,25(OH)₂D₃ was injected locally, at the concentration of 10^–10M, once every 3 days in the submucosal palatal area of the root bifurcation of the molar on the right side. Histomorphometric analysis revealed that tooth movement without application of 1,25(OH)₂D₃ decreased the mineral appositional rate (MAR) on the compression area at 7 days. Repeated injections of 1,25(OH)₂D₃ in the orthodontically treated animals distinctly stimulated alveolar bone formation on the mesial side at 14 days. There was a significant increase in MAR associated with elevated osteoblast surface (Ob.S/BS) value on the tension surface. These findings suggest that local application of 1,25(OH)₂D₃ enhances the reestablishment of supporting tissue, especially alveolar bone of teeth, after orthodontic treatment.     

Regulation of mineral-to-matrix ratio of lumbar trabecular bone in ovariectomized rats treated with risedronate in combination with or without vitamin K<Subscript>2</Subscript>

The relationship between bone turnover and bone tissue and material properties was examined in ovariectomized (OVX) rats treated with risedronate in combination with or without vitamin K₂. Seventy female rats, 18 weeks of age, were assigned to 7 groups (n = 10): sham-operated + vehicle control; OVX + vehicle control; OVX + risedronate 0.1, 0.5, or 2.5mg/kg/day po; OVX + vitamin K₂ 30mg/kg/day po; OVX + vitamin K₂ (30mg/kg/day) and risedronate (0.5mg/kg/day). Treatments were given daily for 9 months. To assess bone turnover, we measured serum osteocalcin and urinary deoxypyridinoline at 0, 3, and 9 months. To assess vertebral and femoral tissue and material properties, bone mass, bone mineral density (BMD by DXA), trabecular bone structure (vertebra: 3D-CT), cortical bone structure (femur: histomorphometry), biomechanical properties, and mineral properties (mineral-to-matrix and carbonate-to-phosphate ratios by Fourier transform infrared microspectroscopy) were measured ex vivo at 9 months. Ovariectomy increased bone turnover and induced significant loss of bone mass/density, structure, mineral properties (mineral-to-matrix ratio), and strength. Risedronate produced dose-dependent inhibition of the ovariectomy-induced increase in turnover and loss of bone mass/density, structure, mineral-to-matrix ratio, and strength, with a lowest effective dose of 0.1–0.5mg/kg/day. High-dose risedronate (2.5mg/kg/day) did not induce increases in any parameter above that of sham control. Vitamin K₂ had no effects. In the OVX groups, urinary deoxypyridinoline at 3 and 9 months correlated significantly with vertebral BMD, trabecular bone volume, ultimate load, stiffness, and mineral-to-matrix ratio, and with femoral BMD, cortical area, and ultimate load. These results support the concept that changes in bone tissue and material properties can result directly from changes in bone turnover. Different effects among different drugs on material properties, including mineral-to-matrix ratio, may reflect differences in the relative rate and magnitude of osteoclastic bone resorption and osteoblastic primary bone mineralization.     

Effects of treadmill exercise on bone mass,bone metabolism,and calciotropic hormones in young growing rats

The aim of the present study was to examine the effects of exercise on bone mass, bone metabolism, and calciotropic hormones in young growing rats. Twenty 6-week-old female Wistar rats were randomized into the following four groups with 5 animals each: 7 weeks of exercise, 7 weeks of sedentary control, 11 weeks of exercise, and 11 weeks of sedentary control. The exercise regimen consisted of running on a treadmill at 25m/min for 1h each day on 5 days a week. After each period of exercise, the bone mineral content (BMC) of the tibia and fifth lumbar spine was measured by dual-energy X-ray absorptiometry, using a Lunar DPX-L instrument. The femoral length and levels of bone markers and calciotropic hormones were also assessed. Seven and 11 weeks of exercise increased the serum osteocalcin and 1,25-dihydroxyvitamin D₃ levels, and decreased the serum parathyroid level. Seven weeks of exercise decreased the urinary deoxypyridinoline level, and 11 weeks of exercise increased the serum alkaline phosphatase level and decreased the serum tartrate-resistant acid phosphatase level. As a result, 7 and 11 weeks of exercise increased the femoral length and tibial BMC, but did not alter the lumbar BMC. The present study demonstrates that treadmill exercise stimulates bone formation and suppresses bone resorption, increases the serum 1,25-dihydroxyvitamin D₃ level, and decreases the serum parathyroid hormone level, resulting in an increase in bone mass with stimulation of longitudinal bone growth, especially at weight-bearing sites, in young growing rats. Further studies with long-term exercise may be needed to obtain a positive effect on the lumbar BMC.     

