Biomedical subjects
Publications and source records attributed to D G Armstrong.
Fractures of the proximal humerus with concomitant vascular injury are rare in children. We describe the presentation, diagnosis, and treatment of a fracture of the proximal humerus in association with an axillary artery injury in a child.
The insulin-like growth factors, IGF-I and -II, have been shown to play a key role in luteal function in some species. The IGF binding proteins, IGFBP-2 and -3, have been shown to inhibit binding of IGF-I and -II to bovine luteal cells and decrease progesterone production. We have recently shown that equine follicles have the genetic capacity to produce IGFBP-2, and that levels decrease in healthy preovulatory follicles. In the present study expression of mRNAs encoding IGFBP-2, as well as the rate-limiting steroidogenic enzyme, P450scc, were studied in equine corpora lutea to investigate whether IGFBP-2 might be involved in luteolysis. Corpora lutea were collected from mares in mid-luteal phase (day 10), at early regression (day 14), late regression (day 17), and 12 and 36 h after intramuscular administration of the PGF(2alpha) analogue, cloprostenol (0.5 microg/kg). During early natural regression, and 12 h after administration of cloprostenol on day 10, steady state levels of mRNAs encoding P450scc had decreased significantly compared with day 10 of dioestrus (P < 0.001). Levels of mRNA encoding IGFBP-2 increased significantly between mid-diestrus and early (P < 0.01) and late (P < 0.001) regression, and 36 h after cloprostenol administration (P < 0.001). We conclude that the genetic capacity for increased IGFBP-2 production in the early stages of natural luteolysis in the mare may act to sequester IGF-I in the CL, assisting in inhibition of progesterone production. However the delay in increase in mRNA encoding IGFBP-2 after cloprostenol administration, combined with the sharp fall in expression of P450scc mRNA, suggests that the luteolytic action of a pharmacological dose of cloprostenol may not be mediated via IGFBP-2 in the mare.
Matrix metalloproteinases (MMPs) degrade the proteinaceous components of the extracellular matrix and are presumably essential for follicular growth culminating in ovulation or atresia. The objectives of this study were to characterize the gelatinolytic and caseinolytic MMPs secreted by cultured bovine thecal and granulosal cells and to determine the effect of luteinizing hormone (LH) on MMP secretion. Thecal and granulosal cells were collected from small bovine follicles (<5 mm) on day 2 or 5 of the estrous cycle (day 0=estrus). A serum-free culture system was utilized in which bovine thecal and granulosal cells do not spontaneously luteinize, but produce androstenedione and estradiol in response to physiological concentrations of LH and follicle-stimulating hormone (FSH) respectively. The effect of LH (0, 1 or 100 ng/ml) on MMP production was determined in conditioned media collected every 48 h for 144 h. MMPs were detected by gelatin and casein zymography and MMP activity was quantified by image analysis. Thecal and granulosal cell conditioned media contained MMPs that had a relative molecular size (Mr) ranging from 53,000 to 200,000 and addition of 1,10 phenanthroline (MMP inhibitor) blocked gelatinolytic and caseinolytic activity. Patterns of gelatinolytic activity in thecal and granulosal cell conditioned media differed over time with the Mr 62,000 and 83,000 MMPs being increased (P <0.05) and the Mr 53,000 MMP being decreased (P <0.05) at 96 h of culture. LH (1 or 100 ng/ml) increased (P <0.05) gelatinolytic activity of the Mr 53,000 and 62,000 gelatinases within thecal cell conditioned media but not granulosal cell conditioned media. The Mr 62,000 and 83,000 gelatinolytic activities corresponded to the active forms of gelatinase A (Mr 62,000) and B (Mr, 83,000) and gelatinase A was detected in thecal cell conditioned media by Western blot analysis. Caseinolytic activity (Mr 83,000) was detected in both thecal and granulosal cell conditioned media and increased from 48 to 96 h. In summary, thecal and granulosal cells secrete gelatinolytic and caseinolytic MMPs and thecal cell production of gelatinase A was stimulated by LH.
