Retrograde tracing with Fluoro-Gold (FG) was used to identify the complete population of knee joint sympathetic postganglionic efferents in the lumbar sympathetic chain of adult female Wistar rats. In 6 rats, the total number and distribution of FG-labelled neurons in the lumbar sympathetic chain was determined. The rat knee joint is supplied by an average of 187±57 sympathetic afferents with the majority at the L3 and L4 levels. Immunohistochemistry using antibodies specific for tyrosine hydroxylase (TH), somatostatin (SS) or vasoactive intestinal polypeptide (VIP) revealed that 33% of knee joint sympathetic afferents contained TH, 42% contained VIP, and none contained somatostatin. Retrograde tracing with FG provided accurate and reproducible labelling of the joint-innervating subpopulation of sympathetic efferent neurons. This model lends itself to the further study of the molecular responses of this neuronal population in the various disorders and conditions affecting joints.

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of ‘pure’ I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n=1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in numerous previous publications based on the use of histochemistry alone. This has important implications in attempts to study the physiological properties of fast fibre types adequately in horses.

As part of a large comparative study on the development of reptilian skin, we provide the first ultrastructural description of differentiation of the epidermis of the carapace and plastron in the Chelonia, using the Australian pleurodiran turtle Emydura macquarii as a model. The epidermis is initially composed of an external flat peridermis and a basal layer of cuboidal cells. During differentiation, the peridermis darkens, flakes off and is partially lost before hatching. Four to 6 layers of flat cells containing lipids and mucus form from the basal layer beneath the external peridermis. Because such cells are found only during embryogenesis, we have referred to these layers as embryonic epidermis. They contain reticulate bodies made of a meshwork of coarse filaments similar to those described in the inner peridermis of lizard and bird embryos. In advanced embryos, cells of the embryonic epidermis condense into a thin dark stratum which is subsequently lost after hatching. The lowermost 2 layers of the embryonic epidermis keratinise, as for a typical lepidosaurian α-layer. A splitting zone is progressively formed beneath the α-layer to separate the embryonic epidermis from the underlying β-layer. Patterns of cytodifferentiation of the β-synthesising cells over the carapace and plastron essentially resemble those of the lepidosaurian epidermis. The β-keratin matrix initially accumulates among ribosomes as round bodies not clearly surrounded by a membrane. These bodies appear not to be derived from the Golgi apparatus. Melanosomes and other dark granules of uncertain nature are present among early differentiating β-cells. The round β-keratin bodies merge with the dense bodies to produce the definitive variegated pattern of the mature β-keratin layer. The histochemistry suggests that calcium combines with organic molecules within β-keratinising cells to harden the tissue. In contrast to the β-keratin cells of lizards and snakes, cells of the mature β-keratin layer of E. macquarii maintain their cell boundaries in part or completely, a characteristics shared with the β-keratin layer of Sphenodon and crocodilians.

The majority of studies as to whether gastric and nongastric factors are involved in the ‘rotation’ of the human embryonic stomach around its longitudinal axis have been morphological. The aim of this study was to analyse, with morphometric support, the influence on the angular modifications during gastric rotation of the simultaneous changes of the gastric walls and mesenteric growth, and the volumes of organs adjacent to the stomach (right, left and posterior groups). Computer imaging techniques were applied on cross sections of a graded series of 10 human embryos (from Carnegie stage (CS) 11) and 2 fetuses. A clockwise gastric rotation occurred during the embryonic period. The most rapid angular modifications took place from CS 11 to 16, after the rotation became oscillatory with angular changes in clockwise and anticlockwise directions, reaching the highest value at CS 18 (at the supraomental part of the stomach: 54.37°; at the omental part: 68.03°); after this period the angular values tended to stabilise. The predominant growth of the left gastric wall over the right and the changes in the width of the gastric mesentery were the most persistent factors involved in the modifications of the transverse gastric angle during the embryonic period although without relationship to their direction. During the increasing angular phases, clockwise rotation was promoted by the decrease of the volume of any group of organs adjacent to the stomach. When the volume of the left and posterior groups increased simultaneously, clockwise or anticlockwise angular directional change was related to the respective decrease or increase adjacent organ volume to the right. We conclude that the stomach of the human embryo undergoes heterogeneous and multifactorial rotation as a consequence of the overall increase in gastric wall growth to the left and the increase of gastric mobility produced by the previous mesenteric enlargement, and that the rotational direction results from the forces exerted on the stomach and the gastric mesenteries by the adjacent organs.