Comparison of the effects of room air and N<Subscript>2</Subscript>O + O<Subscript>2</Subscript> used for ProSeal LMA cuff inflation on cuff pressure and oropharyngeal structure

This study aimed to evaluate the effects of different inflating gases used for ProSeal LMA (PLMA) cuff inflation on cuff pressure, oropharyngeal structure, and the incidence of sore throat. Eighty patients (American Society of Anesthesiologists; ASA I–II) were randomly divided into two groups. PLMA cuff inflation was achieved with appropriate volumes of 50% N₂O + 50% O₂ in group I and room air in group II, respectively. When the PLMA was removed, oropharyngeal examination was carried out immediately, using a rigid optical telescope. Patients were asked about sore throat symptoms postoperatively. Cuff pressures were significantly lower in group I, except at the initial pressure measurement. Cuff pressure was positively correlated with the length of the operation in group II, and negatively correlated in group I. PLMA cuff inflation with room air led to increased cuff pressure during the operation, possibly due to the diffusion of N₂O into the cuff. We consider that a PLMA cuff inflated with an N₂O-O₂ mixture is convenient, especially in operations in which N₂O has been used.     

Effects of unilateral arterial infusion of GH and IGF-I on tibial longitudinal bone growth in hypophysectomized rats

Summary We have studied the effect of local arterial infusion of bacterially produced human growth hormone (hGH), insulinlike growth factor I (IGF-I), or pituitary-derived ovine prolactin (oPRL) on longitudinal bone growth of hypophysectomized rats. The substances were infused during a 14-day period by osmotic mini-pumps through a catheter which was implanted into the femoral artery of one hindlimb. Longitudinal bone growth was measured by the intravital marker tetracycline. Infusion of 1 μg hGH per day stimulated bone growth only of the treated limb and not of the uninfused contralateral limb. Infusion of 10 μg hGH per day also stimulated unilateral longitudinal bone growth, but the uninfused contralateral limb also showed a significant growth response, probably because local administration of GH at this dose caused a significant elevation of GH in the systemic circulation. As a result, the differential growth response between the GH-treated and untreated limbs decreased compared to rats that were infused with 1 μg hGH per day. Unilateral arterial infusion of 5 μg human IGF-I or 10 μg oPRL per day did not produce a significant growth response. The results of the present study confirm the observation by Schlechter and co-workers [9, 16], who demonstrated that unilateral arterial infusion of GH maintained tibial cartilage width following hypophysectomy in the rat. The results of Schlechter and co-workers and the results of the present study show that GHin vivo stimulates epiphyseal cartilage growth directly. However, an increased local production of insulinlike growth factors is probably of importance for the expression of the direct effect of GH on longitudinal bone growth. The present results do not completely rule out the possibility that insulinlike growth factors in the circulation might have the growth plate as a target organ.     

