Adaptation of diaphyseal structure with aging and increased mechanical usage in the adult rat: a histomorphometrical and biomechanical study

The experimental increase in mechanical usage or overloading of the left hindlimb was produced by immobilization of the contralateral hindlimb. The right hindlimb was placed in a flexed position against the body and was immobilized using an elastic bandage. Some control animals were sacrificed initially at time zero and increased mechanical usage and age-matched control animals were sacrificed after 2, 10, 18, and 26 weeks of treatment. All animals received double bone fluorochrome labeling prior to sacrifice. Cortical bone histomorphometry and cross-sectional moments of inertia were determined. Marrow cavity enlargement and total cross-sectional area expansion represented the age-related cortical bone changes. Increased mechanical usage enhanced periosteal bone modeling in the formation mode and dampened endocortical bone remodeling and bone modeling in the resorption mode (resorption drift) to create a slight positive bone balance. These observations are in general agreement with Frost's postulate for mechanical effects on bone modeling and remodeling (Frost, H.M. 1987b. Bone "mass" and the "mechanostat." A proposal. Anat. Rec. 219: 1-9). The maximum moment of inertia did not change significantly in either control or overloaded tibial shafts. The minimum and polar moment of inertias in overloaded bones increases over those of controls at 18 and 26 weeks of the experiment.     

Adaptation of cancellous bone to aging and immobilization in the rat: a single photon absorptiometry and histomorphometry study

Nine-month-old female rats were double-labeled with bone markers and subjected to right hindlimb immobilization or served as control for 0, 2, 10, 18, or 26 weeks. The right limb was immobilized against the abdomen, thus unloading it, while the left limb was overloaded during ambulation. Single photon absorptiometry and cancellous bone histomorphometry were performed on dissected intact femur and 20-microns-thick undecalcified specimens of the proximal tibial metaphysis. In the unloaded limb, immobilization-induced muscle and cancellous bone loss occurred rapidly before 10 weeks and stabilized at 50% less bone mass after 18 weeks. Unloading caused a negative bone balance from a combination of elevated bone resorption and depressed bone formation. At 2, 10, and 18 weeks of immobilization, the ratios of bone resorption to bone formation surfaces were 1.6, 1.5, and 1.3, respectively; at 26 weeks, the ratio was 1. The bone loss was accompanied by poorer trabecular architecture (trabecular number decreased and trabecular separation increased), reaching the maximum at 18 weeks and stabilizing thereafter. These observations are in general agreement with Frost's postulate for mechanical effects on lamellar bone remodeling, and the findings on disuse osteoporosis in man. Therefore, the one-legged immobilization model can be useful in studies of the mechanisms of structural adaptation to mechanical usage.     

Adaptation of cancellous bone to overloading in the adult rat: a single photon absorptiometry and histomorphometry study

Nine-month-old female rats were subjected to right hindlimb immobilization or served as controls for 0, 2, 10, 18, and 26 weeks and were double-labeled with bone markers. The right limb was immobilized against the abdomen and considered unloaded, while the left limb was overloaded during ambulation. Single-photon absorptiometry was performed on intact femur; static and dynamic histomorphometry were performed on 20 microns thick undecalcified frontal sections of the proximal tibial metaphysis. Changes in the continuously overloaded limb was compared to that in both limbs of age-matched control animals. Single-photon absorptiometry detected increases of bone mineral density of +6%, +6%, and +5% in the proximal and +9%, +7%, and +10% in the distal femoral metaphyses after 10, 18, and 26 weeks of continuous overloading. Morphometrically, significant changes occurred in proximal tibial metaphyses compared to age-matched controls: trabecular area increased +41% and +45%, trabecular number increased +31% and +32%, and trabecular separation decreased -30% and -31% after 18 and 26 weeks of overloading. A significant increase in mineral apposition rate (+38%) was found only at 26 weeks of overloading. Insignificant decreases in both eroded and labeled bone surfaces occurred at all time periods. The histomorphometric changes indicated that increased cancellous bone mass was caused by an increase in bone formation activity (i.e., increases in mineral apposition and bone formation rates) and a decrease in remodeling space (i.e., decrease in bone eroded surface). These findings indicate that the adult skeleton can quickly adapt to the increased biomechanical needs by increasing its cancellous bone mass with an adequate structural pattern.(ABSTRACT TRUNCATED AT 250 WORDS)     

