Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and fem... more Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and female rats. Whereas it was found that female rats had higher hepatic MT levels than male rats in basal, unstressed conditions, no significant differences were found in serum MT levels. Restraint stress increased both serum and liver MT in both sexes. The increase in serum MT was greater in male than in female rats, whereas no significant differences between sexes were found in liver MT content after restraint stress. It is suggested that MT regulation might be sex-dependent and that MT might play some extrahepatic function during stress.
Recent authors have hypothesized that small-scale deep convective towers possessing intense value... more Recent authors have hypothesized that small-scale deep convective towers possessing intense values of cyclonic vertical vorticity in their cores (vortical hot towers) play a critical role in tropical cyclogenesis via a two stage process: (1) preconditioning the local environment by creating small-scale potential vorticity anomalies and humidifying the lower to middle troposphere, and (2) merger, axisymmetrization and collection of these potential vorticity anomalies to generate the larger scale vortex. In this study we further investigate the role played by vortical hot towers in the upscale growth process. We simulate the evolution of Hurricane Diana in a full-physics numerical model with 1km grid spacing and compare our results to previous, coarser resolution simulations. We employ traditional weather analysis techniques and new innovative means of displaying large and complex datasets to investigate the interaction between the cloud scale features and the larger system scale environment. The results are compared to prior studies to assess if simulated vortical hot tower dynamics exhibit a significant dependence on model resolution. We find the basic physics of the vortical hot tower pathway is largely unchanged as grid-spacing decreases from 3km to 1km for simulations of Hurricane Diana. The differences between our high resolution simulation and coarser resolution simulations are mainly associated with fine scale variability. Our 1km simulation represents nearly an order of magnitude more convective towers with smaller spatial scales than what was observed in previous simulations. We find maximum updraft velocities in our 1km simulation typically between 15ms-1 and 20ms-1 with instantaneous maximum values as high as 35ms-1, though these values typically decrease during the simulation. We also find that, while the cores in the vortical hot towers are significantly moistened by the vertical transport of moisture in the updraft, the larger-scale environment actually dries significantly due to horizontal advection. Lastly, we examine a series of vortex merger events and find that merger activity is a ubiquitous and important aspect of the genesis of Hurricane Diana. Our results broadly confirm previous work using coarser numerical resolution and provide new insights into the hypothesized upscale growth process in incipient hurricanes.
Journal of Neuropathology and Experimental Neurology, Apr 1, 2003
Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillar... more Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) causes significant damage and alters the expression of many genes, including a dramatic upregulation of metallothionein-I (MT-I). The findings in this report support the idea that the upregulation of MT-I observed in GFAP-IL6 mice is an important mechanism for coping with brain damage. Thus, GFAP-IL6 mice that were crossed with TgMTI transgenic mice (GFAP-IL6xTgMTI) and overexpressed MT-I in the brain showed a decreased upregulation of cytokines such as IL-6 and a diminished recruitment and activation of macrophages and T cells throughout the CNS but mainly in the cerebellum. The GFAP-IL6 mice showed clear evidence of increased oxidative stress, which was significantly decreased by MT-I overexpression. Interestingly, MT-I overexpression increased angiogenesis in GFAP-IL6 mice but not in control littermates. Overall, the results strongly suggest that MT-I+II proteins are valuable factors that protect against cytokine-induced CNS injury.
Interleukin-6 (IL-6) is a major cytokine involved in both normal physiological brain functions an... more Interleukin-6 (IL-6) is a major cytokine involved in both normal physiological brain functions and underlying significant neuropathology. IL-6 has been suggested to play a role in the control of body weight but the results are somewhat controversial. In this study we have challenged transgenic mice with astrocytetargeted IL-6 expression (GFAP-IL6 mice) with a high-fat diet (55% kcal from fat) versus a control diet (10%). The results demonstrate that the GFAP-IL6 mice are resistant to high-fat diet-induced increases in body weight and body fat, apparently without altering food intake and with no evidences of increased sympathetic tone. The high-fat diet-induced impaired responses to an insulin tolerance test (ITT), and to an oral glucose tolerance test (OGTT) in both genotypes. The GFAP-IL6 mice did not differ from littermate wild-type (WT) mice in ITT, but they were more glucose intolerant following the high-fat diet feeding. In summary, the present results demonstrate that brain-specific IL-6 controls body weight which may be a significant factor in physiological conditions and/or in diseases causing neuroinflammation.