The effect of vitamin K2 on bone metabolism in aged female rats

Reactive oxygen species (ROS) may contribute to aging and osteoporosis resulting from marked decreases in plasma antioxidants in aged osteoporotic women. On the other hand, high-dose vitamin K₂ (menaquinone-4: menatrenone, MK-4) supplementation has been reported to reduce ovariectomy-induced bone loss in rats and to decrease osteoporotic fracture in postmenopausal women. However, the mechanism by which vitamin K₂ prevents osteoporosis is unclear. Recently, vitamin K₂ has been suggested to preserve antioxidant activity as a novel function. Therefore, we investigated the effect of vitamin K₂ on the osteoporosis of aged rats by evaluating the relationships between serum antioxidant levels and bone metabolism. Aged female rats exhibited significantly lower serum alkaline phosphatase activity and osteocalcin level, together with lower serum levels of antioxidants such as 17-estradiol, macrophage migration inhibitory factor (MIF) and glutathione peroxidase (GPx) activity, as compared with young female rats. On the other hand, vitamin K₂ supplementation (500 mg/kg, food intake) for 98 days led to a significantly increased serum vitamin K₂ level (3,045±915 ng/ml in the vitamin K₂ supplemented group vs. 4.6±3.4 ng/ml in the control diet group; P <0.0001) with increased serum alkaline phosphatase activity and MIF level ( P <0.05). Unexpectedly, however, it failed to increase the serum level of antioxidants such as GPx. Nor did it affect bone metabolism markers such as oteocalcin and osteopontin, which were significantly lower than in the young female rats ( P <0.05). Finally, the histomorphometric properties of the proximal tibia in the femur were not altered by vitamin K₂. These results suggest that high-dose vitamin K₂ supplementation neither improves lowered antioxidant levels nor stimulates bone formation in aged rats.     

Peritoneal and systemic pH during pneumoperitoneum with CO<Subscript>2</Subscript> and helium in a pig model

Background Local peritoneal effects of laparoscopic gases might be important in peritoneal biology during and after laparoscopic surgery. The most commonly used gas, CO₂, is known to be well tolerated, but also causes changes in acid-base balance. Helium is an alternative gas for laparoscopy. Although safe, it is not widely used. In this study a method for monitoring peritoneal pH during laparoscopy was evaluated and peritoneal pH during CO₂ and helium pneumoperitoneum was studied as well as its systemic reflection in arterial pH. Methods For these experiments 20 pigs were used, with ten exposed to pneumoperitoneum with CO₂, and ten to helium. Peritoneal and sub-peritoneal pH were continuously measured before and during gas insufflation, during a 30-minute period with a pneumoperitoneum and during a 30-minute recovery period. Arterial blood-gases were collected immediately before gas insufflation, at its completion, at 30 minutes of pneumoperitoneum and after the recovery period. Results Peritoneal pH before gas insufflation was in all animals 7.4. An immediate local drop in pH (6.6) occurred in the peritoneum with CO₂ insufflation. During pneumoperitoneum pH declined further, stabilising at 6.4, but was restored after the recovery period (7.3). With helium, tissue pH increased slightly (7.5) during insufflation, followed by a continuous decrease during pneumoperitoneum and recovery, reaching 7.2. Systemic pH decreased significantly with CO₂ insufflation, and increased slightly during helium insufflation. Systemic pH showed co-variation with intra-peritoneal pH at the the end of insufflation and after 30 minutes of pneumoperitoneum. Conclusions Insufflation of CO₂ into the peritoneal cavity seemed to result in an immediate decrease in peritoneal pH, a response that might influence biological events. This peritoneal effect also seems to influence systemic acid-base balance, probably due to trans-peritoneal absorption.     

Effect of Vitamins D<Subscript>2</Subscript> and D<Subscript>3</Subscript> Supplement Use on Serum 25OHD Concentration in Elderly Women in Summer and Winter

