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OBJECTIVE: Several studies have demonstrated biologic responses of intervertebral disc (IVD) cells to loading, although the mechanotransduction pathways have not been elucidated. In articular chondrocytes, which have a phenotype similar to that of IVD cells, a number of mechanoreceptors have been identified, with alpha5beta1 integrin acting as a predominant mechanoreceptor. The purpose of this study was to investigate the role of integrin signaling in IVD cells during mechanical stimulation and to determine whether RGD integrins are involved. METHODS: Human nucleus pulposus (NP) cells derived from nondegenerated and degenerated discs were subjected to dynamic compressive loading in the presence of an RGD inhibitory peptide. Expression of the alpha5beta1 heterodimer in IVD tissue was examined by immunohistochemistry and possible alternative mechanoreceptors by real-time quantitative polymerase chain reaction. RESULTS: Aggrecan gene expression was decreased following loading of NP cells from nondegenerated and degenerated discs. This response was inhibited by treatment with an RGD peptide in cells from nondegenerated, but not degenerated, IVDs. Immunohistochemistry demonstrated that expression of the alpha5beta1 heterodimer was unaltered in degenerated IVD tissue as compared with normal IVD tissue. CONCLUSION: Our results indicate that the mechanotransduction pathways are altered in cells from degenerated IVDs. Mechanosensing in NP cells from nondegenerated discs occurs via RGD integrins, possibly via the alpha5beta1 integrin, while cells from degenerated discs show a different signaling pathway that does not appear to involve RGD integrins.

The intervertebral disc (IVD) is one of the body's most important load-bearing structures with the major mechanical force experienced in the nucleus pulposus (NP) being hydrostatic pressure (HP). Physiological levels of HP have an anabolic effect on IVD matrix metabolism in cells derived from non-degenerate animal and herniated IVD while excessive HP has a catabolic effect. However, no studies have investigated the response of non-degenerate and degenerate human disc cells derived from non-herniated discs to HP. Here we investigate the effect of physiological HP on such cells using a novel loading rig. Human IVD cells (both NP and AF) cultured in alginate were subjected to dynamic HP (0.8-1.7 MPa 0.5 Hz) for 2 h. Cell viability was assessed, RNA extracted and qRT-PCR for 18 s, c-fos, Sox-9, collagen type II, aggrecan and MMP-3 performed. Cell viability was unaffected by the loading regime. In non-degenerate NP cells, HP increased c-fos, aggrecan, Sox-9 and collagen type II (significantly so in the case of c-fos and aggrecan), but not MMP-3 gene expression. In contrast, application of HP to AF or degenerate NP cells had no effect on target gene expression. Our data shows that cells obtained from the healthy NP respond to dynamic HP by up-regulating genes indicative of healthy matrix homeostasis. However, responses differed in degenerate NP cells suggesting that an altered mechanotransduction pathway may be operational.

Reactive astrocytes are thought to protect the penumbra during brain ischemia, but direct evidence has been lacking due to the absence of suitable experimental models. Previously, we generated mice deficient in two intermediate filament (IF) proteins, glial fibrillary acidic protein (GFAP) and vimentin, whose upregulation is the hallmark of reactive astrocytes. GFAP−/−Vim−/− mice exhibit attenuated posttraumatic reactive gliosis, improved integration of neural grafts, and posttraumatic regeneration. Seven days after middle cerebral artery (MCA) transection, infarct volume was 210 to 350% higher in GFAP−/−Vim−/− than in wild-type (WT) mice; GFAP−/−, Vim−/− and WT mice had the same infarct volume. Endothelin B receptor (ETBR) immunoreactivity was strong on cultured astrocytes and reactive astrocytes around infarct in WT mice but undetectable in GFAP−/−Vim−/− astrocytes. In WT astrocytes, ETBR colocalized extensively with bundles of IFs. GFAP−/−Vim−/− astrocytes showed attenuated endothelin-3-induced blockage of gap junctions. Total and glutamate transporter-1 (GLT-1)-mediated glutamate transport was lower in GFAP−/−Vim−/− than in WT mice. DNA array analysis and quantitative real-time PCR showed downregulation of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of tissue plasminogen activator. Thus, reactive astrocytes have a protective role in brain ischemia, and the absence of astrocyte IFs is linked to changes in glutamate transport, ETBR-mediated control of gap junctions, and PAI-1 expression.

