Loss of Direct Vascular Contact to Astrocytes in the Hippocampus as an Initial Event in Alzheimer's Disease. Evidence from Patients, In Vivo and In Vitro Experimental Models.

Alzheimer's disease (AD) is characterized by the accumulation of aggregated amyloid peptides in the brain parenchyma and within the walls of cerebral vessels. The hippocampus-a complex brain structure with a pivotal role in learning and memory-is implicated in this disease. However, there is limited data on vascular changes during AD pathological degeneration in this susceptible structure, which has distinctive vascular traits. Our aim was to evaluate vascular alterations in the hippocampus of AD patients and PDAPP-J20 mice-a model of AD-and to determine the impact of Aß40 and Aß42 on endothelial cell activation. We found a loss of physical astrocyte-endothelium interaction in the hippocampus of individuals with AD as compared to non-AD donors, along with reduced vascular density. Astrocyte-endothelial interactions and levels of the tight junction protein occludin were altered early in PDAPP-J20 mice, preceding any signs of morphological changes or disruption of the blood-brain barrier in these mice. At later stages, PDAPP-J20 mice exhibited decreased vascular density in the hippocampus and leakage of fluorescent tracers, indicating dysfunction of the vasculature and the BBB. In vitro studies showed that soluble Aß40 exposure in human brain microvascular endothelial cells (HBMEC) was sufficient to induce NFκB translocation to the nucleus, which may be linked with an observed reduction in occludin levels. The inhibition of the membrane receptor for advanced glycation end products (RAGE) prevented these changes in HBMEC. Additional results suggest that Aß42 indirectly affects the endothelium by inducing astrocytic factors. Furthermore, our results from human and mouse brain samples provide evidence for the crucial involvement of the hippocampal vasculature in Alzheimer's disease.

Interplay between lysosomal, mitochondrial and death receptor pathways during manganese-induced apoptosis in glial cells.

Manganese (Mn) is an essential trace metal which plays a critical role in brain physiology by acting as a cofactor for several enzymes. However, upon overexposure, Mn preferentially accumulates within the basal ganglia leading to the development of a Parkinsonism known as Manganism. Data from our group have proved that Mn induces oxidative stress-mediated apoptosis in astrocytoma C6 cells. In the present study we described how cathepsins impact on different steps of each apoptotic cascade. Evidence obtained demonstrated that Mn generates lysosomal membrane permeabilization (LMP) and cathepsin release. Both cathepsins B (Ca-074 Me) and D (Pepstatin A) inhibitors as well as Bafilomycin A1 prevented caspases-3, -7, -8 and -9 activation, FasL upregulation, Bid cleavage, Δφm disruption and cytochrome c release. Results from in vivo studies showed that intrastriatal Mn injection increased cathepsin D levels from corpus striatum and substantia nigra pars compacta. Our results point to LMP and lysosomal cathepsins as key mediators in the apoptotic process triggered by Mn. These findings highlight the relevance of targeting the lysosomal pathway for Manganism therapy.

Sphingosine-1-phosphate restores endothelial barrier integrity in ovarian hyperstimulation syndrome.