We show how to obtain the probability density function for the amplitude of the curvature perturb... more We show how to obtain the probability density function for the amplitude of the curvature perturbation, \mathcal {R} , produced during an epoch of slow-roll, single-field inflation, working directly from n-point correlation functions of \mathcal {R} . These n-point functions are the usual output of quantum field theory calculations, and as a result we bypass approximate statistical arguments based on the central limit theorem. Our method can be extended to deal with arbitrary forms of non-Gaussianity, appearing at any order in the n-point hierarchy. We compute the probability density for the total smoothed perturbation within a Hubble volume, epsi, and for the spectrum of epsi. When only the two-point function is retained, exact Gaussian statistics are recovered. For when the three-point function is taken into account, we compute explicitly the leading slow-roll correction to the Gaussian result.
The Journal of Comparative Neurology, Sep 20, 1999
Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essentia... more Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essential for brain development. However, because excess Zn may cause neuronal injury, cerebral mechanisms for Zn regulation must operate. The metallothionein isoforms I and II (MT I + II) are putative candidates for chelating unbound Zn released from Zn-containing nerve terminals or transported into the brain. Whether vesicular Zn and MT I + II occur in identical regions of the developing brain is unknown. Accordingly, the developmental distribution of MT I + II and vesicular Zn was mapped. By using double-labeling fluorescence histochemistry, MT I + II immunoreactivity (ir) was attributed to astrocytes and cells of myelomonocytic lineage. The cells of the myelomonocytic lineage shared the morphology of monocytes and macrophages but not of microglia and occurred primarily around vessels and ventricles in the brainstem. By contrast, astrocytes were widespread throughout the developing brain. In embryonic and neonatal brain, MT I + IIir astrocytes were almost selectively observed in the septum and fascia dentate hilus (hi) of the hippocampus. With increasing postnatal age, they also occurred in hippocampal cortex, basal forebrain, neocortex, cerebellar cortex, and cranial nerve nuclei. MT I + II mRNAs were detected in regions of the brain that also displayed MT I + IIir, indicating transcriptional events. Vesicular Zn was recorded in neonatal brain solely in the dentate hi of the hippocampus. With increasing age, the amount of vesicular Zn increased in the hippocampus and other forebrain regions. The presence of MT I + II proteins in the developing brain was confirmed by radioimmunoassay. The regional distribution of astrocytic MT I + IIir and vesicular Zn suggests that MT I + II are implicated in Zn metabolism in the developing forebrain.
Interleukin-6 (IL-6) is a multifunctional cytokine that not only plays major roles in the immune ... more Interleukin-6 (IL-6) is a multifunctional cytokine that not only plays major roles in the immune system, but also serves as a coordinator between the nervous and endocrine systems. IL-6 is produced in multiple cell types in the CNS, and in turn, many cells respond to it. It is therefore important to ascertain which cell type is the key responder to IL-6 during both physiological and pathological conditions. In order to test the role of astrocytic IL-6 in neuroinflammation, we studied an extensively-used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), in mice with an IL-6 deficiency in astrocytes (Ast-IL-6 KO). Results indicate that lack of astrocytic IL-6 did not cause major changes in EAE symptomatology. However, a delay in the onset of clinical signs was observed in Ast-IL-6 KO females, with fewer inflammatory infiltrates and decreased demyelination and some alterations in gliosis and vasogenesis, compared to floxed mice. These results suggest that astrocyte-secreted IL-6 has some roles in EAE pathogenesis, at least in females.