Vitamin D₂ and D₃ are generally considered equipotent in humans. A few studies have reported that serum 25OHD levels are higher in vitamin D₃- compared with vitamin D₂-supplemented subjects. As both vitamin D₂ and D₃ supplements are commonly used by elderly in United States, in the present study we determined the effect of self-reported vitamin D₂ and vitamin D₃ supplement use on serum total 25OHD levels according to season in elderly women aged 65–77 years. Serum total 25OHD levels were determined in winter and summer in unsupplemented women (N = 307) and in women who reported taking vitamin D₂ (N = 56) and vitamin D₃ (N = 55) supplements by competitive protein binding assay. In vitamin D₂-supplemented women, the contribution of vitamin D₂ and D₃ to the mean serum total 25OHD level was assessed by HPLC. In summer, there were no significant differences in the mean total serum 25OHD levels (ng/ml) among the vitamin D₂ (32 ± 2.1), vitamin D₃ (36.7 ± 1.95), and unsupplemented (32.2 ± 0.95) groups. In winter, the mean serum total 25OHD levels were higher in women on vitamin D₂ (33.6 ± 2.34, P < 0.05) and vitamin D₃ (29.7 ± 1.76, NS) supplements compared with unsupplemented women (27.3 ± 0.72). In vitamin D₂-supplemented women, about 25% of the mean serum total 25OHD was 25OHD₂, in both summer and winter. Twelve percent of unsupplemented women and 3.6% of vitamin D-supplemented women had a mean serum total 25OHD level below 15 ng/ml in winter. In elderly subjects, both vitamin D₂ and Vitamin D₃ supplements may contribute equally to circulating 25OHD levels, with the role of vitamin D supplement use being more predominant during winter. This work was presented in part as an abstract at Second Joint Meeting of the American Society for Bone and Mineral Research and The International Bone and Mineral Society, San Francisco, CA, 1–6 December 1998.     

Effects of growth hormone on bone modeling and remodeling in hypophysectomized young female rats: a bone histomorphometric study

Growth hormone (GH) deficiency causes decreased bone mineral density and osteoporosis, predisposing to fractures. We investigated the mechanism of action of GH on bone modeling and remodeling in hypophysectomized (HX) female rats. Thirty female Sprague–Dawley rats at age 2 months were divided into three groups with 10 rats each: control (CON) group, HX group, and HX + GH (3 mg/kg daily SC) group, for a 4-week study. Hypophysectomy resulted in cessation of bone growth and decrease in cancellous bone mass. Periosteal bone formation decreased and bone turnover rate of endocortical and trabecular surfaces increased as compared to the CON group. GH administration for 4 weeks restored weight gain and bone growth and mitigated decrease in bone density after hypophysectomy. However, trabecular bone mass in the proximal tibial metaphysis remained lower in group HX + GH than in group CON. Dynamic histomorphometric analysis showed that bone modeling of periosteal bone formation and growth plate elongation was significantly higher in group HX + GH than in group HX. New bone formed beneath the growth plate was predominately woven bone in group CON and group HX + GH. Bone remodeling and modeling–remodeling mixed modes in the endocortical and PTM sites were enhanced by GH administration; both bone formation and resorption activities were significantly higher than in group HX. In conclusion, GH administration to HX rats reactivated modeling activities in modeling predominant sites and increased new bone formation. GH administration also increases remodeling activities in remodeling predominant sites, giving limited net gain in the bone mass.     

甲状旁腺激素联合降钙素对大鼠骨质疏松性骨折骨生长因子的影响

目的探讨甲状旁腺素联合降钙素对大鼠骨质疏松性骨折骨相关生长因子及骨折愈合的影响。方法75只雌性SD大鼠随机分为:假手术组、去势组、甲状旁腺素组、降钙素组、联合用药组,15只/组,首先构建去卵巢大鼠骨质疏松性骨折动物模型。观察并评估骨折愈合,测定骨痂BMD和BMC,检测血清BMP-2、VEGF、TGF-β、IGF-1水平和骨痂蛋白表达,观察骨痂形态结构变化。结果去势组较假手术组骨折愈合评分、骨痂BMD和BMC、血清BMP-2、VEGF、TGF-β、IGF-1水平和骨痂蛋白表达均显著降低(P均<0.05),而联合用药组较去势组上述指标均显著升高(P均<0.05)。去势组骨痂骨小梁明显减少,生长稀疏,排列紊乱,大量间充质干细胞及纤维软骨细胞,成骨细胞及微血管少见;联合用药组骨痂大量骨小梁,生长旺盛,互相交错网状,排列致密有序,可见较多成熟的骨细胞及成骨细胞。结论甲状旁腺素联合降钙素通过介导提高相关骨生长因子表达,提高骨质疏松性骨折骨密度和矿物含量,改善骨组织形态学,加快骨折愈合。     