STUDY QUESTION: Are follicular fluid (FF) sphingosine-1-phosphate (S1P) levels in patients at risk of developing ovarian hyperstimulation syndrome (OHSS) altered and in part responsible for the high vascular permeability observed in these patients. STUDY ANSWER: FF S1P levels are lower in FF from patients at risk of OHSS and treatment with S1P may reduce vascular permeability in these patients. WHAT IS KNOWN ALREADY: Although advances have been made in the diagnosis, and management of OHSS and in basic knowledge of its development, complete prevention has proven difficult. STUDY DESIGN, SIZE, DURATION: A total of 40 FF aspirates were collected from patients undergoing ART. The women (aged 25-39 years old) were classified into a control group (n = 20) or a group at risk of OHSS (n = 20). The EA.hy926 endothelial cell line was used to assess the efffects of FF from patients at risk of OHSS with or without the addition of S1P. An animal model that develops OHSS in immature Sprague-Dawley rats were also used. PARTICIPANTS/MATERIALS, SETTING, METHODS: Migration assays, confocal microscopy analysis of actin filaments, immunoblotting and quail chorioallantoic membrane (CAM) assays of in-vivo angiogenesis were performed and statistical comparisons between groups were made. MAIN RESULTS AND THE ROLE OF CHANCE: The S1P concentration was significantly lower in FF from patients at risk of OHSS (P = 0.03). The addition of S1P to this FF decreased cell migration (P < 0.05) and prevented VE-cadherin phosphorylation in endothelial cells (P < 0.05). S1P in the FF from patients at risk of OHSS increased the levels of VE-cadherin (P < 0.05), N-cadherin (P < 0.05) and ß-catenin (P < 0.05), and partially reversed actin redistribution in endothelial cells. The addition of S1P in FF from patients at risk of OHSS also decreased the levels of vascular endothelial growth factor (VEGF121; P < 0.01) and S1P lyase (SPL; P < 0.05) and increased the levels of S1PR1 (P < 0.05) in endothelial cells. In CAMs incubated with FF from patients at risk of OHSS with S1P, the number of vessel branch points decreased while the periendothelial cell coverage increased. Additionally, in a rat OHSS model, we demonstrated that vascular permeability and VEGF121 and its receptor KDR expression were increased in the OHSS group compared to the control group and that S1P administration decreased these parameters. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The results of this study were generated from an in-vitro system. This model reflects the microvasculature in vivo. Even though the ideal model would be the use of human endothelial cells from the ovary, it is obviously not possible to carry out this kind of approach in ovaries of patients from ART. More studies will be necessary to delineate the effects of S1P in the pathogenesis of OHSS. Hence, clinical studies are needed in order to choose the most appropriate method of prevention and management. WIDER IMPLICATIONS OF THE FINDINGS: The use of bioactive sphingolipid metabolites may contribute to finding better and safer therapeutic strategies for the treatment of OHSS and other human diseases that display aberrant vascular leakage. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants ANPCyT (PICT 2012-897), CONICET (PIP 5471), Roemmers and Baron Foundation, Argentina. The authors declare no conflict of interest.

The endometrium of cycling cows contains populations of putative mesenchymal progenitor cells.

Endometrial stem cells have been identified in humans, mice and pigs. This study was designed to determine whether the uterine endometrium of cycling cows contains such cells, to identify markers of stemness and ultimately to isolate putative stem/progenitor cell and evaluate their capability to differentiate into mesodermal derivatives. Uteri from healthy cows in the early (days 1-5) and late luteal phases (days 13-18) of the oestrous cycle were collected. Total RNA and proteins were isolated and searched for gene markers of embryonic (OCT4, NANOG, SOX2) and mesenchymal (CD44, STAT3, CD-117) stem cells and for protein markers (Oct4, Sox2, Cd44) in Western blots or immunostaining of paraffin-embedded tissue. Primary cell cultures were isolated; characterized in terms of morphology, colony formation and gene/protein expression; and induced osteogenic and chondrogenic differentiation. We identified expression of embryonic (OCT4 and SOX2, but not NANOG) and mesenchymal (STAT3, CD44 and c-KIT) gene markers in the endometrium of cycling cows and the encoded proteins (Oct4, Sox2 and Cd44) in both stages of the oestrous cycle. Derived cell lines displayed essentially the same gene expression pattern; however, at the protein level, Oct4 was not detected. No clear influence of the stage of the oestrous cycle was found. Cell lines from late luteal phase displayed osteogenic and chondrogenic differentiation potential upon chemical stimulation. In this research, we demonstrated the presence of mesenchymal progenitor cell populations of apparently mesenchymal origin in the endometrium of cycling cows, in both the early and late phases of the oestrous cycle. The cells isolated from the late luteal phase were more acquiescent to differentiate into mesodermal derivatives than cells in the early luteal phase. Our findings might have implications for the understanding of uterine stem cell biology in cows and other farm animal species.

Follicular dynamics, interval to ovulation and fertility after AI in short-term progesterone and PGF2α oestrous synchronization protocol in sheep.