Through its involvement in inflammation, opsonization, and cytolysis, the complement protects against infectious agents. Although most of the complement proteins are synthesized in the central nervous system (CNS), the role of the complement system in the normal or ischemic CNS remains unclear. Here we demonstrate for the first time that neural progenitor cells and immature neurons express receptors for complement fragments C3a and C5a (C3a receptor (C3aR) and C5a receptor). Mice that are deficient in complement factor C3 (C3(-/-)) lack C3a and are unable to generate C5a through proteolytic cleavage of C5 by C5-convertase. Intriguingly, basal neurogenesis is decreased both in C3(-/-) mice and in mice lacking C3aR or mice treated with a C3aR antagonist. The C3(-/-) mice had impaired ischemia-induced neurogenesis both in the subventricular zone, the main source of neural progenitor cells in adult brain, and in the ischemic region, despite normal proliferative response and larger infarct volumes. Thus, in the adult mammalian CNS, complement activation products promote both basal and ischemia-induced neurogenesis.

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

The normal aged brain undergoes pro-inflammatory changes. We investigated the effect of injecting a potential inflammatory stimulus, an adenoviral vector, on the response of microglia and astroglia in the aged brain. Groups of young (4 months) and old (31 months) male C57BL/Icrfa t mice received a unilateral injection into the striatum of adenoviral vector encoding the LacZ gene. After 48 h, the mice were killed and the brains analysed for numbers of activated microglia and macrophages using the biotinylated lectin Griffonia simplicifolia as a marker; astroglia were identified by immunohistochemistry for glial fibrillary acidic protein (GFAP). The cell counts were analysed using two-way analysis of variance (ANOVA). Transgene expression was assessed by β-galactosidase histochemistry. The numbers of activated microglia in the striatum increased in response to the adenovirus in both young [contralateral 19.5 (3. 7), ipsilateral 36 (3.0)] and old [contralateral 23.1 (9.6), ipsilateral 40.8 (6.9)] mice (two-way ANOVA; P < 0.0001), but there was no significant difference between the two age groups. There was a significant age-related increase in the number of GFAP-positive astroglia in the uninjected, contralateral striatum [4 months, 2.5 (1.4); 31 months, 29.7 (9.3)] (two-way ANOVA; P < 0.0001). However, there was no difference in response to the adenovirus in both young [contralateral 2.5 (1.4), ipsilateral 3.2 (1.2)] and old [contralateral 29.7 (9.3), ipsilateral 28.9 (8.2)] mice. We conclude that even though it has been argued that the aged brain is in a pro-inflammatory state, under the experimental conditions used in this study, there was no difference in the nature of the immune response between young and old mice of this strain to an adenoviral load.

This study examines the effect of age on oedema and brain swelling, and associated glial cell involvement on the size of the lesion in two models of permanent, focal cerebral ischaemia. Ischaemia was induced in male C57BL/Icrfat mice (4-6 and 26-31-month-old) by middle cerebral artery (MCA) occlusion using either electrocoagulation after craniotomy (MCA/craniotomy), or by an intraluminal filament through the carotid artery (MCA/icf). Twenty-four hours after inducing ischaemia, brain swelling and lesion size were measured in young and aged mice, and cerebral oedema by wet/dry brain weights. Histopathology and immunocytochemistry were performed on a separate set of perfusion fixed brains. The MCA/icf technique produced a significantly larger lesion than MCA/craniotomy in both age groups. The percentage of water taken into the brain was significantly greater after MCA/icf, with aged mice showing the greatest increase. When lesion size was corrected for brain swelling there was no age-related increase in the size of the lesion. The numbers of microglia and astroglia increased significantly in the parietal cortex of aged control animals, and there were qualitative differences in the glial response between the two stroke models. This study emphasizes the importance of age in models of permanent focal ischaemia, with oedema clearly being a significant factor. Differ-ences in the responsiveness of the glial cell population with age may be of fundamental importance in the progress of ischaemic brain damage.

Urine concentrating ability declines with increasing age, partly due to an impaired response of kidney medullary collecting ducts to the antidiuretic hormone, vasopressin. We investigated this change in isolated mouse medullary collecting ducts by measuring the activity of adenylate cyclase and phosphodiesterase, which catalyse the formation and hydrolysis of cAMP, respectively. Adenylate cyclase activity was measured in the presence of vasopressin (which stimulates adenylate cyclase via the receptor) or forskolin (which directly stimulates the catalytic subunit). We showed an age-related decrease in the catalytic subunit of adenylate cyclase, and no difference in the activity of phosphodiesterase, indicating that a reduction in the catalytic subunit of adenylate cyclase contributes towards the age-related decrease in cyclic AMP response of kidney to vasopressin.