The receptor, c-kit, and its ligand, stem cell factor (SCF), are important regulators of ovarian follicle growth and development. The aim of this study was to identify the sites of expression of mRNA for c-kit and SCF in prepubertal and mature (pregnant and non-pregnant) animals. Ovaries were recovered from prepubertal animals, non-pregnant sows and five sows at approximately 3 months of gestation. Ovine SCF and c-kit DNA were cloned into plasmid vectors to produce RNA probes. Expression of mRNA encoding SCF and c-kit were detected via in situ hybridization. Both mRNA were detected throughout ovaries from all animals. This study provides evidence that the growth-factor complex is required throughout follicle development, and also for continued maintenance of the corpus luteum (CL) in the mature animal. SCF mRNA was localized to the granulosa cell layer and was also extensively expressed in endothelial tissue and throughout the CL. c-kit mRNA was detected in the theca layer, oocytes and also in CL. In conclusion, expression of SCF and c-kit mRNA in granulosa and theca cells, respectively, indicate an important interaction between somatic cells throughout follicle development and that in the mature animal, SCF and c-kit potentially have a role in maintaining progesterone secretion by the CL. The observations of continued expression of SCF and c-kit throughout development suggest that there may be differences in the role of this receptor-ligand complex between large mono- vs. poly ovulatory species, such as the pig.
The period between seasonal anoestrus and cyclicity is characterized in many mares by cyclical growth and regression of large dominant follicles. The insulin-like growth factor (IGF) system plays a key role in follicular growth and regression; therefore, we hypothesized that changes in the IGF system and its binding proteins would modulate onset of cyclicity in mares. Ovaries were obtained from pony mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle, and also at the second or third oestrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter. Size of dominant follicles at the time of removal was similar in transition (32 +/- 0.8 mm) and at oestrus (34 +/- 0.6 mm). IGF-I mRNA was present in granulosa cells, with low thecal expression, whereas IGF-II mRNA was confined to the theca layer. Expression of IGF-I and -II mRNAs, and intrafollicular concentrations of oestradiol, were lower (P < 0.01; paired t test) in transitional anovulatory follicles than in preovulatory follicles. Messenger RNA encoding IGFBP-2 was present in both theca and granulosa layers. Steady-state concentrations of mRNA encoding IGFBP-2 mRNA increased (P < 0.001) in theca in preovulatory follicles. Intrafollicular concentrations of IGFBP-2 were higher (P < 0.001) in transitional than in preovulatory follicles. The similarity in circulating concentrations of IGF-I in transitional and cyclic mares, suggested that the somatotrophic axis is not involved in transition from anovulatory to ovulatory cycles. The results suggest that the increased expression of IGF-I and -II mRNAs in preovulatory follicles, along with the decrease in IGFBP-2 concentrations, could increase the bioavailability of intrafollicular IGF in large follicles during the breeding season, and support our hypothesis that intrafollicular IGF bioavailability must exceed a threshold level before ovulation can occur.
Regulation of ovarian activity is an integrated process encompassing both extraovarian signals and intrafollicular factors. Initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotropins, but FSH may affect the rate of preantral follicle growth. Antral follicle development from 1 to 4 mm in sheep and cattle is completely gonadotropin dependent. These recruited follicles express a range of mRNA encoding steroidogenic enzymes, gonadotropin receptors, and local regulatory factors and their receptors. As follicles continue to mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism involved in selection of follicles for continued growth. Locally produced growth factors, such as the IGF and members of the transforming growth factor-beta superfamily, work in concert with gonadotropins throughout the follicular growth continuum and can have significant effects on follicle selection. Environmental influences, such as changes in nutrition, also have an effect on ovarian activity. This can occur without significant variation in circulating gonadotropin concentrations and can be correlated with changes in circulating concentrations of metabolic hormones, including insulin, IGF-I, GH, and leptin. Nutrition can also affect the expression of mRNA encoding components of the ovarian IGF system to regulate the sensitivity/response of follicles toward gonadotropins. Hence, the roles of growth factors in follicular development and survival depend on gonadotropin status and differentiation state of the follicle. In conclusion, it is the integration of these extraovarian signals and intrafollicular factors that determine whether a follicle will continue to develop or be diverted into atretic pathways.