Mucous and oxynticopeptic cells in the gastric mucosa of the seps, Chalcides chalcides (Linnaeus, 1758) were examined by standard histochemical staining methods and by lectin histochemistry. The epithelial mucous cells lining the surface of the stomach and the mucous cells of the fundic glands elaborated mainly neutral glycoproteins with β(1,4)GlcNAc oligomers, GalNAc glycosidic residues and Galβ1,3GalNAc terminal sequences. The mucous cells of the fundic glands were stained specifically with the Paradoxical Con A method. The mucosecreting cells of the pyloric glands produced neutral glycoproteins, with β(1,4)GlcNAc oligomers, GalNAc residues and Galβ1,3GalNAc terminal sequences. Terminal L-fucose bound to the penultimate GlcNAc residues, and/or difucosylated oligosaccharides were also present. The pyloric glands did not stain with the Paradoxical Con A procedure. The morphology of the oxynticopeptic cells changes from the oral to the aboral region of the fundic mucosa. In the oral fundic tract the oxynticopeptic cells showed cytoplasm filled with zymogen granules, while in the aboral fundic region these cells contained few zymogen granules and showed cytoplasm full of empty vesicles, typical of the acid secreting cells. A secretion gradient of proteolytic enzymes and hydrochloric acid along the fundic mucosa of the seps can be hypothesised.

Neuronal nitric oxide synthase immunoreactivity (NOS1-ir) in sacral motor neurons of normal adult cats was compared with that in cats surviving 1–10 wk after unilateral transection and ligation of the pudendal nerve. Levels of immunostaining were measured by microdensitometry. In nonoperated cats 60% of motor neurons in the ventrolateral nucleus (VL) and Onuf's nucleus (ON) showed high levels of NOS1-ir with lower NOS1-ir in 40%. Following axotomy, motor neurons in ON on both sides of the cord showed an acute rise in mean level of NOS1-ir at 1 wk, with a further increase at 2 wk. Mean levels of NOS1-ir in the ipsilateral and contralateral ON remained elevated at 10 wk after axotomy. Elevation of NOS1-ir occurred in the VL with a similar time-course to that in ON, implying a wider response in motor nuclei synaptically coupled to ON. Measurements of neuronal size in ON and VL revealed an increase in neuronal size in ON but not VL, indicating increased NOS1-ir in ON was not an artifact of neuronal atrophy. The proportion of motor neurons in ON and VL possessing higher levels of NOS1-ir increased from 60% in controls to 100% at 2–3 wk postaxotomy. The proportion slightly declined by 8 wk due to re-emergence of motor neurons exhibiting low NOS1-ir, but remained greater than normal at 10 wk in both nuclei. Based on evidence from related analyses of synaptology, we argue that acute axotomy induced alterations in presynaptic complement which increased overall Ca2+ influx and thereby stimulated NOS1-ir.

The pig is often used in experimental studies on the significance of bronchial artery circulation, but the anatomy of this artery is only poorly described. The purpose of this study was to improve the anatomical basis for experimental studies on the porcine bronchial artery circulation. The origin of the artery from the aorta is described in 32 pigs. Heart–lung blocks were perfused with saline and removed in 16 pigs, and the broncho-oesophageal orifice was identified and cannulated. In these 16 specimens the intrapulmonary ramification was studied by angiography, and the extrapulmonary distribution and supply area by injection of Evans Blue. The broncho-oesophageal artery originated from the aorta as a single trunk in 91%. Angiography showed that each principal bronchus was accompanied by 2 bronchial artery branches far into the lung parenchyma. The central branching pattern of the artery between the aorta and the principal bronchi was divided into 3 subtypes. Evans Blue showed communication with the whole mediastinum. The anatomical relations are described. It is concluded that the broncho-oesophageal artery divides to follow each bronchus with 2 bronchial branches. A nomenclature for these branches is suggested. The pig anatomy is suited for experimental investigations on the bronchial circulation.