Low back pain (LBP) is a major cause of disability worldwide that has been linked to intervertebral disc (IVD) degeneration. An improved understanding of the pathogenesis of disc degeneration is now developing, which is leading to the development of a number of possible future therapies targeted at the underlying pathology and regeneration strategies. Although results thus far are promising, the investigation of such therapies in an environment that mimics the mechanical environment of the human disc in vivo is problematic. The development of an in vitro model system that can maintain metabolically active IVD tissue within a loading environment pertaining to that of the human spine is crucial for testing the efficacy of future cell-based and tissue-engineering therapies for IVD degeneration. Here, using our novel loading rig, capable of mimicking the loading environment experienced within the human spine, we have cultured nucleus pulposus tissue explants, applied a daily hydrostatic loading regime for up to 2 weeks and investigated proteoglycan retention, metabolic activity and cellular phenotype. IVD tissue cultured under a loading environment pertaining to the in vivo loading environment maintained metabolic cell activity, proteoglycan content and cellular phenotype. Indeed, all parameters were improved in IVD tissue cultured with load compared to unloaded controls. Such a model is invaluable for investigations assessing the feasibility and efficacy of future therapeutic approaches to inhibiting degeneration or stimulating regeneration of the IVD, where the in vivo loading environment may be crucial to their success or failure.

In this study, we investigated age-associated changes in neuroaxonal transport of the hormone vasopressin (AVP) and its associated neurophysin (NPII), from the supraoptic nucleus (SON) of the hypothalamus to the neurohypophysis. C57BL/Icrfa1 male mice of 6 and 28 months of age were injected in the hypothalamus with l -[35S]cysteine. Animals were killed up to 2.25 h after injection and NPII and AVP from the SON and neurohypophysis were separated using HPLC, and the fractions counted for radioactivity. In the SON, radiolabelled NPII and AVP were first detected after 0.50 h in both young and old mice. There was no significant difference between the age-groups in the incorporation of radiolabel over the time course studied. Radiolabelled NPII in the neurohypophysis was significantly above background after 1.25 h in the young, and after 1.50 h in the old mice. The differences between the two age groups was significant (P =0.05). Radiolabelled AVP followed a similar trend, but was not significantly above background until 1.50 h in the young and 1.75 h in the old. The differences between the two age groups was on the point of significance (P = 0.056). These results indicate a significant reduction of up to 25% in the rate of axonal transport of neurohypophyseal peptides with advancing age.

The intervertebral disc (IVD) is one of the body's most important load-bearing structures with the major mechanical force experienced in the nucleus pulposus (NP) being hydrostatic pressure (HP). Physiological levels of HP have an anabolic effect on IVD matrix metabolism in cells derived from non-degenerate animal and herniated IVD while excessive HP has a catabolic effect. However, no studies have investigated the response of non-degenerate and degenerate human disc cells derived from non-herniated discs to HP. Here we investigate the effect of physiological HP on such cells using a novel loading rig. Human IVD cells (both NP and AF) cultured in alginate were subjected to dynamic HP (0.8-1.7 MPa 0.5 Hz) for 2 h. Cell viability was assessed, RNA extracted and qRT-PCR for 18 s, c-fos, Sox-9, collagen type II, aggrecan and MMP-3 performed. Cell viability was unaffected by the loading regime. In non-degenerate NP cells, HP increased c-fos, aggrecan, Sox-9 and collagen type II (significantly so in the case of c-fos and aggrecan), but not MMP-3 gene expression. In contrast, application of HP to AF or degenerate NP cells had no effect on target gene expression. Our data shows that cells obtained from the healthy NP respond to dynamic HP by up-regulating genes indicative of healthy matrix homeostasis. However, responses differed in degenerate NP cells suggesting that an altered mechanotransduction pathway may be operational.

The nuclei of cells from the supraoptic nucleus (SON) of virgin female C57BL/lcrfat mice, ranging from eight to thirty-two months of age, were studied by electron microscopy. A quantitative study has shown that the frequency of membranous and fibrillar inclusions within the cell nuclei increase with age, but the membranous inclusions decline at extreme age, (32 months). Inclusions of either type were rarely encountered in young or old male mice.