增龄及去睾丸大鼠的骨形态计量学研究

目的探讨4月龄雄性大鼠增龄及去睾丸后不同部位骨量的变化,了解两者对成熟期大鼠骨代谢的影响。方法30只4月龄SD雄性大鼠,随机分为基础对照组、年龄对照组和去睾丸组,同等条件下饲养90d。90d后取胫骨上段、腰椎和胫骨中段行不脱钙制片进行骨形态计量学观察。结果年龄对照组(7月龄)与基础对照组(4月龄)比较,胫骨上段骨形成和骨吸收都下降(骨小梁面积百分率减少28.6%);腰椎骨形成和骨吸收有下降趋势。去睾丸组与年龄对照组比较,胫骨上段骨量进一步丢失(骨小梁面积百分率减少57.1%);腰椎骨量也下降,表现为骨形成下降,骨吸收增加;胫骨中段皮质骨外膜的骨形成动态参数如矿化沉积率、骨形成率下降(P﹤0.05),内膜骨形成和吸收均有增加趋势。结论4月龄大鼠增龄及去睾丸90d均能引起大鼠骨量丢失。胫骨上段出现较早和更严重,腰椎和胫骨中段皮质骨丢失表现滞后。去睾丸后的大鼠主要表现为松质骨的骨量在增龄基础上进一步丢失。     

Experimental renal failure and iron overload: a histomorphometric study in rat tibia

Renal failure (RF) is a serious disease of relatively high incidence, known to cause bone alterations. RF patients frequently suffer anemia, which is usually treated with iron. Given that iron overload inhibits bone formation, the aim of the present study was to evaluate the effect of iron on the subchondral bone of rat tibiae, using a model of renal failure. Male Wistar rats were subjected to experimental nephrectomy in order to induce renal failure and to iron overload by daily intraperitoneal injections of 88 mg/kg body weight of iron-dextran for 16 days. Tetracyclines were injected intraperitoneally to evaluate dynamic parameters of bone. Undecalcified histological sections of the tibiae were obtained. Serum urea, creatinine, and paratohormone (PTH) levels were evaluated 30 days after the onset of the experiment. Static and dynamic histomorphometric measurements were performed. Iron overload modified the response of the animals with renal failure: a reduction in bone forming activity compatible with adynamic bone disease and a decrease in peritrabecular fibrosis were observed. Our results suggest that iron is yet one more factor involved in the imbalance in bone metabolism typically found in renal failure patients treated with iron, rendering diagnosis and treatment of bone disease in these patients more complex.     

Ovarian histomorphometric changes in adult offspring of diabetic rat mothers

In pregnant mothers, maternal diabetes occurs when the pancreas cannot produce enough insulin, which leads to increase blood glucose concentration in the mother and consequently in the foetus, causing various neonatal problems. This study was conducted to evaluate the effects of maternal diabetes on foetal ovarian structure. Sixteen adult female rats were allocated in two equal groups. Diabetes was induced in one group by alloxan. Both groups became pregnant by natural matting. Ninety days after birth, the female offspring were terminated, the body weight and blood glucose of the animals measured and their ovaries removed. Various histological parameters were determined using histological techniques. Results revealed a significant increase in body weight and blood glucose in the offspring of diabetic mothers (ODM) compared to that of controls. The weight, volume and diameter of the ovary and ovarian capsule thickness were decreased in the ODM group. The number and diameter of primary, preantral, antral and preovulatory follicles and corpora lutea were decreased in ovaries in the ODM. Maternal hyperglycaemia exhibited deleterious effects on the reproductive system of their offspring     

The effect of thionaphthene-2-carboxylic acid (TNCA) on bone structure in the rat: a histomorphometric study.

Thionaphthene-2-carboxylic acid (TNCA) has been shown to decrease osteoclast-mediated bone resorption in vitro and has shown efficacy in animal models of hypercalcaemia of malignancy. In this study, the effects of TNCA on the tibial proximal epiphysis and the sternum have been quantified by image analysis in rats administered the drug orally for 90 days at 75, 125 or 250 mg/kg/day. The amount of bone in the subepiphyseal area and sternum was increased in a dose-related manner. TNCA exerted a biphasic effect on the height of the epiphyseal plate. It was stimulatory at the low dosage and inhibitory at the high dose. This study constitutes the first of its kind to demonstrate the in-vivo effect of TNCA on the bones of rats.     

Effects of thyroxine on cortical bone remodeling in adult dogs: a histomorphometric study.