The study was aimed to assess the influence that short-term progesterone treatments have on follicular dynamics, oestrus and ovulation in sheep. The treatment was tested thereafter in a field trial to assess its fertility after AI with fresh semen. In a first experiment, 12 ewes without CL were grouped to receive a new (n = 6) or used CIDR (n = 6) for 7 days and blood samples were obtained to follow plasma progesterone profiles. In a second experiment, 39 cycling ewes were synchronized by a 7-day P4+PGF2α protocol using a new (n = 20) or a 7-day used CIDR (n = 19). Half of both groups received 400 IU eCG and half remained untreated as controls. Ultrasound ovarian examination and oestrous detection were used to compare follicular dynamics, oestrus and ovulation in both groups. In a third experiment, 288 ewes in 3 farms were synchronized by the short-term P4+PGF2α+eCG protocol and ewes were AI with fresh semen 24 h after oestrous detection. Lambing performance was used to test the fertility of the treatment. In Experiment 1, ewes with new inserts presented higher P4 concentration than ewes with used inserts throughout the sampling period (p < 0.05) and exhibited a P4 peak at days 1-2 of the treatment that was not observed in ewes with used inserts. In Experiment 2, ewes treated with new and used inserts show similar ovarian and behavioral traits (p > 0.10). However, ewes treated with eCG show shorter interval to oestrus (p = 0.004) and tend to have larger mature CL (p = 0.06). In Experiment 3, oestrous presentation and lambing performance after AI with fresh semen was considered normal compared to published results. Results suggest that the oestrous synchronization protocol based on P4+PGF2α allows little control of follicular dynamics without compromising fertility after AI with fresh semen provided that eCG is added at the end of the treatment.

Brain alterations in autoimmune and pharmacological models of diabetes mellitus: focus on hypothalamic-pituitary-adrenocortical axis disturbances.

Type 1 diabetes (T1D) is linked to an 'encephalopathy' explained by some features common to the aging process, degenerative and functional disorders of the central nervous system. In the present study we describe a manifest hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis in two different experimental mouse models of T1D including the pharmacological one induced by streptozotocin and the spontaneous NOD (nonobese diabetic mice). The high expression of hypothalamic hormones like oxytocin and vasopressin were part to this alteration, together with elevated adrenal glucocorticoids and prominent susceptibility to stress. In the hippocampus of diabetic animals a marked astrogliosis, often associated with neural damage, was present. Dentate gyrus neurogenesis was also affected by the disease: proliferation and differentiation measured by bromodeoxyuridine immunodetection were significantly reduced in both experimental models used. Several facts, including changes associated with chronic hyperglycemia, hyperstimulation of the HPA axis, increased levels of circulating glucocorticoids in combination with brain inflammation and low production of new neurons, contribute to emphasize the impact of diabetes on the central nervous system.

Prominently decreased hippocampal neurogenesis in a spontaneous model of type 1 diabetes, the nonobese diabetic mouse.

In human diabetes, degenerative and functional disorders of the central nervous system, including depression, are common findings. Defective dentate gyrus (DG) neurogenesis is associated with affective-related disorders and depression. We previously demonstrated reduced DG neurogenesis in a pharmacological type 1 diabetes model, the streptozotocin (STZ)-treated mouse. Here, we explored DG neurogenesis in a spontaneous T1D model, the nonobese diabetic (NOD) mouse, at prediabetic and diabetic stages. Cell proliferation was assessed in the DG of 5, 8 and 12-week-old control C57BL/6 and BALB/c strains and NOD mice, killed 2 h after bromodeoxyuridine (BrdU) administration. Survival of the newly generated cells was studied in 15-week-old animals that were killed 21 days after BrdU injection. The number of proliferative BrdU-positive cells in the DG was, regardless of age, constantly and significantly lower in NOD than in control strains, showing the presence of hippocampal alterations far before clinical diabetes onset in NOD mice. Diabetes also strongly decreased cell survival in NOD DG. However, cell phenotype proportion, as assessed by co-localization with neuronal or glial markers and confocal microscopy, was not modified. Hippocampal neurogenesis is strongly diminished in the spontaneous NOD model, like in the STZ model. Notably, NOD hippocampal DG cell proliferation defect takes place during the prediabetic stage. Whether this early alteration might result, in this autoimmune strain, from hypothalamo-pituitary adrenal axis alterations and/or ongoing brain inflammatory process sharing many characteristics of aging is discussed and deserves further investigation.

GABAB receptors and glucose homeostasis: evaluation in GABAB receptor knockout mice.