Observations on the age-related volume composition of lipofuscin and hormone-containing organelles in the neurosecretory neurones of the supraoptic nucleus of the hypothalamo-neurohypophyseal system of the ageing C57BL mouse are presented. There is an age-related accumulation of lipofuscin in these neurones with age and although there are changes in the relative volumes of hormone containing structures as determined by quantitative ultrastructural techniques, these are not necessarily connected with lipofuscin accumulation. Under conditions of osmotic stress, the hormone containing organelles remain at a static level whereas lipofuscin pigment is significantly reduced. Evidence is put forward for a turnover of lipofuscin in these cells, and for the presence of this pigment being an indicator of intracellular damage. It is argued that there is insufficient knowledge available concerning the role of lipofuscin in ageing cells and that under these circumstances, it is a matter of urgency to determine this prior to the irresponsible use of drug treatments to reduce pigment content.

The effect of a dietary deficiency of vitamin E on the concentrations of lipofuscin in the hippocampus and the supraoptic nucleus (SON) of the hypothalamus, and in adrenal cortical cells was assessed in male mice. The animals were fed either a control diet or the vitamin E deficient diet after 2 months of age for a period of 6 months. There was no significant difference in the growth curves of the 2 groups of animals over the period studied. Fluorescence microscopy and transmission electron microscopy were used to assess the effect of the diet on lipofuscin in the different tissues. Quantitative morphological techniques were used to determine the relative volumes of lipofuscin in the neurons from the SON and in the adrenal cortical cells. There was no significant difference in the concentration of lipofuscin in the SON neurons after vitamin E deficiency but there was a significant increase in the adrenal cortical cells. There was a clear difference in the effect of the deficiency on mitotic and fixed post-mitotic cells over the period investigated but further studies would be necessary to determine whether or not there was a critical period in the life span where vitamin E deficiency may induce changes in all cell types.

A quantitative study of various morphological parameters in endocrine cells of the neuroendocrine region of the laboratory mouse was carried out. The supraoptic nucleus of the hypothalamo-neurohypophyseal system synthesises the hormones vasopressin and, to a lesser extent, oxytocin, and transports them to the posterior pituitary. Female C57BL/Icrfa t mice at 8 and 26 months of age, free of macroscopic pathology, were sampled when in a physiologically defined resting state. No significant differences in the volume fractions of most cell and subcellular components could be detected at the two ages studied; however, significant increases in the volume fractions of hormone-containing granules and lipofuscin (aging pigment) were demonstrated in older animals. These observations are similar to those made on rat endocrine pancreas also in the resting state. The notion that these hormone-secreting cells are “protected” to some extent from the aging process, and may have some of the qualities of “pace-maker” cells, is discussed.

The neurons of the supraoptic nucleus of male C57BL/Icrfa 1 mice at 6 or 28 months of age were examined from normally hydrated, osmotically loaded and osmotically loaded-rehydrated animals. Using quantitative morphological techniques, a reduction in the concentration of lipofuscin in the neurons was observed in osmotically loaded mice at both ages, and these levels were restored to control values during rehydration. In addition, there was a significant difference in the pattern of response of lipofuscin levels between the two age groups during the experiment. The concentration of hormone containing neurosecretory granules in the neurons of the supraoptic nucleus did not differ significantly between the two age groups during the experiment. However, the surface area of rough endoplasmic reticulum per unit volume of the supraoptic nucleus cell did differ significantly between the two age groups over the course of the experiment. It is concluded that increasing concentrations of lipofuscin do not affect the ability of the cell to control the concentration of neurosecretory granules or rough endoplasmic reticulum. The simplistic view that lipofuscin accumulates with age to the detriment of cell function must be revised.

