Glucocorticoid-treated piglet model as a model for the steroid-treated premature baby

Our laboratory has developed the steroid-treated piglet model in order to elucidate the mechanisms of glucocorticoid action on overall growth and bone metabolism, and to evaluate potential adjunctive therapies that could attenuate the glucocorticoid effects on growth and skeletal development. Piglets have a metabolic response to steroids that closely mimics the response observed in infants and children receiving long-term glucocorticoid therapy (Ward et al. 1998, Weiler et al. 1995). Both piglets and developing humans experience significant weight and length growth delay. As well, both display a delay in bone development as demonstrated by a lower BMC and BMD in dexamethasone-treated piglets compared to control (Weiler et al. 1995, Ward et al. 1998) and in treated infants (Weiler et al. 1997, Ward et al. 1998a).

In the piglet model, a two week tapering course of dexamethasone (5 days of each of 0.5, 0.3 and 0.2 mg/kg/d), resulted in a 15% lower whole body BMD and femur BMD, compared to non- steroid-treated littermates (Ward et al. 1998). Measurement of BMC and BMD of a developing long bone (femur) at the 1/2, 1/3, and 1/4 site distal to the proximal femur demonstrated that dexamethasone affects both trabecular bone and cortical bone to a similar extent (Weiler et al. 1995, Ward et al. 1998). The BMD of glucocorticoid-treated piglets at the 1/2, 1/3, and 1/4 site distal to the proximal femur was 25%, 20% and 33% lower in treated versus untreated piglets. Similarly, the biochemical markers of bone turnover, plasma osteocalcin and urinary N-telopeptide, were reduced after 5 days of the highest dose of dexamethasone (Ward et al. 1998). The reduction in plasma osteocalcin and urinary N-telopeptide provides evidence that the activities of both the osteoblast (bone formation) and the osteoclast (bone resorption) are adversely altered by glucocorticoids.

The effects of dose of steroid on growth and bone mass were assessed in two ways. First, we investigated the response to the tapering doses (0.5 to 0.3 to 0.2 mg dexamethasone/kg/d). With frequent assessment of plasma osteocalcin (after each change in dexamethasone dose) and daily monitoring of weight and length growth, we observed that the reductions in growth velocity and plasma osteocalcin were dose-dependent, persisting only during the two highest doses of dexamethasone (Ward et al. 1998). During the lowest tapering dose of dexamethasone, both circulating osteocalcin and the rate of weight and length growth returned to control values. However, due to the insult that incurred during steroid treatment, absolute weight and length, as well as BMC and BMD were significantly less than controls after the two week study.

A second approach to assessing the influence of dose of steroid was to investigate whether a circadian rhythm occurs in bone formation during early development; and if so, whether low evening doses of dexamethasone might attenuate the steroid effects on bone turnover. Piglets were randomized to dexamethasone (0.5 mg/kg/d) given either as 50% daily dose in the morning and 50% in the evening or as 70% daily dose in the morning and 30% in the evening. Steroid-induced reductions in weight, length, bone mineral content, femoral length and plasma osteocalcin occurred with both dosing regimens (Guo et al. 1998). However, the regimen of low evening dose resulted in significantly less of a reduction in osteocalcin and a later onset of weight reduction compared the 50/50 dose (Guo et al. 1998). Thus it appears that minimizing the steroid dose or giving it to compliment circadian rhythms of bone turnover may be important in order to lessen the negative side-effects of glucocorticoid therapy. The therapeutic efficacy of such dosing regimens in the target clinical populations require investigation before adopting for use in infants.

The potential mechanisms whereby glucocorticoids stunt growth and impair normal bone development are currently not fully understood. Both GH and IGF-I are positive and critical modulators of bone growth via endocrine and autocrine regulation, respectively. More specifically, GH and IGF-I are potent stimuli of growing long bones; both are essential for the complex regulation of bone modeling and turnover (Canalis et al. 1997). Bone tissue expresses all the components of the GH-IGF-I system (Rosen et al. 1994). Since the GH-IGF-I axis has many different components, there are many potential sites for interference with exogenous glucocorticoids. Exogenous glucocorticoids may impair release of GH from the anterior pituitary, reduce circulating or tissue levels of IGF-I, or modulate the activity of IGF-I by altering the circulating or tissue-specific insulin-like growth factor binding protein (IGFBP) profile. Indeed, there is mounting evidence from steroid-treated animals (Altman et al. 1992), children (Hyams et al. 1988) and adults (Reid et al. 1989) that glucocorticoids mediate the negative side-effects by reducing circulating GH and IGF-I, and by altering the circulating IGFBP profile and tissue expression of IGFBPs.