GABA has been proposed to inhibit insulin secretion through GABAB receptors (GABABRs) in pancreatic beta-cells. We investigated whether GABABRs participated in the regulation of glucose homeostasis in vivo. The animals used in this study were adult male and female BALB/C mice, mice deficient in the GABAB1 subunit of the GABABR (GABAB(-/-)), and wild types (WT). Blood glucose was measured under fasting/fed conditions and in glucose tolerance tests (GTTs) with a Lifescan Glucose meter, and serum insulin was measured by ELISA. Pancreatic insulin content and islet insulin were released by RIA. Western blots for the GABAB1 subunit in islet membranes and immunohistochemistry for insulin and GABAB1 were performed in both genotypes. BALB/C mice preinjected with Baclofen (GABABR agonist, 7.5 mg/kg ip) presented impaired GTTs and decreased insulin secretion compared with saline-preinjected controls. GABAB(-/-) mice showed fasting and fed glucose levels similar to WT. GABAB(-/-) mice showed improved GTTs at moderate glucose overloads (2 g/kg). Baclofen pretreatment did not modify GTTs in GABAB(-/-) mice, whereas it impaired normal glycemia reinstatement in WT. Baclofen inhibited glucose-stimulated insulin secretion in WT isolated islets but was without effect in GABAB(-/-) islets. In GABAB(-/-) males, pancreatic insulin content was increased, basal and glucose-stimulated insulin secretion were augmented, and impaired insulin tolerance test and increased homeostatic model assessment of insulin resistance index were determined. Immunohistochemistry for insulin demonstrated an increase of very large islets in GABAB(-/-) males. Results demonstrate that GABABRs are involved in the regulation of glucose homeostasis in vivo and that the constitutive absence of GABABRs induces alterations in pancreatic histology, physiology, and insulin resistance.

Abnormalities of the hippocampus are similar in deoxycorticosterone acetate-salt hypertensive rats and spontaneously hypertensive rats.

Hippocampal neuropathology is a recognised feature of the brain in spontaneously hypertensive rats (SHR), but similar studies are lacking in another model of hypertension, the mineralocorticoid-salt-treated rat. The present study aimed to compare changes in hippocampal parameters in 16-week-old male SHR (blood pressure approximately 190 mmHg) and their normotensive Wistar-Kyoto controls, with those of male Sprague-Dawley rats receiving (i) 10 mg deoxycorticosterone acetate (DOCA) every other day during 3 weeks and drinking 1% NaCl solution (blood pressure approximately 160 mmHg) and normotensive controls treated with (ii) DOCA and drinking water, (iii) drinking water only or (iv) 1% NaCl only. In these experimental groups, we determined: (i) cell proliferation in the dentate gyrus (DG) using the 5-bromo-2'-deoxyuridine-labelling technique; (ii) the number of glial fibrillary acidic protein (GFAP) positive astrocytes under the CA1, CA3 and DG; (iii) the number of apolipoprotein E (ApoE) positive astrocytes as a marker of potential neuronal damage; and (iv) the number of neurones in the hilus of the DG, taken as representative of neuronal density in other hippocampal subfields. Changes were remarkably similar in both models, indicating a decreased cell proliferation in DG, an increased number of astrocytes immunopositive for GFAP and ApoE and a reduced number of hilar neurones. Although hypertension may be a leading factor for these abnormalities, endocrine mechanisms may be involved, because hypothalamic-pituitary function, mineralocorticoid receptors and sensitivity to mineralocorticoid treatment are stimulated in SHR, whereas high exogenous mineralocorticoid levels circulate in DOCA-treated rats. Thus, in addition to the deleterious effects of hypertension, endocrine factors may contribute to the abnormalities of hippocampus in SHR and DOCA-treated rats.

Oestradiol restores cell proliferation in dentate gyrus and subventricular zone of streptozotocin-diabetic mice.

Type 1 diabetes mellitus correlates with several brain disturbances, including hypersensitivity to stress, cognitive impairment, increased risk of stroke and dementia. Within the central nervous system, the hippocampus is considered a special target for alterations associated with diabetes. Neurogenesis is a plastic event restricted to few adult brain areas: the subgranular zone of the dentate gyrus and the subventricular zone (SVZ). First, we studied the ability for neurogenesis in the dentate gyrus and SVZ of chronic diabetic mice induced by streptozotocin (STZ). Using bromodeoxyuridine (BrdU) labelling of cells in the S-phase, we observed a strong reduction in cell proliferation rate in both brain regions of diabetic mice killed 20 days after STZ administration. Second, because oestrogens are active neuroprotective agents, we investigated whether 17beta-oestradiol (200 micro g pellet implant in cholesterol during 10 days) restored brain cell proliferation in the diabetic mouse brain. Our results demonstrated a complete reversibility of dentate gyrus cell proliferation in oestrogen-treated diabetic mice. This plasticity change was not exclusive to the hippocampus because oestrogen treatment restored BrdU incorporation into newborn cells of the SVZ region of diabetic animals. Oestrogen treatment did not alter the hyperglycemic status of STZ-diabetic mice. Moreover, oestrogen did not modify BrdU incorporation in control animals. These data show that oestrogen treatment strongly stimulates brain neurogenesis of diabetic mice and open up new venues for understanding the potential neuroprotective role of steroid hormones in diabetic encephalopathy.