The aim of the present study was to test the hypothesis that peroxisome proliferator activated re... more The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor co-activator (PGC)-1 is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1 knock-out (KO) and littermate wild-type (WT) mice performed a single treadmill running exercise bout. Soleus and white gastrocnemius (WG) were obtained immediately, 2h or 6h after exercise. Another group of PGC-1 -KO and WT mice performed 5 weeks exercise training. Soleus, WG and quadriceps were obtained ~37 hours after the last training session. Resting muscles of the PGC-1 -KO mice had lower (~ 20%) cytochrome c (cyt c), cytochrome oxidase (COX)I and aminolevulinate synthase (ALAS)1 mRNA and protein levels than WT, but similar levels of AMP activated protein kinase (AMPK) 1, AMPK 2 and hexokinase (HK) II compared with WT mice. A single exercise bout increased phosphorylation of AMPK and acetyl-CoA carboxylase-and the level of HKII mRNA similarly in KO and WT. In contrast cyt c mRNA in soleus was up regulated in WT muscles only. Exercise training increased cyt c, COXI, ALAS1 and HKII mRNA and protein levels equally in WT and KO animals, but cyt c, COXI and ALAS1 expression remained ~20% lower in KO animals. In conclusion, lack of PGC-1 reduced resting expression of cyt c, COXI and ALAS1 and exercise-induced cyt c mRNA expression. However PGC-1 is not mandatory for training-induced increases in ALAS1, COXI and cyt c expression showing that other factors than PGC-1 can exert these adaptations.
is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cyto... more is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣) are considered important for induction and pathogenesis of EAE/MS disease, which is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE. However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-␣ during EAE. Zn-MT-II treatment could also significantly reduce apoptotic cell death of neurons and oligodendrocytes during EAE, as judged by using TUNEL and immunoreactivity for cytochrome c and caspases 1 and 3. In contrast, the number of apoptotic lymphocytes and macrophages was less affected by Zn-MT-II treatment. The Zn-MT-II-induced decrease in proinflammatory cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE in vivo and that MT-II might be a potentially useful factor for treatment of EAE/MS.
In addition to detrimental inflammation, widespread axon degeneration is an important feature of ... more In addition to detrimental inflammation, widespread axon degeneration is an important feature of multiple sclerosis (MS) pathology and a major correlate for permanent clinical deficits. Thus, treatments that combine immunomodulatory and neuroprotective effects are beneficial for MS. Using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG)-induced experimental autoimmune encephalomyelitis (EAE) as a model of MS, we recently showed that daily treatment with the phosphodiesterase 5 (PDE5) inhibitor sildenafil at peak disease rapidly ameliorates clinical symptoms and neuropathology . We have now investigated the immunomodulatory and neuroprotective actions of sildenafil treatment from the onset of EAE when the immune response prevails and show that early administration of the drug prevents disease progression. Ultrastructural analysis of spinal cord evidenced that sildenafil treatment preserves axons and myelin and increases the number of remyelinating axons. Immunostaining of oligodendrocytes at different stages of differentiation showed that sildenafil protects immature and mature myelinating oligodendrocytes. Brain-derived neurotrophic factor (BDNF), a recognized neuroprotectant in EAE, was up-regulated by sildenafil in immune and neural cells suggesting its implication in the beneficial effects of the drug. RNA microarray analysis of spinal cord revealed that sildenafil up-regulates YM-1, a marker of the alternative macrophage/microglial M2 phenotype that has neuroprotective and regenerative properties. Immunostaining confirmed up-regulation of YM-1 while the classical macrophage/microglial activation marker Iba-1 was down-regulated. Microarray analysis also showed a notable up-regulation of several members of the granzyme B cluster (GrBs). Immunostaining revealed expression of GrBs in Foxp3+-T regulatory cells (Tregs) suggesting a role for these proteases in sildenafil-induced suppression of T effector cells (Teffs). In vitro analysis of splenocytes from sildenafil-treated animals showed down-regulation of Th1/Th2/Th17 responses while Tregs were up-regulated. Additionally, sildenafil treatment prevented MOGspecific IgG2b accumulation in serum. Taken together these data demonstrates that daily sildenafil treatment from the initiation of EAE symptoms prevents further clinical deterioration by stimulating immunomodulatory and neuroprotective mechanisms. Importantly, we also show here that sildenafil enhances the ability of human Tregs from healthy donors to down-regulate the proliferation of Teffs in vitro, strongly supporting the potential of sildenafil for therapeutic intervention in MS.