The effects of thyroxine on cortical bone remodeling were studied under conditions of normal and accelerated remodeling induced by surgical elevation of the periosteum. Six adult female beagle dogs were administered orally 1.0 mg of L-thyroxine per kg body weight daily for 60 days. Static and dynamic changes were evaluated using tetracycline and DCAF (2,4-bis-N,N'-dicarboxymethyl aminomethyl fluorescein) in vivo double labeling of rib specimens taken before treatment and after 60 days. Thyroxine administered in moderate pharmacologic doses increased the activation frequency, number of bone-forming and -resorbing sites, and the osteoid seam circumference in unaltered bone. Thyroxine, by activating skeletal remodeling, increased bone turnover and both formation and resorption at the tissue level. In ribs with periosteal elevation and accelerated remodeling, thyroxine increased the activation frequency, number of bone resorption and formation sites, and ratio of bone resorptive-to-formative surfaces. In addition, thyroxine under these conditions resulted in an increase in the osteoid seam circumference, radial closure rate, and bone formation rate at the tissue level but decreased the osteon formation time. Under conditions of accelerated remodeling, thyroxine increased osteoblastic and resorptive activity to a greater degree than in unaltered bone and resulted in bone changes similar to that described in human beings with thyrotoxicosis. The increased serum calcium and phosphorus levels and urinary hydroxyproline excretion at several intervals during thyroxine administration were consistent with the morphometric evidence of increased bone turnover and resorption. These findings suggest that thyroxine is a potent activator of skeletal remodeling under conditions of both normal and accelerated remodeling.     

Sensitivity of rat soleus muscle to a mechanical stimulus is decreased following hindlimb unweighting

Mechanical loading is thought to be an important stimulus regulating muscle mass. However, the responsiveness of a muscle atrophied by a period of mechanical unloading to a subsequently imposed mechanical challenge is not well understood. This study examined the phosphorylation of the mechanically sensitive p54 c-jun NH₂-terminal kinase (JNK) signaling protein in atrophied rat soleus muscle in response to a mechanical challenge in situ (isometric contractions; 100 Hz, 150 ms, once every 1 s for 5 min). Rats underwent either 7 or 14 days of hindlimb suspension (HLS) following which phosphorylation of JNK was measured biochemically. Immunofluorescence analysis revealed that phosphorylated JNK was localized in myonuclei. Baseline JNK phosphorylation measured in non-stimulated soleus muscles of 7- and 14-day HLS groups was 3.0- and 2.8-fold, respectively, the baseline phosphorylation measured in muscle of weight-bearing control animals (CTL). Following a mechanical challenge, JNK phosphorylation in stimulated CTL and 7-day HLS groups was significantly increased by 3.2- and 1.8-fold at the non-stimulated baseline levels, respectively. In stimulated muscle of 14-day HLS, JNK phosphorylation levels did not significantly differ from the baseline levels suggesting that the ability to elicit a mechanically induced phosphorylation of the JNK signaling protein gradually decreases with unweighting and is attenuated after 14-day HLS. Changes in the responsiveness of mechanically sensitive intracellular signaling pathways in atrophic muscle may contribute to the functional impairment experienced by muscle in the absence of weight bearing for prolonged periods.     

Gastric epithelial mucus--a densitometric histochemical study of aspirin-induced damage in the rat

In an attempt to elucidate which mechanisms of mucus barrier breakdown are important in the formation of gastric erosions, changes occurring in the epithelial mucin content of gastric mucosa exposed to a damaging agent were studied quantitatively. Male Wistar rats (n = 30) were starved for 24 h, then dosed orally with aspirin suspension (300 mg/kg body weight). Animals were killed after 3, 5 or 9 h. Paraffin sections of gastric mucosa were prepared from each and were stained with Alcian blue pH 2.5 (for acidic glycoproteins) or PAS (neutral glycoproteins). Control animals (n = 15) were treated identically, apart from the omission of aspirin from the suspension. The mean integrated optical density (IOD) of each slide was measured using a Vickers M85 Microdensitometer. The mean IOD for PAS-stained sections dropped to 50% of the control value in the 3-h group (P less than 0.001), returning to 84% of the control value in the 5-h group and normal in the 9-h group. In contrast, mean IOD for Alcian blue stained sections was only marginally reduced after 3 h, but showed considerable reduction in the 5-h (57% of control value) and 9-h (61% of control value) groups. It is suggested that this diminished epithelial mucin content is a consequence of cellular exfoliation. Such depletion of epithelial mucin content represents a diminished source of supply for the surface gel, the reduced thickness of which would compromise the 'mucus-bicarbonate' barrier.     