Changes in Fos expression in various brain regions during deoxycorticosterone acetate treatment: relation to salt appetite, vasopressin mRNA and the mineralocorticoid receptor.

Salt appetite, a conditioning factor for hypertension and cardiovascular diseases, is produced when high doses of mineralocorticoids are given to experimental animals. A commonly used procedure to identify neuronal activation is to determine the number of Fos-immunoreactive cells. In rats with established salt appetite after 8 days of deoxycorticosterone acetate (DOCA) treatment, Fos-positive cells were studied in seven brain areas. Significant increases in Fos activity were recorded in the paraventricular (PVN) and supraoptic (SON) nuclei, median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), preoptic area (POA), bed nucleus of the stria terminalis (BNST) and amygdala (AMYG). In most of these areas, increased Fos expression was also observed early (2 h) after a single DOCA injection, well before salt appetite develops. Using a mineralocorticoid receptor (MR) antibody, we studied whether Fos-active regions also expressed MR. MR-positive cells were found in the OVLT, MnPO, AMYG and BNST, but not in the POA, PVN and SON. In the PVN and SON, nevertheless, prolonged or single DOCA treatment increased expression of mRNA for arginine vasopressin (AVP). The present demonstration of Fos activation, in conjunction with differential expression of MR and stimulation of AVP mRNA, suggests that a neuroanatomical pathway comprising the AMYG, osmosensitive brain regions and magnocellular nuclei becomes activated during DOCA effects on salt appetite. It is recognized, however, that DOCA effects may also depend on mechanisms and brain structures other than those considered in the present investigation. Since some Fos-positive regions were devoid of MR, a comprehensive view of DOCA-induced salt appetite should consider nongenomic pathways of steroid action, including the role of reduced DOC metabolites binding to GABAergic membrane receptors.

Glucocorticoid effects on Fos immunoreactivity and NADPH-diaphorase histochemical staining following spinal cord injury.

Glucocorticoids (GC) provide neuroprotection and early recovery after spinal cord injury (SCI). While several mechanisms were proposed to account for these effects, limited information exists regarding GC actions in sensory areas of the spinal cord. Presently, we studied the time course of Fos expression, and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical staining to monitor neuronal responses to SCI with or without GC treatment. Rats with sham-operation or transection at the thoracic level (T7-T8) received vehicle or 5 mg/kg of the GC dexamethasone (DEX) at 5 min post-lesion and were sacrificed 2 or 4 h after surgery. Another group of SCI rats received vehicle or intensive DEX treatment (5 min, 6 h, 18 h and 46 h post-lesion) and were sacrificed 48 h after surgery. The number of NADPH-d positive neurons or Fos immunoreactive nuclei was studied by computer-assisted image analysis in superficial dorsal horn (Laminae I-III) and central canal area (Lamina X) below the lesion. While constitutive Fos immunoreactive nuclei were sparse in controls, SCI increased Fos expression at 2 and 4 h after injury. DEX treatment significantly enhanced the number of Fos positive nuclei in Laminae I-III by 4 h after transection, although the response was not maintained by intensive steroid treatment when tested at 48 h after SCI. NADPH-d positive neurons in Laminae I-III increased at 2 and 4 h after SCI while a delayed increased was found in central canal area (Lamina X). DEX treatment decreased NADPH-d positive neurons to sham-operated levels at all time points examined. Thus, while GC stimulation of Fos suggests activation of neurons involved in sympathetic outflow and/or pain, down-regulation of NADPH-d indicates attenuation of nociceptive outflow, considering the role of enzyme-derived nitric oxide in pain-related mechanisms. Differential hormonal effects on these molecules agree with their localization in different cell populations.