Journal of Neuropathology and Experimental Neurology, May 1, 1999
The balance between trophic factors and inhibitory molecules is likely to determine the outcome o... more The balance between trophic factors and inhibitory molecules is likely to determine the outcome of neural tissue damage. The growth inhibitory factor (GIF), a member of the metallothionein family of proteins named metallothionein-III (MT-III), has been suggested to play an important role in tissue repair after adult brain injury. Because no information is available on this factor in relation to immature brain damage, we examined the chronological changes of GIF (MT-III) mRNA and protein following excitotoxic lesions to the postnatal day 9 brain using in situ hybridization and immunocytochemical techniques. We observed a significant decrease of neuronal GIF (MT-III) mRNA and protein levels between 4 and 24 hours postinjury and an increase in glial GIF (MT-III) levels. Double immunocytochemical techniques showed GIF (MT-III) and GFAP positive astrocytes from 2-4 hours postinjury. From 3 days postinjury strongly reactive astrocytes expressed strong levels of both GIF (MT-III) mRNA and protein, which were maintained in the glial scar formed at longer times. These results show the expression of an inhibitory molecule by postnatal reactive astrocytes. Glial GIF (MT-III) expression may play an important role in the tissue reconstruction after immature brain damage.
Background and Purpose-Exposure of animals for a few hours to moderate hypoxia confers relative p... more Background and Purpose-Exposure of animals for a few hours to moderate hypoxia confers relative protection against subsequent ischemic brain damage. This phenomenon, known as hypoxic preconditioning, depends on new RNA and protein synthesis, but its molecular mechanisms are poorly understood. Increased expression of IL-6 is evident, particularly in the lungs of animals subjected to hypoxic preconditioning. Stanniocalcin-1 (STC-1) is a 56-kDa homodimeric glycoprotein originally discovered in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. We originally reported expression of mammalian STC-1 in brain neurons and showed that STC-1 guards neurons against hypercalcemic and hypoxic damage. Methods-We treated neural Paju cells with IL-6 and measured the induction of STC-1 mRNA. In addition, we quantified the effect of hypoxic preconditioning on Stc-1 mRNA levels in brains of wild-type and IL-6 deficient mice. Furthermore, we monitored the Stc-1 response in brains of wild-type and transgenic mice, overexpressing IL-6 in the astroglia, before and after induced brain injury. Results-Hypoxic preconditioning induced an upregulated expression of Stc-1 in brains of wild-type but not of IL-6 -deficient mice. Induced brain injury elicited a stronger STC-1 response in brains of transgenic mice, with targeted astroglial IL-6 expression, than in brains of wild-type mice. Moreover, IL-6 induced STC-1 expression via MAPK signaling in neural Paju cells. Conclusion-These findings indicate that IL-6 -mediated expression of STC-1 is one molecular mechanism of hypoxic preconditioning-induced tolerance to brain ischemia.
The metallothionein (MT) gene family consists of four members (MT-I through -IV) that are tightly... more The metallothionein (MT) gene family consists of four members (MT-I through -IV) that are tightly regulated during development. Whereas MT-I and MT-II are widely expressed isoforms, MT-III has been found to be mainly expressed in the central nervous system in adult animals, and is the only isoform that inhibits survival and neurite formation of cortical neurons in vitro. A number of models of brain injury have been shown to affect MT-III mRNA levels, which has been suggested to be related to the putative neurotrophic role of this protein. However, a stress response will presumably be associated to the brain injury which could, in turn, drive MT-III regulation. In the present report the effect of a classical stress model, immobilization stress, on brain MT regulation has been studied in rats. MT-I+II protein levels were measured by radioimmunoassay in up to eight brain areas and, as expected, it was found that stress increased selectively MT-I+II levels. Adrenalectomy (ADX) had a general decreasing effect on basal MT-I+II levels; however, ADX blunted the MT-I+II response to stress in cerebellum and presumably in frontal cortex and medulla plus pons but not in the hypothalamus. MT-I mRNA measurements were in accordance with the MT-I+II protein levels in the brain areas studied. In contrast to MT-I mRNA, MT-III mRNA levels of brain cortex tended to decrease during stress, although this effect was not statistically significant. ADX also tended to decrease basal MT-III mRNA levels. Northern blot assays of pooled mRNAs suggested similar differential regulation of these two brain MT isoforms in the cerebellum. These results indicate that glucocorticoids mediate brain MT-I+II response to stress in some but not all brain areas, that a role of these hormones is likely also for MT-III, and that the regulation of MT isoforms differs substantially in the brain.
Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and fem... more Sex differences in serum and liver metallothionein (MT) levels were studied in adult male and female rats. Whereas it was found that female rats had higher hepatic MT levels than male rats in basal, unstressed conditions, no significant differences were found in serum MT levels. Restraint stress increased both serum and liver MT in both sexes. The increase in serum MT was greater in male than in female rats, whereas no significant differences between sexes were found in liver MT content after restraint stress. It is suggested that MT regulation might be sex-dependent and that MT might play some extrahepatic function during stress.
Recent authors have hypothesized that small-scale deep convective towers possessing intense value... more Recent authors have hypothesized that small-scale deep convective towers possessing intense values of cyclonic vertical vorticity in their cores (vortical hot towers) play a critical role in tropical cyclogenesis via a two stage process: (1) preconditioning the local environment by creating small-scale potential vorticity anomalies and humidifying the lower to middle troposphere, and (2) merger, axisymmetrization and collection of these potential vorticity anomalies to generate the larger scale vortex. In this study we further investigate the role played by vortical hot towers in the upscale growth process. We simulate the evolution of Hurricane Diana in a full-physics numerical model with 1km grid spacing and compare our results to previous, coarser resolution simulations. We employ traditional weather analysis techniques and new innovative means of displaying large and complex datasets to investigate the interaction between the cloud scale features and the larger system scale environment. The results are compared to prior studies to assess if simulated vortical hot tower dynamics exhibit a significant dependence on model resolution. We find the basic physics of the vortical hot tower pathway is largely unchanged as grid-spacing decreases from 3km to 1km for simulations of Hurricane Diana. The differences between our high resolution simulation and coarser resolution simulations are mainly associated with fine scale variability. Our 1km simulation represents nearly an order of magnitude more convective towers with smaller spatial scales than what was observed in previous simulations. We find maximum updraft velocities in our 1km simulation typically between 15ms-1 and 20ms-1 with instantaneous maximum values as high as 35ms-1, though these values typically decrease during the simulation. We also find that, while the cores in the vortical hot towers are significantly moistened by the vertical transport of moisture in the updraft, the larger-scale environment actually dries significantly due to horizontal advection. Lastly, we examine a series of vortex merger events and find that merger activity is a ubiquitous and important aspect of the genesis of Hurricane Diana. Our results broadly confirm previous work using coarser numerical resolution and provide new insights into the hypothesized upscale growth process in incipient hurricanes.
Journal of Neuropathology and Experimental Neurology, Apr 1, 2003
Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillar... more Transgenic expression of interleukin-6 (IL-6) in the CNS under the control of the glial fibrillary acidic protein (GFAP) gene promoter (GFAP-IL6 mice) causes significant damage and alters the expression of many genes, including a dramatic upregulation of metallothionein-I (MT-I). The findings in this report support the idea that the upregulation of MT-I observed in GFAP-IL6 mice is an important mechanism for coping with brain damage. Thus, GFAP-IL6 mice that were crossed with TgMTI transgenic mice (GFAP-IL6xTgMTI) and overexpressed MT-I in the brain showed a decreased upregulation of cytokines such as IL-6 and a diminished recruitment and activation of macrophages and T cells throughout the CNS but mainly in the cerebellum. The GFAP-IL6 mice showed clear evidence of increased oxidative stress, which was significantly decreased by MT-I overexpression. Interestingly, MT-I overexpression increased angiogenesis in GFAP-IL6 mice but not in control littermates. Overall, the results strongly suggest that MT-I+II proteins are valuable factors that protect against cytokine-induced CNS injury.