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The purpose of this manuscript was to describe a classification of diabetic foot surgery performed in the absence of critical limb ischaemia. The basis of this classification is centered on three fundamental variables which are present in the assessment of risk and indication: 1) the presence or absence of neuropathy (loss of protective sensation); 2) the presence or absence of an open wound; 3) the presence or absence of acute, limb-threatening infection. The conceptual framework for this classification is to define distinct classes of surgery in an order of theoretically increasing risk for high-level amputation. These classes include: Class I: Elective Diabetic Foot Surgery (procedures performed to treat a painful deformity in a patient without loss of protective sensation); Class II: Prophylactic (Procedure performed to reduce risk of ulceration or reulceration in person with loss of protective sensation but without open wound); Class III: Curative (Procedure performed to assist in healing open wound) and Class IV: Emergent (Procedure performed to limit progression of acute infection). The presence of critical ischaemia in any of these classes of surgery should prompt a vascular evaluation to consider a) the urgency of the procedure being considered and b) possible revascularization prior or temporally concomitant with the procedure. It is our hope that this system begins a dialogue amongst physicians and surgeons which can ultimately facilitate communication, enhance perspective, and improve care.
Reproductive function is an integrated process encompassing both extra-ovarian signals, such as gonadotrophins, and intrafollicular factors, such as locally produced growth factors. Initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotrophins. However, in vivo and in vitro studies indicate that FSH may stimulate the rate of preantral follicle growth and that it can take only 3 months for a primordial follicle to reach the ovulatory stage. Antral follicle development from 2 and 4 mm in diameter in sheep and cattle, respectively, is gonadotrophin dependent. During the oestrous cycle a transient increase in circulating FSH precedes the recruitment of a group of follicles. Recruited follicles are characterized by induction of expression of mRNAs encoding a range of steroidogenic enzymes, gonadotrophin receptors and local regulatory factors. As follicles continue to mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism involved in selection of follicles for continued growth. The mechanism of selection of the ovulatory follicle seems to be linked to the timing of mRNA expression encoding LHr and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in granulosa cells. Locally produced growth factors, such as the insulin-like growth factors (IGFs) and members of the transforming growth factor beta (TGFbeta) superfamily (inhibins, activins and bone morphogenetic proteins (BMPs)), work in concert with gonadotrophins throughout the follicular growth continuum. The roles of growth factors in follicular development and survival are dependent on gonadotrophin status and differentiation state, including morphology. In conclusion, it is the integration of extraovarian signals and intrafollicular factors that determine whether a follicle will continue to develop or be diverted into atretic pathways, as is the case for most of the follicles in monovulatory species, such as cattle.
The effects of acute changes in dietary intake on ovarian activity can be correlated with changes in circulating concentrations of metabolic hormones including insulin, insulin-like growth factor I (IGF-I), growth hormone and leptin. There is no corresponding change in circulating gonadotrophin concentrations and it is proposed that the dietary induced changes in ovarian activity, resulting from acute changes in dietary intake, are a result of direct actions of these metabolic hormones on the ovary. Changes in the peripheral concentrations of insulin, IGF-I and leptin were also associated with the initiation of a synchronized wave of follicle growth and it is hypothesized that oestrogen secreted by the developing follicle is involved in regulating the secretion of these metabolic hormones. At the cellular level, physiological concentrations of insulin and IGF-I interact to stimulate oestradiol production by granulosa cells. In contrast, leptin inhibits FSH-stimulated oestradiol production by granulosa cells and LH-stimulated androstenedione production by theca cells. At the molecular level, dietary energy intake affects the expression of mRNA encoding components of the ovarian IGF system and these changes can directly influence the bioavailability of intrafollicular IGF. This, in turn, can increase the sensitivity or response of follicles to FSH and is one mechanism through which nutrition can directly affect follicle recruitment. Dietary induced increases in intrafollicular IGF bioavailability also have a negative effect on oocyte quality, and diets that are optimal for follicle growth may not necessarily be optimal for oocyte maturation.