A quantitative study of various morphological parameters of neuroendocrine cells from the hypothalamo-neurohypophyseal system has been carried out. The cells of the supraoptic nucleus (SON) of male C57BL/ICrfat mice at 6 and 28 months of age were examined from both normally hydrated and osmotically loaded animals. The only significant age-related difference between the principal components of the SON (i.e. SON cells, glia, capillary and neuropil) from normally hydrated mice was an increase in the relative volume fraction of the capillary lumen in the SON of old animals. No significant differences could be detected in the above-mentioned components of the SON from young and old salt-stressed mice. There were differences relating to age and to salt stress in the volume fractions of several sub-cellular components of SON cells. Under the influence of osmotic stress there was an increase in the relative volume of the SON cell occupied by cytoplasm when compared to the controls; the response was similar at both ages tested. Similarly for the nucleus, the relative volume of the SON cell occupied at both ages in control and salt-stressed animals was of the same order. The relative volume of SON cells occupied by mitochondria did not alter at either age during salt stress; however, the number of mitochondria per unit area of SON cell cytoplasm fell significantly in the old salt-stressed mice. The volumes of the Golgi and the hormone-containing granules (neurosecretory granules) of these cells remained the same with both age and salt stress. The surface area of the rough endoplasmic reticulum, however, was similar in SON cells from old and young controls, but there was significantly less rough endoplasmic reticulum in cells from the old when compared with the young salt-stressed animals. There was a significant increase in the volume of the soncell occupied by lipofuscinnin old animals, and the relative volume of age pigment was significantly reduced in the cells of salt-stressed animals at both ages. The morphological correlates of functional activity in the SON cells from young, osmotically loaded mice are similar to those seen in other laboratory rodents under the same conditions. In the old animal, the overall response to osmotic stress by the SON as an organ, and also by the individual cells (i.e. cytoplasm and nucleus), are similar to those in young animals. However, there are significant differences in detail, particularly with respect to mitochondrial numbers, surface area of rough endoplasmic reticulum and the relative volumes of lipofuscin and “lipid bodies” in these cells. The morphological correlates of hormone production (neurosecretory granules) do not show age-related changes. The findings are discussed with respect to the efficiency of function of the aged neuroendocrine cell under osmotic stress conditions.

The influence of age on the response of the supraoptic nucleus (SON) of the hypothalamo-neurohypophyseal system to physiological stress has been studied by means of the electron microscope. An osmotic load was applied to male C57BL/Icrfa t mice at 6 and 28 months of age and the resulting changes in the ultrastructure of SON cells in response to this stress analysed. In the young animals the differences in morphology observed between the SON cells from control and from those of salt-loaded mice were very similar to those seen in salt-stressed rats at a similar age. Qualitative differences were noted in several sub-cellular components of old, salt-stressed mice. The mitochondria showed evidence of ultrastructural damage in cells from the old, stressed animals. The Golgi system and the rough endoplasmic reticulum both showed heterogeneity in structure when compared with cells from young, salt-stressed mice. In addition, there was a decrease in the lipofuscin content of old, salt-stressed mice. These changes are discussed with respect to the overall efficiency of cellular activity in old, physiologically stressed animals.

The neurons of the supraoptic nucleus (SON) of male C57BL/Icrfa t mice at 6 or 28 months of age were examined from normally hydrated, osmotically loaded and osmotically loaded/rehydrated animals.Using quantitative morphological techniques it was shown that the majority of the ultrastructural variables investigated were controlled in the same way in the SON neurons of young and old mice. The major exception was the mitochondrial compartment which although maintaining a constant proportion of the volume of the SON cell did show a significant reduction in the number of mitochondria in the old group, particularly during the osmotic challenge period of the experiment. The “lipid” body compartment of the SON neuron also behaved differently in the cells from the older age group. This study when viewed in conjunction with previous investigations suggests that these SON neuroendocrine neurons from old animals are able to produce hormone until late in the life-span. However, other aspects of cellular activity appeared to be altered when judged by morphological criteria. It is concluded thatthe SON neurons from these old mice are able to synthesize hormone-containing organelles to the same concentration as the cells from a younger animal. However, the efficiency of the process, or at least the efficiency of concomitant cellular processes, must be questioned in view of the alterations in the rough endoplasmic reticulum, mitochondrial and lysosomal systems.

Age-related changes in the intake of food and water, and the output of faeces and urine were investigated in C57BL/Icrfat mice of 6 and 24 months of age. Animals were singly housed in a metabolic cage for a period of 30 days. 14 days were allowed for acclimatization before the animals were dehydrated for 24 hours. 10 days of rehydration were allowed prior to a hyperosmotic challenge with 3% sodium chloride in the drinking water. The animals were then observed for 5 more days of rehydration. Urine was collected and analysed with regard to sodium, potassium, urea and vasopressin output/24 hours (/100g body weight), and the osmotic pressure of the urine was determined. Data were analysed by a 2 factor analysis of variance with repeated measures on one factor. Significant changes were detected in the control of body weight, potassium, sodium and urea outputs. No age-differences were detected in the intake of food or water, the output of faeces or urine, the urine osmotic pressure or the excretion of vasopressin. However, significant changes in these variables were detected in both age groups on the days of physiological challenge. The conclusion drawn is that in the mouse strain studied, and for the period of the lifespan investigated, there is no age related defect in the secretion of vasopressin. However, there are trends in the data suggesting a decreased responsiveness of the kidney with age.