Osteosynthesis of diaphyseal fracture by Ossatite experimental study in rat

In order to develop a biodegradable interlocking nail for fracture fixation, hydoxylapatite pins and paste were implanted in the femoral bone of rats. A distal fracture was performed. The union and the tissue reaction to hydroxylapatite versus stainless-steel rods were studied after 15 days, 1, 2 and 6 months implantation. Metal pins induced a union. Hydroxylapatite pins (Ossatite) did not prevent callus formation, but did not lead to consolidation in all cases due to weakness of gelatin matrix binding the apatite particles together. The biocompatibility of material is satisfactory and the osteo-inductive properties of hydroxylapatite was confirmed. With injectable Ossatite , we could not obtain rat femoral fracture consolidation. We can confirm good biomaterial tolerance in bone which contrasts with important soft tissue reactions. Use of such material should be carefully limited to filling intra-osseous cavities.     

Gastric epithelial mucus--a densitometric histochemical study of aspirin-induced damage in the rat.

In an attempt to elucidate which mechanisms of mucus barrier breakdown are important in the formation of gastric erosions, changes occurring in the epithelial mucin content of gastric mucosa exposed to a damaging agent were studied quantitatively. Male Wistar rats (n = 30) were starved for 24 h, then dosed orally with aspirin suspension (300 mg/kg body weight). Animals were killed after 3, 5 or 9 h. Paraffin sections of gastric mucosa were prepared from each and were stained with Alcian blue pH 2.5 (for acidic glycoproteins) or PAS (neutral glycoproteins). Control animals (n = 15) were treated identically, apart from the omission of aspirin from the suspension. The mean integrated optical density (IOD) of each slide was measured using a Vickers M85 Microdensitometer. The mean IOD for PAS-stained sections dropped to 50% of the control value in the 3-h group (P less than 0.001), returning to 84% of the control value in the 5-h group and normal in the 9-h group. In contrast, mean IOD for Alcian blue stained sections was only marginally reduced after 3 h, but showed considerable reduction in the 5-h (57% of control value) and 9-h (61% of control value) groups. It is suggested that this diminished epithelial mucin content is a consequence of cellular exfoliation. Such depletion of epithelial mucin content represents a diminished source of supply for the surface gel, the reduced thickness of which would compromise the ''mucus-bicarbonate'' barrier.     

Bone histomorphometric changes in the femoral neck of aging and ovariectomized rats

Background: This study characterizes the changes in cortical and cancellous bone and cross sectional moment of inertia of the femoral neck from aging and ovariectomized (ovx'd) rats to determine their role in the previously reported ovx-induced reduction of mechanical strength in the femoral neck. Methods: Undecalcified double-fluorescent lableled cross sections of femoral neck of 3.5-, 5.5-, 6.5-, and 8.5-month-old female rats and rats ovx'd at 3.5 months for 2, 3, and 5 months of 45 rats were studied. The estimated endocortical and trabecular surfaces, cortical and cancellous bone histomorphometry, and cortical moment of inertia were determined. Results: The femoral neck was adding cortical bone between 3.5 and 5.5 months of age by increasing cortical thickness and decreasing marrow cavity area. No change of cortical bone mass was found between 5.5 and 8.5 months and the same amount of cancellous bone was observed between 3.5 and 8.5 months of age. Ovariectomy-induced cancellous, but not cortical bone loss. The loss was due to a transient ovx-induced negative bone balance that by 5 months post-ovx produced a 42% loss in trabecular bone while the histomorphometry profiles were the same as controls. The crosssectional moment of inertia increased with age but did not differ significantly between ovx'd and controls. Conclusions: Our findings suggest that the ovx-induced cancellous bone loss could be a contributing factor to the reduced mechanical strength in the femoral neck of ovx'd rats reported previously. © 1995 Wiley-Liss, Inc.     

Spinal tanycytes in the adult rat: a correlative Golgi gold-toning study

In Golgi impregnated transverse sections through the cervical spinal cord of the 7-12-week-old adult rat, numerous tanycytes were observed radiating from the ependyma into the gray matter that surrounds the central canal. The tail processes of these tanycytes terminated as foot processes in association with blood vessels. Spinal tanycytes were classified into ependymal (E) and subependymal (S) types on the basis of the shape and position of the soma. The soma of the E tanycyte was shaped as a column and was entirely located within the ependyma. In contrast, the soma of the S tanycyte was flask shaped, with the widest portion of the flask located subependymally and the elongated portion extending through the ependyma ultimately reaching the luminal surface. Selected Golgi impregnated sections were gold toned and deimpregnated for direct correlative analysis at the ultrastructural level. Gold-toned tanycytes contained the fine clusters of gold particles underlying the plasma membrane of the cell body and coarse clusters of gold particles throughout the tail and foot processes. The apical surface of tanycytes was characterized by numerous microvilli and large cytoplasmic protrusions that evaginated from the apical surface into the lumen of the central canal. At the luminal surface, adjacent tanycytes were joined laterally by junctional complexes with punctate tight junctions and zonulae adhaerentes associated with fibrils and microtubules. In contrast, gap junctions, hemidesmosomes, and puncta adhaerentia were found between adjacent tail processes of tanycytes. The foot processes interdigitated with one another and abutted the basal lamina around the perivascular space of blood vessels. The basal lamina was continuous around the lateral walls of foot processes and filled the spaces between membranous infoldings of the lateral walls. These basal membrane labyrinths were continuous with the basal lamina of the blood vessel and may provide an extensive surface relation between the perivascular space and the neighboring extracellular compartment. The findings of the present study support the contention that tanycytes may modify the composition of substances moving between the perivascular and extracellular spaces.     