We have previously shown that the number of ovarian follicles <4 mm in diameter can be increased by enhanced dietary intake in heifers. This study investigated the effect of the same dietary treatment on superovulatory response. The estrous cycles of 24 mature Hereford x Friesian heifers were synchronized by a standard progesterone plus prostaglandin protocol. The animals were fed with either 100% (group M, n = 12) or 200% (group 2M, n = 12) maintenance requirements for a 3-week period. Starting from day 4 of the synchronized estrous cycle, all the animals were superovulated using a standard 4-day FSH regime followed by an injection of GnRH analogue (GnRHa) to induce ovulation. Rectal ultrasound scanning was carried out to assess ovarian follicular populations at the start of FSH treatment and on the day of GnRHa injection, and to determine the number of corpora lutea 5 days after GnRHa injection. The body weight (BW) and body condition score (BCS) were recorded weekly and plasma samples were collected throughout the experimental period. There were no differences in either BW or BCS between two groups at the start of the experiment. The BW and BCS were maintained during the experiment in the group M, whilst animals in the group 2M showed a non-significant (P > 0.05) increase in BW and BCS. Circulating concentrations of insulin were significantly (P < 0.01) higher in heifers from the group 2M throughout the controlled feeding period. The group 2M had significantly (P < 0.05) more follicles 2-4 mm in diameter at the start of FSH treatment and more (P < 0.01) follicles >9 mm in diameter on the day of GnRHa injection, when compared with the group M. Similarly, 5 days after GnRHa injection there were significantly (P < 0.01) more corpora lutea in the group 2M (18.1+/-2.2) than in the group M (10.6+/-3.0). In addition, plasma progesterone concentrations following GnRHa injection were significantly (P < 0.01) higher in heifers from the group 2M. In conclusion, these results confirm that increased dietary intake can enhance the recruitment of ovarian follicles in heifers. This treatment may provide a valuable approach to improving superovulatory response in cattle.
LH and PGF(2alpha) are the principal luteotrophic and luteolytic hormones in domestic animals, however, it is becoming increasingly apparent that intra-ovarian factors can modulate luteal function. For example, the insulin-like growth factors (IGF-I and -II) can regulate ovarian function, and have direct effects on ovarian cells. An important role for the IGFs in regulating ovarian function is suggested by the multiple effects of IGFs on both follicular and luteal steroidogenesis. Expression of mRNA encoding IGF-I, IGF-II and the type 1 IGF receptor has also been detected in the ruminant CL and is suggestive of autocrine/paracrine roles for both IGF-I and -II in the regulation of luteal function. The actions of the IGFs are further modulated by their association with specific binding proteins (IGFBPs), which regulate the transport of IGFs and their presentation to specific receptors. IGFBPs have been detected in the CL of domestic animals, and inhibitory effects on IGF-I-stimulated progesterone production have been demonstrated. The rapid cyclical changes in luteal growth and regression are associated with rapid changes in vasculature. The principle angiogenic factors include the fibroblast growth factors (FGFs), vascular endothelial growth factor (VEGF) and the angiopoietins (Ang). Other locally produced factors include cytokines such as TNF-alpha and IL-1beta. One such factor is monocyte chemoattractant protein (MCP-1), which increases after exogenous PGF(2alpha). An influx of macrophages takes place in the CL around luteolysis, possibly in response to MCP-1 release, but these changes are not observed in cattle when luteolysis is inhibited. In conclusion locally produced factors are important in the control of luteal function, although their roles have yet to fully elucidated.
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The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.