Diabetes increases the expression of hypothalamic neuropeptides in a spontaneous model of type I diabetes, the nonobese diabetic (NOD) mouse.

1. Synthesis of oxytocin (OT) and arginine-vasopressin (AVP) is increased in induced models of Type I diabetes, such as the streptozotocin model. However, these parameters have not yet been evaluated in spontaneous models, such as the nonobese diabetic mouse (NOD). Therefore, we studied in the magnocellular cells of the paraventricular nucleus (PVN) of nondiabetic and diabetic 16-week-old female NOD mice and control C57B1/6 mice, the immunocytochemistry of OT and AVP peptides and their mRNA expression, using nonisotopic in situ hybridization (ISH). 2. In nondiabetic and diabetic NOD female mice, the number of OT- and AVP-immunoreactive cells were similar to those of the controls, whereas immunoreaction intensity was significantly higher for both peptides in diabetic NOD as compared with nondiabetic NOD and control C57B1/6 mice. 3. ISH analysis showed that the number of OT mRNA-containing cells was in the same range in the three groups, whereas higher number of AVP mRNA expressing cells was found in diabetic NOD mice. However, the intensity of hybridization signal was also higher for both OT and AVP mRNA in the diabetic group as compared with nondiabetic NOD and control mice. 4. Blood chemistry demonstrated that haematrocrit, total plasma proteins, urea, sodium, and potassium were within normal limits in diabetic mice. Thus, NOD mice were neither hypernatremic nor dehydrated. 5. We suggest that upregulation of OT and AVP reflects a high-stress condition in the NOD mice. Diabetes may affect neuropeptide-producing cells of the PVN, with the increased AVP and OT playing a deleterious role on the outcome of the disease.

Changes of hypothalamic and plasma vasopressin in rats with deoxycorticosterone-acetate induced salt appetite.

Mineralocorticoids play a predominant role in development of salt appetite and hypertension. Since vasoactive peptides could mediate the central effects of mineralocorticoids, we evaluated changes of immunoreactive (IR) arginine vasopressin (AVP) in the paraventricular (PVN) and supraoptic (SON) hypothalamic nucleus during DOCA-induced salt appetite. In one model, rats having free access to water and 3% NaCl during 9 (prehypertensive stage) or 21 days (hypertensive stage) received DOCA (s.c., 10 mg/rat/in alternate days). A decrease in the IR cell area, number of IR cells and staining intensity was obtained in magnocellular PVN of rats treated during 9 days. After 21 days IR cell area and number of cells in the PVN also decreased, but staining intensity of remaining cells was normal. The same parameters were unchanged in the SON. In another model, animals treated with DOCA during 9 days had only access to 3% NaCl or water. The IR cell area in PVN and SON significantly increased in mineralocorticoid-treated and control animals, both drinking 3% NaCl. Staining intensity (PVN and SON) and number of IR cells (PVN) also augmented in DOCA-treated animals drinking salt respect of a group drinking water. Plasma AVP in rats treated with DOCA and offered salt and water, exhibited a 2-2.5 fold increase at the time of salt appetite induction. Plasma AVP was substantially higher in rats drinking salt only, while the highest levels were present in salt-drinking DOCA-treated rats. Thus, peptide depletion in the PVN may be due to increased release, because reduced levels of hypothalamic and posterior pituitary AVP were measured in this model. In rats drinking salt only the substantial increase of IR AVP in the PVN and SON, may be due to dehydration and hyperosmosis. Because DOCA-salt treated rats showed higher AVP levels in the PVN compared to untreated rats drinking salt only, it is possible that DOCA sensitized PVN cells to increase AVP production. The results suggest the vasopressinergic system could mediate some central functions of mineralocorticoids.

Glucocorticoid regulation of motoneuronal parameters in rats with spinal cord injury.