The effect of systemic NGF treatment on loss and shrinkage of dorsal root ganglion cells was studied in adult male rats after permanent axotomy. Nineteen 16 to 18-wk-old rats had their right 5th lumbar spinal nerve ligated and cut approximately 7 mm peripheral to the ganglion. Two days before the operation, treatment with subcutaneous injections of human recombinant NGF (1.0–0.5 mg/kg/day) was started in 9 test rats; 10 controls were given saline injections. After 1 mo the levels of substance P (SP) and calcitonin gene related peptide (CGRP) were significantly increased in intact sciatic nerve. The number and mean volume of perikarya were estimated using assumption-free stereological techniques including vertical sections, the Cavalieri principle, optical disectors, the planar rotator and systematic sampling techniques. Systemic NGF administration had no influence on survival of primary sensory neurons after axotomy. The number of perikarya was 14300 (S.D.=1800) in axotomised ganglia in control rats versus 14700 (S.D.=2100) in axotomised ganglia of NGF treated rats. The reduction of perikarya volume after axotomy was significantly less after NGF treatment (11600 μm3 in the control group versus 8000 μm3 in the NGF treated group). However, the apparent protection of NGF-treatment on perikaryal volume is explained by a hitherto unrecognised size effect on nonaxotomised dorsal root ganglion cells. The untreated rats had a mean volume of 24700 μm3 (S.D.=2700 μm 3) whereas rats treated with NGF had a volume of 20400 μm3 (S.D.=1700 μm3) on the nonaxotomised side. In conclusion, systemic NGF treatment in adult rats has no effect on dorsal root ganglion cell loss in permanent axotomy whereas perikaryal size of intact nonaxotomised cells is reduced.

Muscle spindles in 2 synergistic avian skeletal muscles, the anterior (ALD) and posterior (PLD) latissimus dorsi, were studied by light and electron microscopy to determine whether morphological or quantitative differences existed between these sensory receptors. Differences were found in the density, distribution and location of muscle spindles in the 2 muscles. They also differed with respect to the morphology of their capsules and intracapsular components. The slow ALD possessed muscle spindles which were evenly distributed throughout the muscle, whereas in the fast PLD they were mainly concentrated around the single nerve entry point into the muscle. The muscle spindle index (number of spindles per gram wet muscle weight) in the ALD was more than double that of its fast-twitch PLD counterpart (130.5±2.0 vs 55.4±2.0 respectively, n=6). The number of intrafusal fibres per spindle ranged from 1 to 8 in the ALD and 2 to 9 in the PLD, and their diameters varied from 5.0 to 16.0 μm and 4.5 to 18.5 μm, respectively. Large diameter intrafusal fibres were more frequently encountered in spindles of the PLD. Unique to the ALD was the presence of monofibre muscle spindles (12.7% of total spindles observed in ALD) which contained a solitary intrafusal fibre. In muscle spindles of both the ALD and PLD, sensory nerve endings terminated in a spiral fashion on the intrafusal fibres in their equatorial regions. Motor innervation was restricted to either juxtaequatorial or polar regions of the intrafusal fibres. Outer capsule components were extensive in polar and juxtaequatorial regions of ALD spindles, whereas inner capsule cells of PLD spindles were more numerous in juxtaequatorial and equatorial regions. Overall, muscle spindles of the PLD exhibited greater complexity with respect to the number of intrafusal fibres per spindle, range of intrafusal fibre diameters and development of their inner capsules. It is postulated that the differences in muscle spindle density and structure observed in this study reflect the function of the muscles in which they reside.