A study was conducted to determine the effects of FSH and bovine somatotrophin on the expression of mRNA encoding the gonadotrophin receptors and steroidogenic enzymes in ovarian follicles of cattle rendered hypogonadotrophic by treatment with a GnRH agonist. Hereford x Friesian heifers were allotted into two pretreatment groups: controls (n = 10) and GnRH agonist-treated (n = 20). Ovaries of control cows were removed on day 2 of the first follicular wave after synchronized oestrus. GnRH agonist-treated heifers were given either FSH or no FSH. FSH was infused at 50 microg h(-1) for 48 h. Ovaries in GnRH agonist-treated heifers were removed at the end of exogenous hormone treatment. The control, GnRH agonist and GnRH agonist plus FSH treatment groups were divided further into bovine somatotrophin or no bovine somatotrophin treatments (n = 5 per treatment). Bovine somatotrophin (25 mg day(-1) by s.c. injection) was administered for 3 days. Ovaries were scanned once a day by ultrasonography. Blood samples for hormone measurements were collected three times a day from oestrus until the time of removal of ovaries. Expression of mRNAs for the FSH and LH receptors and cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase (P450arom) enzymes was localized by in situ hybridization and quantified by image analysis. Ovarian follicular growth was arrested at < or = 4.5 mm in diameter in GnRH agonist-treated heifers. There was no effect of bovine somatotrophin on follicular dynamics, gonadotrophin secretion or expression of mRNA for either the gonadotrophin receptors or steroidogenic enzymes. Infusion of FSH to GnRH agonist-treated heifers increased FSH concentrations in serum to the physiological concentrations observed in controls and stimulated growth of follicles to a size similar (5.5-8.0 mm in diameter) to recruited follicles in control cows. FSH induced mRNA expression of P450scc and P450arom in granulosa cells of follicles at a smaller size (< or = 4.5 mm in diameter) than in controls and increased (P < 0.001) expression in larger (> 4.5 mm in diameter) follicles. Expression of mRNAs for P450scc and P450c17 increased (P < 0.001) with increasing follicle size and was higher (P < 0.01) in theca cells of GnRH agonist plus FSH-treated heifers than in the other groups. There were no treatment differences in expression of FSH receptor in granulosa cells or LH receptor in theca cells, but expression of both receptors increased with follicle size. There was no expression of LH receptor in the granulosa cells of cows from any treatment group. In conclusion, FSH treatment in GnRH agonist-treated heifers induced similar changes in follicular growth to those observed during the first follicular wave, but despite similar peak concentrations, prolonged exposure to high FSH induced precocious expression of mRNAs for P450scc and P450arom in granulosa cells from small follicles and markedly upregulated expression of these enzymes in granulosa cells from recruited follicles. The results of this study demonstrate the key role that FSH plays in the induction of follicular growth and differentiation.
Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.
UThe insulin-like growth factor binding proteins (IGFBPs) bind IGFs with high affinity and so regulate their access to the type 1 and 2 IGF receptors. This is the principal mechanism involved in regulating IGF bioavailability during folliculogenesis. IGFBPs undergo a number of post-translational modifications, including proteolytic cleavage, phosphorylation and glycosylation, which can regulate the affinity of IGFBPs for IGFs. However, the post-translational changes to IGFBPs that occur during folliculogenesis have not been fully characterized. The charge and size variants of the IGFBPs in bovine follicular fluid were examined by two-dimensional non-reducing SDS-PAGE followed by non-isotopic western ligand blot analysis, and immunoblot analysis during follicular development. The results demonstrate the presence of at least 51 IGFBP isoforms corresponding to IGFBP-1 to -6 in bovine follicular fluid from subordinate follicles, many of which were phosphorylated. The total number of IGFBPs was reduced in dominant follicles, whereas no gross changes in isoforms were observed during follicular development. These results demonstrate the high degree of conservation of IGFBP post-translational modifications between species, and from the in vitro dephosphorylation of these proteins it is hypothesized that these modifications may result in changes to IGF binding or susceptibility to proteolytic cleavage.
A classification system should be thought of as a diagnostic language. Ideally, this language should be used by a large, diverse population of clinicians to guide therapy and ultimately predict outcome. The diabetic foot ulcer is widely known to be the prime precipitant of lower extremity amputations. Its description and classification is the first step toward widespread standardization of treatment and prevention. This article chronologically describes the major diabetic foot wound classification systems devised over the previous generation, discussing each of their attributes and their shortcomings, with an emphasis on an evidence basis for care. It is our hope that discussions such as this one will ultimately lead to a diagnostic and therapeutic lingua franca for diabetic foot wounds and a commensurate reduction in the unconscionably high prevalence of amputations, which we now currently face.