Marta Fernández

Different bone growth rates are associated with changes in the expression pattern of types II and X collagens and collagenase 3 in proximal growth plates of the rat tibia.

Skeletal growth depends on endochondral ossification in growth plate cartilage, where proliferation of chondrocytes, matrix synthesis, and increases in chondrocyte size all contribute to the final length of a bone. To learn more about the potential role of matrix synthesis/degradation dynamics in the determination of bone growth rate, we investigated the expression of matrix collagens and collagenase 3 in tibial growth plates in three age groups of rats (21, 35, and 80 days after birth), each characterized by specific growth rates. By combining stereological and in situ hybridization techniques, it was found that the expression of matrix collagens and collagenase 3 was specifically turned on or off at specific stages of the chondrocyte‐differentiation cycle, and these changes occurred as a temporal sequence that varied depending of animal growth rate. Furthermore, the expression of these matrix proteins by a growth plate chondrocyte was found to be sped up or slowed down depending of the growth rate. In addition to expression of types II and X collagen, collagenase‐3 expression was found to constitute a constant event in the series of changes in gene expression that takes place during the chondrocyte‐differentiation process. Collagenase‐3 expression was found to show a biphasic pattern: it was intermittently expressed at the proliferative phase and uniformly expressed at the hypertrophic stage. An intimate relationship between morphological and kinetic changes associated with chondrocyte hypertrophy and changes in the expression pattern of matrix collagens and collagenase 3 was observed. Present data prove that the matrix synthesis/degradation dynamics of the growth plate cartilage varied depending on growth rate; these results support the hypothesis that changes in matrix degradation and synthesis are a critical link in the sequence of tightly regulated events that lead to chondrocytic differentiation.

Resistance to growth hormone and insulin-like growth factor-I in acidotic rats

Abstract Growth impairment induced by chronic metabolic acidosis is associated with an abnormal growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis. To examine the potentially beneficial effects of IGF-I on acidosis-induced growth impairment and the influence of GH and IGF-I treatment on the GH/IGF-I axis, three groups of acidotic young rats (untreated, AC, n=12; treated with recombinant human GH, GH, n=8; treated with recombinant human IGF-I, IGF-I, n=8) were studied, and compared with nonacidotic rats fed ad libitum (C, n=9)) or pair-fed with the AC group (PF, n=12). After 14 days of acidosis and 7 days of treatment, growth rate, hepatic abundance of 4.7-kilobase (kb) and 1.2-kb GH receptor transcripts and 7.5-kb and 1.8- to 0.8-kb IGF-I transcripts, serum GH-binding protein (GHBP), and IGF-I concentrations (mean±SEM) were analyzed. Significant decreases of 4.7-kb GH receptor [26±2 vs. 49±6 arbitrary densitometry units (ADU)] and 7.5 kb IGF-I (41±3 vs. 104±10 ADU) transcripts and low serum GHBP (25±1 vs. 32±1 ng/ml) and IGF-I (279±50 vs. 366±6 nmol/l) levels were found in the AC compared with the C rats. The majority of these alterations were also observed in PF rats. Compared with acidotic untreated rats, GH and IGF-I therapy produced no improvement in growth rate. GH treatment normalized the levels of IGF-I mRNA, aggravated the acidosis-related inhibition of the GH receptor gene, and did not modify the serum levels of GHBP and IGF-I. In contrast, IGF-I administration depressed the hepatic expression of all GH and IGF-I transcripts and normalized serum IGF-I concentrations. Our results confirm that sustained metabolic acidosis alters the GH/IGF-I axis, in part because of associated malnutrition, and induced growth retardation that is resistant to GH therapy. Our study also shows that administration of IGF-I does not accelerate the growth of acidotic rats, suggesting a peripheral mechanism, at the level of target tissues, is responsible for the resistance to the growth-promoting actions of GH and IGF-I.