Comparison of the effects of 1,25-dihydroxycholecalciferol,prostaglandin E2, and osteoclast-activating factor with parathyroid hormone on the ultrastructure of osteoclasts in cultured long bones of fetal rats

Summary The effects of 1,25-dihydroxy vitamin D₃ [1,25(OH)₂D₃], prostaglandin (PGE₂), and osteoclast-activating factor (OAF) on the size of osteoclasts, nuclei, ruffled borders, and clear zones in cultured long bones of fetal rats were quantitated. In addition, the number of osteoclasts in the bones was counted and the release of calcium from the bone into the culture medium was determined. These data were compared with the corresponding effects of parathyroid hormone (PTH). All agents tested increased the size of the ruffled borders significantly after 3 h, the size of the clear zones after 12 h, and the size of the cells after 12–24 h. No important differences in sizes were noticed between the agents tested or between the agents and PTH. The number of osteoclasts was increased after 24 h of treatment with PTH, but not after the other agents. Calcium release was significantly increased for all agents between 12 and 24 h. It is concluded that bone resorption by 1,25(OH)₂D₃, OAF, and PGE₂ is mediated primarily by increased activity of existing osteoclasts similar to PTH activation.     

Increased bone growth by local prostaglandin E2 in rats

Summary The effects of prostaglandin E₂ (PGE₂) on bone growth were investigated in rats. Daily injection of PGE₂ (1, 10, and 100 pmol) was given via local intraosseous route into the metaphysis of the left tibia for 14 days. The contralateral right tibia injected with vehicle and saline was for the control. The rats receiving no injection provided as normal control. The results obtained indicated that PGE₂ slightly but significantly decreased the body weight increment without effect on tibial length. The most prominent effect of PGE₂ was the increase of metaphyseal bone trabeculae by 45–81% in a dose-dependent manner. The microscopic examination revealed that PGE₂ unequivocally increased the new woven bone formation. The bone cell population study showed no difference between the number of osteoblasts and osteoclasts in primary spongiosa of the PGE₂-injected limbs and those of contralateral limbs. However, the numbers of osteoblasts and osteoclasts were markedly increased in secondary spongiosa in the PGE₂-injected limbs. This finding confirmed a stimulatory role of PGE₂ in the bone formation. The local intraosseous injection of PGE₂ was proven to be a good model for the study of local growth factors on bone metabolism with a lower effective dose which eliminates the systemic side effects.     

p53 expression and apoptosis in liver and spleen during CO<Subscript>2</Subscript> pneumoperitoneum

Backgrounds and aims  Laparoscopic surgery techniques have been increasingly preferred to classic laparotomy by surgeons since 1987. However, this method has some important adverse effects on intra-abdominal organs. The aim of this study is to evaluate the effects of different pressures of CO₂ on apoptosis and p53 expression in cells in liver and spleen. Methods  In total, 30 male Sprague–Dawley rats were used in the study. CO₂ was insufflated into the intra-abdominal cavity via angiocatheter cannule by an insufflator in two different pressures of 10 and 20 mm Hg for 60 min. However, in the control group, only cannule was inserted into the intra-abdominal cavity, but no gas was insufflated. After 60 min, the rats were killed and laparotomy was applied. The liver and spleen were excised. The samples were histologically processed and immunohistochemistry was applied. Results  All the data revealed that the number of apoptotic cells in liver and spleen increases in proportion to CO₂ pressure level. No p53 expression was detected in both organs. Conclusion  CO₂ pressure level and application time may affect on cells living in liver and spleen. High pressure and/or long application time may cause releasing of cytokines and superoxide radicals from these organs’ cells, and transient or serious organ dysfunctions may occur.     