Treatment with either GH or IGF-I has the potential to stimulate linear growth and bone mass accretion in infants and children receiving glucocorticoid treatment. Descriptive studies in children, largely limited to kidney transplant recipients who receive prolonged low dose glucocorticoid treatment, have shown that adjunctive GH therapy improves weight and height growth velocities, elevates circulating C-terminal type 1 procollagen and improves bone formation rates (Allen and Goldberg 1992, Van Drop et al. 1992, Benfield et al. 1993, Fine et al. 1991).

From our recent studies it appears that piglets are an appropriate model in which to test the effectiveness of anabolic agents to attenuate the deleterious effects of steroids since they respond to adjunctive therapies such as growth hormone (GH) and insulin-like growth factor-I (IGF-I). Using this animal model, we demonstrated that the negative side-effects of glucocorticoids are at least partially mediated by interference with one or more components or aspects of the GH-IGF-I axis (Ward et al. 1998). Although circulating IGF-I was not reduced by glucocorticoid treatment, both circulating IGFBP-2 and IGFBP-3, the predominant circulating IGFBPs during early development, were significantly reduced. Approximately 99% of IGF-I circulates bound to one of six different IGFBPs (IGFBP-1 through 6). The IGFBPs bind IGF-I with a high affinity, controlling IGF-I activity and extending the half-life. IGFBP-3 binds 95% of circulating IGF-I. IGFBP-3 forms a 150 kda complex with an acid labile subunit. Due to the size of this complex, it has limited permeability across the capillary and extends the half-life of IGF-I from minutes to hours. The increased half-life may heighten the actions of IGF-I by providing a larger pool of slow-release IGF-I to the IGF-I receptor. The precise function of IGFBP-2 is less clear. It may regulate the passage of IGF-I from the intravascular to the extravascular space or exert IGF-independent effects on target cells by binding to IGFBP-specific receptors (integrin receptors). Thus, the reduction in IGFBP-2 and IGFBP-3 suggests that IGF-I activity may have been modified with glucocorticoid treatment. Moreover, tissue mRNA expression of IGFBP-2 and IGFBP-4 was altered even after the lowest dose of dexamethasone (Ward et al. 1998). Thus, although some growth recovery occurred during the lowest dose of dexamethasone treatment, impaired mRNA expression of specific IGFBPs by the liver was sustained.

Adjunctive administration of GH alone, or in combination with IGF-I, during dexamethasone treatment partially attenuated the dexamethasone-induced reductions in whole body growth and bone mass (Ward et al. 1998). All measures of BMD (whole body, whole femur, and at the 1/2, 1/3, and 1/4 sites of the femur) were intermediate between the control and dexamethasone-treated groups. This finding provided evidence that the piglet’s bones could respond to GH or GH+IGF-I treatment. The fact that the BMD of piglets receiving adjunctive GH or GH+IGF-I was not significantly higher than piglets receiving dexamethasone alone, revealed that these therapies could only partially attenuate the insult on bone mass. This finding emphasizes the potent effects of glucocorticoid drugs on developing bone.

A curious finding was that there was no additional benefit on growth or bone mass with combined administration of GH+IGF-I compared to treatment with GH alone. It appears that the observed improvements were mediated directly by GH. Our observation that neither adjunctive GH or GH+IGF-I countered the changes in the circulating IGFBP profile or tissue mRNA expression of the IGFBPs, suggests that both adjunctive treatments were acting by autocrine or paracrine regulatory mechanisms rather than via endocrine regulation. In order to understand more fully the mechanisms of action, quantification of IGF-I, the IGFBPs and other growth factors in the bone is required. Such information will help to delineate the interaction of glucocorticoids, GH and IGF-I in the local environment of the bone. Further investigation is also required to determine the most effective timing of GH administration. Our current research suggests that it is prudent to administer adjunctive GH during the lowest dose of a tapering course of glucocorticoid. However, intervention with GH after termination of glucocorticoid therapy may be even more effective to promote ‘catch- up’ growth.

Further research is needed to identify the potential of other anabolic agents (for example, estrogen analogues) or bone-specific agents like bisphosphonates that can attenuate the deleterious delay in bone development by glucocorticoids. The potential for administering estrogen analogues like Tamoxifen was studied by our group using the glucocorticoid-treated piglet model (Fritz et al. 1998). Adjunctive tamoxifen attenuated the reduction in axial skeleton growth (snout to rump length) as well as BMD. The mechanism of this favourable effect remains unclear, but two mechanisms have been suggested. Tamoxifen may either up-regulate transforming growth factor- ß production or antagonize estrogen receptor-dependent gene activation (Fritz et al. 1998). The potential for estrogen analogues to counter the negative side-effects of glucocorticoid treatment requires further investigation.

Acknowledgement: The research conducted in the laboratory of SAA is funded by the Medical Research Council of Canada and The Hospital for Sick Children Foundation.

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