1. Glucocorticoids exert beneficial effects after acute CNS injury in humans and experimental animals. To elucidate potential mechanisms of glucocorticoid action in the lesioned spinal cord, we have studied if treatment with dexamethasone (DEX) modulated the neurotrophin binding receptor p75 (p75NTR) and choline acetyltransferase (ChAT), a marker of neuronal functional viability. 2. Rats with a sham operation or with spinal cord transection at the thoracic level received vehicle or DEX several times postlesion and were sacrificed 48 hr after surgery. The lumbar region caudal to the lesion was processed for p75NTR and ChAT immunoreactivity (IR) using quantitative densitometric analysis. 3. We observed that p75NTR-IR was absent from ventral horn motoneurons of sham-operated rats, in contrast to strong staining of neuronal perikaryon in TRX rats. Administration of DEX to TRX rats had no effect on the number of neuronal cell bodies expressing p75NTR-IR but significantly increased the number and length of immunostained neuronal processes. 4. Furthermore, spinal cord transection reduced ChAT immunostaining of motoneurons by 50%, whereas DEX treatment reverted this pattern to cells with a strong immunoreaction intensity in perikaryon and cell processes. 5. It is hypothesized that increased expression of p75NTR in cell processes and of ChAT in motoneurons may be part of a mechanism by which glucocorticoids afford neuroprotection, in addition to their known antiinflammatory effects.

Regulation of gene expression by corticoid hormones in the brain and spinal cord.

Glucocorticoids (GC) and mineralocorticoids (MC) have profound regulatory effects upon the central nervous system (CNS). Hormonal regulation affects several molecules essential to CNS function. First, evidences are presented that mRNA expression of the alpha3 and beta1-subunits of the Na,K-ATPase are increased by GC and physiological doses of MC in a region-dependent manner. Instead, high MC doses reduce the beta1 isoform and enzyme activity in amygdaloid and hypothalamic nuclei, an effect which may be related to MC control of salt appetite. The alpha3-subunit mRNA of the Na,K-ATPase is also stimulated by GC in motoneurons of the injured spinal cord, suggesting a role for the enzyme in GC neuroprotection. Second, we provide evidences for hormonal effects on the expression of mRNA for the neuropeptide arginine vasopressin (AVP). Our data show that GC inhibition of AVP mRNA levels in the paraventricular nucleus is sex-hormone dependent. This sexual dimorphism may explain sex differences in the hypothalamic-pituitary-adrenal axis function between female and male rats. Third, steroid effects on the astrocyte marker glial fibrillary acidic protein (GFAP) points to a complex regulatory mechanism. In an animal model of neurodegeneration (the Wobbler mouse) showing pronounced astrogliosis of the spinal cord, in vivo GC treatment down-regulated GFAP immunoreactivity, whereas the membrane-active steroid antioxidant U-74389F up-regulated this protein. It is likely that variations in GFAP protein expression affect spinal cord neurodegeneration in Wobbler mice. Fourth, an interaction between neurotrophins and GC is shown in the injured rat spinal cord. In this model, intensive GC treatment increases immunoreactive low affinity nerve growth factor (NGF) receptor in motoneuron processes. Because GC also increases immunoreactive NGF, this mechanism would support trophism and regeneration in damaged tissues. In conclusion, evidences show that some molecules regulated by adrenal steroids in neurons and glial cells are not only involved in physiological control, but additionally, may play important roles in neuropathology.

Increased expression of magnocellular vasopressin mRNA in rats with deoxycorticosterone-acetate induced salt appetite.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated in the genesis of hypertension due to deoxycorticosterone acetate (DOCA)-salt treatment of uninephrectomized rats. In this work, we studied if DOCA treatment of intact rats in doses arousing a salt appetite (a prehypertensive state), modulated mRNA for AVP and OT in the hypothalamus. Male Sprague-Dawley rats were offered both tap water and 3% NaCl in separate bottles and received vehicle or subcutaneous injections of 10 mg DOCA on alternate days for 7 days (4 injections) or 17 days (9 injections). They developed a preference for 3% NaCl solutions 24-48 h after treatment. Brain slices from rats killed on the 8th or 18th day were exposed to 35S-labeled probes encoding prepro-AVP mRNA or OT mRNA, respectively. Expression of these mRNAs was measured in the magnocellular and parvocellular divisions of the paraventricular nucleus (PVN) and magnocellular cells of the supraoptic nucleus (SON). No changes were obtained in neuropeptide mRNA levels in the parvocellular division of the PVN between control and the two groups of DOCA-treated rats. However, DOCA-treated animals presented an increased number of grains per cell for AVP mRNA in the magnocellular division of the PVN and in magnocellular cells of the SON, as shown by group mean comparisons and frequency histograms. No changes were detected for OT mRNA. In a second series of studies, control or DOCA-treated rats were offered 3% NaCl or water as the only choice. Animals drinking 3% NaCl showed increased AVP and OT mRNA levels, whether they received DOCA or not. However, AVP mRNA levels in both nuclei were higher in DOCA-treated rats drinking 3% NaCl than in controls drinking salt solution. In comparison, control and DOCA-treated rats drinking water showed lower levels of AVP mRNA. OT mRNA levels in the SON remained unchanged in the same groups. The results suggest that in the magnocellular cells of the PVN and SON, increments in AVP mRNA are obtained following increments in salt intake produced by either mineralocorticoid treatment or exclusive salt drinking. In rats offered salt solution and water to drink, DOCA effects on AVP mRNA developed before changes occurred in serum sodium levels. Because combined DOCA + salt treatment induced a higher response in terms of AVP mRNA expression, we suggest that AVP could be a target of the central effects of the mineralocorticoid.