Glutamine synthetase (GS) is a glucocorticoid-inducible enzyme that has a key role for glutamate metabolism in the central and peripheral nervous system. In this study GS activity was measured and the amount of immunoreactive GS (ir-GS) cells in the rat anterior pituitary gland was quantified as a function of age. In addition, the effects of GS inhibitors, glucocorticoid administration, and adrenalectomy on GS activity were examined. Some of the ir-GS cells were also immunoreactive for S100 protein (ir-S100) which is a known marker for folliculostellate cells (FS) in the anterior pituitary. FS cells expressing GS were first detected in 3-d-old rats, and this cell population, expressed as the immunostained cell area divided by a standard unit area, increased as a function of age. The percentages of FS cells also expressing GS were 0.2, 6.4, 25 and 74% at 3 d, 30 d, 60 d and 2 y of age, respectively. GS enzyme activity also increased in parallel with the increase of ir-GS cell population maturation. The subcutaneous injection of methionine sulphoximine, a GS and γ-glutamylcysteine synthetase inhibitor, reduced pituitary GS activity by 83%, but increased the population of ir-GS cells 3.5-fold in 30-d-old rats. Buthionine sulphoximine, a specific inhibitor of γ-glutamylcysteine synthetase, had little effect on GS activity or the ir-GS cell population. Neither methionine sulphoximine nor buthionine sulphoximine changed the population of ir-S100 protein cells (FS cells). Dexamethasone and hydrocortisone increased the population of ir-GS cells by 3.1 and 4.2-fold, respectively, within 12 h after administration. A significant increase of GS activity due to the injection of glucocorticoids was observed in the anterior pituitary, but not in the brain, retina or liver of immature rats. Adrenalectomy did not cause decrease of pituitary GS activity, and dexamethasone administration increased GS activity in both adrenalectomised and intact rats. In the monolayer culture of anterior pituitary cells, glucocorticoids increased GS activity by ×1.5, and methionine sulphoximine reduced the activity by over 94%. These results demonstrate that GS in folliculostellate cells is a glucocorticoid-inducible enzyme in vivo and in vitro, and that the age-dependent increase of GS activity is independent of endogenous adrenal glucocorticoids.

The shapes of cervical (C1–C7) and upper thoracic (T1, T2) vertebrae from the rat and the grey-headed flying fox have been analysed by Fourier analysis to investigate the types of variation present and to try to isolate bones according to position along the vertebral column and species. It was found that the T2 vertebrae of the rat are very different from all others in the study, that C2 and C6 vertebrae are very similar and that the remaining vertebrae split according to species.

In numerical simulations of skeletal muscle contractions, geometric information is of major importance. The aim of the present study was to determine whether the diffusion tensor imaging (DTI) technique is suitable to obtain valid input with regard to skeletal muscle fibre direction. The accuracy of the DTI method was therefore studied by comparison of DTI fibre directions in the rat tibialis anterior muscle with fascicle striation patterns visible on high-resolution magnetic resonance imaging (MRI) and with fibre directions in an actual longitudinal section (ALS) through the same muscle. The results showed an excellent qualitative agreement between high-resolution MRI and DTI. Despite less accurate quantitative comparison with ALS, it was concluded that DTI does indeed measure skeletal muscle fibre direction. After the experiment, it was possible to determine an appropriate voxel size (0.9 mm3) that provided enough resolution and acceptable accuracy (5°) to use DTI fibre directions in biomechanical analyses. Muscle deformation during contraction, resulting from a finite element simulation with a mesh that was directly generated from the experimental data, has been presented.

The pain of a migraine attack is often described as unilateral, with a throbbing or pulsating quality. The middle meningeal artery (MMA) is the largest artery supplying the dura mater, is paired, and pain-producing in humans. This artery, or its branches, and other large intracranial extracerebral vessels have been implicated in the pathophysiology of migraine by theories suggesting neurogenic inflammation or cranial vasodilatation, or both, as explanations for the pain of migraine. Having previously studied in detail the distribution of the second order neurons that are involved in the transmission of nociceptive signals from intracranial venous sinuses, we sought to compare the distribution of second order neurons from a pain-producing intracranial artery in both monkey and cat. By electrically stimulating the middle meningeal artery in these species and using immunohistochemical detection of the proto-oncogene Fos as a marker of neuronal activation, we have mapped the sites of the central trigeminal neurons which may be involved in transmission of nociception from intracranial extracerebral arteries. Ten cats and 3 monkeys were anaesthetised with α-chloralose and the middle meningeal artery was isolated following a temporal craniotomy. The animals were maintained under stable anaesthesia for 24 h to allow Fos expression due to the initial surgery to dissipate. Following the rest period, the vessel was carefully lifted onto hook electrodes, and then left alone in control animals (cat n = 3), or stimulated (cat n = 6, monkey n = 3). Stimulation of the left middle meningeal artery evoked Fos expression in the trigeminocervical nucleus, consisting of the dorsal horn of the caudal medulla and upper 2 divisions of the cervical spinal cord, on both the ipsilateral and contralateral sides. Cats had larger amounts of Fos expressed on the ipsilateral than on the contralateral side. Fos expression in the caudal nucleus tractus solitarius and its caudal extension in lamina X of the spinal cord was seen bilaterally in response to middle meningeal artery stimulation. This study demonstrates a comparable anatomical distribution of Fos activation between cat and monkey and, when compared with previous studies, between this arterial structure and the superior sagittal sinus. These data add to the overall picture of the trigeminovascular innervation of the intracranial pain-producing vessels showing marked anatomical overlap which is consistent with the often poorly localised pain of migraine.