Collagen Metabolism Is Markedly Altered in the Hypertrophic Cartilage of Growth Plates from Rats with Growth Impairment Secondary to Chronic Renal Failure

Skeletal growth depends on growth plate cartilage activity, in which matrix synthesis by chondrocytes is one of the major processes contributing to the final length of a bone. On this basis, the present work was undertaken to ascertain if growth impairment secondary to chronic renal insufficiency is associated with disturbances of the extracellular matrix (ECM) of the growth plate. By combining stereological and in situ hybridization techniques, we examined the expression patterns of types II and X collagens and collagenase‐3 in tibial growth plates of rats made uremic by subtotal nephrectomy (NX) in comparison with those of sham‐operated rats fed ad libitum (SAL) and sham‐operated rats pair‐fed with NX (SPF). NX rats were severely uremic, as shown by markedly elevated serum concentrations of urea nitrogen, and growth retarded, as shown by significantly decreased longitudinal bone growth rates. NX rats showed disturbances in the normal pattern of chondrocyte differentiation and in the rates and degree of substitution of hypertrophic cartilage with bone, which resulted in accumulation of cartilage at the hypertrophic zone. These changes were associated with an overall decrease in the expression of types II and X collagens, which was especially marked in the abnormally extended zone of the hypertrophic cartilage. Unlike collagen, the expression of collagenase‐3 was not disturbed severely. Electron microscopic analysis proved that changes in gene expression were coupled to alterations in the mineralization as well as in the collagen fibril architecture at the hypertrophic cartilage. Because the composition and structure of the ECM have a critical role in regulating the behavior of the growth plate chondrocytes, results obtained are consistent with the hypothesis that alteration of collagen metabolism in these cells could be a key process underlying growth retardation in uremia.

Hard-Rock Stability Analysis for Span Design in Entry-Type Excavations with Learning Classifiers

The mining industry relies heavily on empirical analysis for design and prediction. An empirical design method, called the critical span graph, was developed specifically for rock stability analysis in entry-type excavations, based on an extensive case-history database of cut and fill mining in Canada. This empirical span design chart plots the critical span against rock mass rating for the observed case histories and has been accepted by many mining operations for the initial span design of cut and fill stopes. Different types of analysis have been used to classify the observed cases into stable, potentially unstable and unstable groups. The main purpose of this paper is to present a new method for defining rock stability areas of the critical span graph, which applies machine learning classifiers (support vector machine and extreme learning machine). The results show a reasonable correlation with previous guidelines. These machine learning methods are good tools for developing empirical methods, since they make no assumptions about the regression function. With this software, it is easy to add new field observations to a previous database, improving prediction output with the addition of data that consider the local conditions for each mine.

Prepubertal Rats Are More Resistant to Ischemic Renal Injury and Recover More Rapidly than Adult Rats

Bilateral clamping of renal pedicles during 60, 75 or 90 min was used to characterize the evolution of ischemic acute renal failure (ARF) in prepubertal rats. To verify the existence of age-conditioned differences in the evolution of ARF, adult rats were exposed to 40, 60 or 75 min of clamping. After 7 days, survival rate was significantly better in young than adult rats for identical times of clamping (89 vs. 35% for 60 min and 69 vs. 35% for 75 min). Young rats largely died within the first 24 h following ischemia while the risk of death extended until the 4th day after ischemia in adult rats. Peak values of serum urea nitrogen and creatinine were observed on the 1st and 3rd day after ischemia in young and adult rats, respectively. In young rats, these markers of renal function returned to normal on days 5 and 6 whereas they remained elevated at the end of the study in adult animals. Growth curves of young rats paralleled those of sham-operated animals from the 3rd day of clamping whereas adult rats did not even reach the initial weight at the end of the study. Analysis of kidneys obtained 7 days after clamping revealed more severe histopathological lesions in adult rats as well as a higher proliferative activity (10–40 times the value of sham-operated animals versus 2–4 times the control value in young rats). Our findings indicate that kidneys from young rats are more resistant to ischemia and recover more quickly from the ischemic insult. Therefore, the experimental model of ischemic ARF is clearly different in young and adult rats.