Differential response to a stress stimulus of proenkephalin peptide content in immune cells of naive and chronically stressed rats.

Proenkephalin peptides produced by endocrine and nervous tissues are involved in stress-induced immunosuppression. However, the role of peptides produced by immune cells remains unknown. The present study examines the effect of acute and chronic foot-shock stress on proenkephalin peptide content in bone marrow (BMMC), thymus (TMC), and spleen (SMC) rat mononuclear cells. Proenkephalin was not processed to met-enkephalin in BMMC, while in TMC and SMC met-enkephalin represented 10% and 26% of total met-enkephalin-containing peptides, respectively. Naive rats receiving a stress stimulus showed a significant decrease of proenkephalin derived peptides in BMMC, TMC and SMC. However, in chronically stressed rats that already showed basal low peptide levels, a new stress stimulus produced a differential response in each immune tissue. That is, in BMMC peptide levels reached control rats values; in TMC remained unmodified; and in SMC, although precursors content increased, met-enkephalin levels were even lower than those observed in acutely stressed rats. Free synenkephalin content paralleled met-enkephalin changes in SMC of acutely and chronically stressed rats. The in vitro release of met-enkephalin and free synenkephalin increased in SMC of stressed rats. Met-enkephalin produced in SMC and partially processed proenkephalin peptides detected in BMMC, were only found in macrophages. However, met-enkephalin only appeared in bone marrow macrophages after at least 4 h of cell culture. Altogether, these results suggest that a stress stimulus induced proenkephalin peptide release from immune tissue macrophages. The differential response observed in chronically stressed rats suggest an alternative activation of heterogeneous proenkephalin-storing macrophage subpopulations.

Antibodies against the amino-terminal portion of pro-enkephalin inhibit DNA synthesis in human peripheral mononuclear cells.

Pro-enkephalin (PENK) mRNA and PENK-derived peptides have been reported in lymphocytes, monocytes, and macrophages. Met-enkephalin (ME) and/or synenkephalin (SYN)-containing peptides are produced and released by human peripheral blood lymphocytes (HPBL) activated with phytohemagglutinin (PHA). Furthermore, SYN (PENK 1-70) was cleaved to low-molecular-mass peptides in HPBL. In this work we studied the effect of a mouse monoclonal antibody (mAb) and a rabbit antiserum (pAb) against the C-terminal portion of SYN on DNA synthesis in PHA-activated HPBL. [3H]Thymidine incorporation into HPBL incubated with 0.1 microgram/ml of PHA was tested in the presence of different concentrations of mAb immunoglobulin (Ig) G or different dilutions of pAb. mAb induced a concentration-dependent decrease of [3H]thymidine incorporation into HPBL: 7%, 19%, 28%, and 35% of inhibition was observed with 0.1, 1, 1.5, and 2 micrograms IgG, respectively, reaching values of 65% with 10 micrograms IgG. Similarly, pAb dilutions of 1/500, 1/1000, 1/2000 and 1/4000 inhibited DNA synthesis by 63%, 61%, 43%, and 30%, respectively. The inhibitory effect of mAb and pAb was specific since it was not produced by non-immune mouse IgG or several non-immune rabbit sera and was completely reversed by 1 microM of the synthetic peptide [Tyr63](syn 63-70) synenkephalin. These results suggest that low-molecular-mass SYN-derived peptides released by PHA-activated HPBL may participate in the proliferative response of these cells. This is further evidence that the non-opioid portion of PENK--that is, SYN-derived peptides--may be involved in tissue development.