Thymic epithelial cells are an important source of cytokines and other regulatory peptides which guide thymocyte proliferation and maturation. Parathyroid hormone-related protein (PTHrP), a cytokine-like peptide, has been reported to affect the proliferation of lymphocytes in vitro. The studies presented here were undertaken to test the hypotheses that PTHrP is produced locally within the thymus where it could influence thymocyte maturation and, more specifically, that thymic epithelial cells (TEC) could be the intrathymic source of PTHrP expression. To this end, immunohistochemical studies were performed to localise PTHrP and the PTH/PTHrP receptor within the adult rat thymus. Antibodies directed against 2 different PTHrP epitopes, PTHrP(1–34) and PTHrP(34–53), demonstrated prominent specific PTHrP immunoreactivity in both subcapsular and medullary TEC. In addition, faint but specific staining for PTHrP was seen in the cortex, interdigitating between cortical lymphocytes while sparing epithelial-free subcapsular areas, thus suggesting that cortical TEC could also be a source of PTHrP immunoreactivity. In contrast, PTH/PTHrP receptor immunoreactivity was only seen in medullary and occasional septal TEC; no evidence of cortical or lymphocytic PTH/PTHrP receptor immunoreactivity was detected. Immunohistochemical studies of cultured cytokeratin-positive rat TEC confirmed the results of these in situ studies as cultured TEC were immunoreactive both for PTHrP and the PTH/PTHrP receptor. Thus these results demonstrate that PTHrP is produced by the epithelial cells of the mature rat thymus. This suggests that PTHrP, a peptide with known cytokine, growth factor and neuroendocrine actions, could exert important intrathymic effects mediated by direct interactions with TEC, or indirect effects on PTH/PTHrP receptor-negative thymocytes.

Conventional histochemistry and the binding patterns of 22 biotinylated lectins were examined for characterisation of glycoconjugates in the components of the olfactory mucosa of the armadillo Chaetophractus villosus. The mucous lining the olfactory epithelium showed binding sites for DSL, WGA, STL, LEL, PHA-E and JAC. Only the basilar processes of the supporting cells stained for Con-A and S-Con A. The olfactory receptor neurons stained with LEL, LCA, Con A, S-Con A, JAC and PNA. The layer of basal cells did not react with any of the lectins studied. Bowman's glands in the lamina propria showed subpopulations of acinar cells reacting with SBA, S-WGA, WGA, STL, Con A, PSA, PNA, SJA, VVA, JAC and S-Con A, but in our optical studies with lectins we were unable to differentiate between mucous and serous cells in the way that is possible on electron microscopy. The ducts of Bowman's glands were labelled with S-WGA, STL, LEL, PHA-E, BSL-I and JAC. This histochemical study on the glycoconjugates of the olfactory mucosa in the order Xenarthra provides a basis for further experimental investigations.

The retinal vasculature of postmortem normal human and diabetic eyes was studied using an immunohistochemical technique in conjunction with confocal laser scanning microscopy. The technique, which stained for von Willebrand factor, allowed both large areas of the retinal vasculature to be visualised and abnormalities to be studied in detail without disturbing the tissue architecture. Only one microaneurysm, defined as any focal capillary dilation, was observed in 10 normal eyes but numerous microaneurysms were seen in 4 out of 5 diabetic retinas; counts varied between 0 and 26 per 0.41 mm2 sample area. Microaneurysms were classified into 3 categories according to morphology: saccular, fusiform and focal bulges. Most were saccular, these having no preferred orientation. The majority of microaneurysms were associated with just 2 vessels suggesting they were unlikely to develop at vascular junctions. The majority were observed to originate from the inner nuclear layer and were therefore in the deeper part of the inner retinal capillary plexus. Variation in the staining of microaneurysms may correlate with endothelial dysfunction seen clinically as dye leakage during fluorescein angiography.