The effect of different levels of peritoneal CO<Subscript>2</Subscript> pressure on bleeding time of spleen capsule injury

Aims: The authors, in contrast to similar injuries with open surgery, had observed spontaneous hemostasis of relatively large spleen capsule injuries during laparoscopic surgery. Methods: Standard spleen injuries were carried out in 5 anesthetized mongrel dogs at different CO₂ pressures and in open surgery. Bleeding was checked every minute by wiping around the injury but not removing the clot. Bleeding time was measured until no more oozing was detected. At every pressure level 3-3 measurements were done and analyzed. Parenchymal pressure of the spleen and systemic blood pressure were detected with direct catheter implantation. Results: In open surgery the average bleeding time was 15.2 min; at 5, 15, and 25 mmHg CO₂ pressures bleeding times were 12.3, 10.6, and 9.8 min, respectively. The parenchymal pressure of the spleen (5–6 mmHg) rose synchronously with peritoneal pressure, but no significant changes in systemic blood pressure were seen. Conclusion: Peritoneal CO₂ pressure may counterweight the parenchymal pressure of the spleen, thus helping hemostasis. There seems to be an inverse proportion between peritoneal pressure and bleeding time. In case of spleen capsule injury during laparoscopic surgery, chances for spontaneous hemostasis are much better compared to open surgery. Attention must be paid to maintain adequate peritoneal pressure.     

Relevance of ICP and ptiO<Subscript>2</Subscript> for indication and timing of decompressive craniectomy in patients with malignant brain edema

Summary Background. The exact effects of decompressive craniectomy on intracranial pressure (ICP) and cerebral tissue oxygenation (ptiO₂) are still unclear. Therefore, we have monitored ICP and ptiO₂ intra-operatively and correlated these values to different operative steps during craniectomy.Methods. ICP and ptiO₂ values have been monitored both, simultaneously and continuously, in 15 patients with cerebral edema due to posttraumatic or postischemic brain swelling. Indications for craniectomy were an increase in ICP above 25 mmHg or a decrease in ptiO₂ below 10 mmHg resistant to conservative treatment (e.g. mannitol, hyperventilation, adequate arterial blood oxygenation, etc.). In all cases, we performed a fronto-temporo-parietal craniectomy (15×12 cm) and dura enlargement with galea-periosteum. During craniectomy, monitoring of ICP and ptiO₂ in the affected hemisphere was continued. Values were recorded and correlated with the different operative steps.Findings. We performed craniectomy according to our treatment protocol in 5 patients. Prior to surgery, mean ICP values were 25.6 mmHg (range: 23–29 mmHg), mean ptiO₂ values were 5.9 mmHg (range: 2.4–9.5 mmHg), and mean CPP values were 66 mmHg (range: 60–70 mmHg). After removing the bone flap, ICP values dropped to physiological values (mean: 7.4 mmHg), whereas ptiO₂ values increased only slightly (mean: 11 mmHg). Opening of the dura resulted in a further decrease of ICP (mean 4.8 mmHg) and an increase of ptiO₂ to normal limits (mean: 18.8 mmHg). After skin closure, mean ICP was 6.8 mmHg and mean ptiO₂ was 21.7 mmHg, respectively. We found a significant decrease of ICP after craniectomy (p<0.042) and after dura enlargement (p<0.039) as well as a statistically significant increase in ptiO₂ after craniectomy (p<0.043) and after dura enlargement (p<0.041).Conclusion. As a large bone flap in decompressive craniectomy is essential for adequate ICP reduction, the results of the presented cases suggest that dura enlargement is the crucial step to restore adequate brain tissue oxygenation and that ptiO₂ monitoring could be an important tool for timing craniectomy in the future.