Sequential analysis of juvenile isolation-induced decreased social behavior in the adult rat.

The consequences of juvenile isolation on adult social behavior were studied in detail using two different analysis methods: frequency, duration, and latency of behavioral elements, and sequential analysis. Rats were either isolated or socially housed during weeks 4 and 5 of age, and after the isolation period housed in pairs with a rat of identical housing condition until the time of testing at 12 weeks of age. Juvenile isolation caused marked effects on the frequency, duration, and latency of various social behavioral elements, whereas the non-social activities such as ambulation, rearing, and self-grooming were hardly affected. Juvenile isolation reduced social exploration, anogenital sniffing, and approach/following and increased the latency to the first occurrence of these social behavioral elements. In contrast, the sequential analysis revealed that the structure of social behavior was barely affected by juvenile isolation. Some transitions were less pronounced in juvenile isolated rats compared to non-isolated rats, but no significant differences were observed in transitions between social elements. Thus, juvenile isolation bisected the time spent on adult social interactions, whereas it did not disrupt the sequential structure of social behavior. The present data suggest that juvenile isolation reduced the motivation for adult social behavior, but when social contact is initiated, a relatively normal social behavioral pattern is displayed.     

A histomorphometric study on the hepatoprotective effects of a green rooibos extract in a diet-induced obese rat model

Obesity, type two diabetes mellitus and insulin resistance are associated with increased oxidative stress and inflammation. Unfermented green rooibos is an aspalathin rich variant of traditional fermented rooibos (Aspalathus linearis) and has a high polyphenol content. The present study aimed to determine the histologically observable effects of a commercially produced, aspalathin-rich green rooibos extract, Afriplex GRT? (GRE) in a diet-induced obese rat model. Male Wistar rats (N = 28) were randomly assigned to four study groups (n = 7): control (C), green rooibos (GRT), high-fat diet (HFD) and experimental (HFD-GRT) group. Body mass was determined prior to euthanasia and liver mass was determined after death. The left lateral lobe of the liver was processed to wax and stained using haematoxylin and eosin (H & E), Masson’s trichrome stain, Gordons and Sweet’s reticulin impregnation and periodic acid-Schiff stain. Frozen liver tissue sections were used for Oil red O staining. Morphometric quantification of steatosis, semiquantitative pathology grading and scoring were performed and verified by a veterinary histopathologist. A significant increase in body and liver mass was observed in the HFD groups while co-treatment with green rooibos significantly reduced both. The volume and area of steatosis were significantly increased in the HFD groups while the area of steatosis significantly reduced with green rooibos co-treatment. The percentage, location and type of steatosis as well as presence of inflammation and hepatocellular injury were reduced in the HFD group co-treated with GRE. These findings suggest that a GRE has potential as an anti-steatotic, anti-inflammatory and weight reducing agent in vivo.     

Remodelling of subchondral bone in osteoarthritis: a histomorphometric study.

AIMS: To determine whether remodelling of subchondral bone in osteoarthritis is related to anatomical and mechanical factors operating on the joint surface. METHODS: Ten femoral heads were examined. Patients due to have the femoral head removed because of osteoarthritis were given a double tetracycline label before surgery. The specimens were divided in three roughly equal parts, corresponding to the medial, central (weight bearing), and lateral aspects of the femoral heads. Undecalcified methylmethacrylate embedded sections were prepared. The subchondral bone was assessed for appositional bone formation by ultraviolet light microscopy, for resorptive activity by osteoclast count, and for trabecular bone volume by semiautomatic computerised image analysis. Appositional bone rate was also correlated with the presence or absence of overlying cartilage. RESULTS: Bone density was reduced in the medial aspect of the femoral heads but there was no significant difference between the appositional bone rate or the resorptive activity in the three areas. Nor was there any correlation between appositional bone rate in the subchondral bone and the presence or absence of overlying cartilage. CONCLUSION: Remodelling of subchondral bone in osteoarthritis, at least in the short term, is fairly constant and is not related to weight bearing.