A joint meeting of the Anatomical Society of Great Britain and Ireland and the British Biophysical Society was held at the School of Biomedical Sciences, University of Leeds, from 5th to 7th January 1999. It included a symposium on ‘Structure and function of molecular motors’ and the Annual General Meetings of both Societies. The following are abstracts of communications and posters presented at the meeting.

Twenty-four human legs were dissected macroscopically to study the morphological details of the accessory deep peroneal nerve. This nerve arose from the superficial peroneal nerve and descended in the lateral compartment of the leg, deep to peroneus longus along the posterior border of peroneus brevis. Approaching the ankle joint, this nerve passed through the peroneal tunnels to wind around the lateral malleolus; it then crossed beneath the peroneus brevis tendon anteriorly to reach the dorsum of the foot. The accessory deep peroneal nerve was found in every case examined and constantly gave off muscular branches to peroneus brevis and sensory branches to the ankle region. In addition, this nerve occasionally had muscular branches to peroneus longus and extensor digitorum brevis, and sensory branches to the fibula and the foot. The anomalous muscles around the lateral malleolus were also innervated by this nerve. Neither cutaneous branches nor communicating branches with other nerves were found. The present study reveals that the accessory deep peroneal nerve is consistently present and possesses a proper motor and sensory distribution in the lateral region of the leg and ankle. It is not an anomalous nerve as has previously been suggested.

It is widely accepted that Meckel's cartilage in mammals is uncalcified hyaline cartilage that is resorbed and is not involved in bone formation of the mandible. We examined the spatial and temporal characteristics of matrix calcification in Meckel's cartilage, using histochemical and immunocytochemical methods, electron microscopy and an electron probe microanalyser. The intramandibular portion of Meckel's cartilage could be divided schematically into anterior and posterior portions with respect to the site of initiation of ossification beneath the mental foramen. Calcification of the matrix occurred in areas in which alkaline phosphatase activity could be detected by light and electron microscopy and by immunohistochemical staining. The expression of type X collagen was restricted to the hypertrophic cells of intramandibular Meckel's cartilage, and staining with alizarin red and von Kossa stain revealed that calcification progressed in both posterior and anterior directions from the primary centre of ossification. After the active cellular resorption of calcified cartilage matrix, new osseous islands were formed by trabecular bone that intruded from the perichondrial bone collar. Evidence of such formation of bone was supported by results of double immunofluorescence staining specific for type I and type II collagens, in addition to results of immunostaining for osteopontin. Calcification of the posterior portion resembled that in the anterior portion of intramandibular Meckel's cartilage, and our findings indicate that the posterior portion also contributes to the bone formation of the mandible by an endochondral-type mechanism of calcification.

Pregnancy and lactation make demands on maternal calcium homeostasis which may affect bone strength. Recently, changes in cancellous architecture have been described in iliac crest bone biopsies from normal pregnant women but the rarity of such human material means an animal model is essential. The microanatomy of cancellous bone was compared in uniparous and multiparous rats using undecalcified histological sections of lumbar and caudal vertebrae and also proximal femora. An automated trabecular analysis system (TAS) measured a comprehensive range of structural variables including the trabecular number, connectivity and width. In the first pregnancy cycle an early stimulation of bone formation (which quadrupled at some sites) was indicated by an increase in the skeletal uptake and spacing of double calcein labels and the immediate generation of thicker more numerous and interconnected trabeculae. A 40% increase in cancellous bone volume was observed in the lumbar spine in comparison with age-matched virgin controls. In contrast, a rapid succession of 3 pregnancy cycles (including lactation) culminated in cancellous atrophy of 15% at the same site, with a loss in trabecular number ranging from 20% (caudal vertebra) to 30% (lumbar vertebrae). In comparison, the proximal femur lost 40% of its struts but, nevertheless, uniquely sustained its cancellous bone volume. When lactation was excluded the number of struts lost was halved although trabecular thinning then took place which was sufficient to maintain the previous 15% deficit in bone volume. It was concluded that a single pregnancy strengthens the cancellous component of the maternal skeleton while a quick succession of pregnancies weakens it. Lactation influences the pattern of bone loss but not its amount.