Interleukin-6 (IL-6) is a major cytokine involved in both normal physiological brain functions an... more Interleukin-6 (IL-6) is a major cytokine involved in both normal physiological brain functions and underlying significant neuropathology. IL-6 has been suggested to play a role in the control of body weight but the results are somewhat controversial. In this study we have challenged transgenic mice with astrocytetargeted IL-6 expression (GFAP-IL6 mice) with a high-fat diet (55% kcal from fat) versus a control diet (10%). The results demonstrate that the GFAP-IL6 mice are resistant to high-fat diet-induced increases in body weight and body fat, apparently without altering food intake and with no evidences of increased sympathetic tone. The high-fat diet-induced impaired responses to an insulin tolerance test (ITT), and to an oral glucose tolerance test (OGTT) in both genotypes. The GFAP-IL6 mice did not differ from littermate wild-type (WT) mice in ITT, but they were more glucose intolerant following the high-fat diet feeding. In summary, the present results demonstrate that brain-specific IL-6 controls body weight which may be a significant factor in physiological conditions and/or in diseases causing neuroinflammation.
We show how to obtain the probability density function for the amplitude of the curvature perturb... more We show how to obtain the probability density function for the amplitude of the curvature perturbation, \mathcal {R} , produced during an epoch of slow-roll, single-field inflation, working directly from n-point correlation functions of \mathcal {R} . These n-point functions are the usual output of quantum field theory calculations, and as a result we bypass approximate statistical arguments based on the central limit theorem. Our method can be extended to deal with arbitrary forms of non-Gaussianity, appearing at any order in the n-point hierarchy. We compute the probability density for the total smoothed perturbation within a Hubble volume, epsi, and for the spectrum of epsi. When only the two-point function is retained, exact Gaussian statistics are recovered. For when the three-point function is taken into account, we compute explicitly the leading slow-roll correction to the Gaussian result.
The Journal of Comparative Neurology, Sep 20, 1999
Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essentia... more Because zinc (Zn) is a co-factor in enzymes and participates in neurotransmission, it is essential for brain development. However, because excess Zn may cause neuronal injury, cerebral mechanisms for Zn regulation must operate. The metallothionein isoforms I and II (MT I + II) are putative candidates for chelating unbound Zn released from Zn-containing nerve terminals or transported into the brain. Whether vesicular Zn and MT I + II occur in identical regions of the developing brain is unknown. Accordingly, the developmental distribution of MT I + II and vesicular Zn was mapped. By using double-labeling fluorescence histochemistry, MT I + II immunoreactivity (ir) was attributed to astrocytes and cells of myelomonocytic lineage. The cells of the myelomonocytic lineage shared the morphology of monocytes and macrophages but not of microglia and occurred primarily around vessels and ventricles in the brainstem. By contrast, astrocytes were widespread throughout the developing brain. In embryonic and neonatal brain, MT I + IIir astrocytes were almost selectively observed in the septum and fascia dentate hilus (hi) of the hippocampus. With increasing postnatal age, they also occurred in hippocampal cortex, basal forebrain, neocortex, cerebellar cortex, and cranial nerve nuclei. MT I + II mRNAs were detected in regions of the brain that also displayed MT I + IIir, indicating transcriptional events. Vesicular Zn was recorded in neonatal brain solely in the dentate hi of the hippocampus. With increasing age, the amount of vesicular Zn increased in the hippocampus and other forebrain regions. The presence of MT I + II proteins in the developing brain was confirmed by radioimmunoassay. The regional distribution of astrocytic MT I + IIir and vesicular Zn suggests that MT I + II are implicated in Zn metabolism in the developing forebrain.
Interleukin-6 (IL-6) is a multifunctional cytokine that not only plays major roles in the immune ... more Interleukin-6 (IL-6) is a multifunctional cytokine that not only plays major roles in the immune system, but also serves as a coordinator between the nervous and endocrine systems. IL-6 is produced in multiple cell types in the CNS, and in turn, many cells respond to it. It is therefore important to ascertain which cell type is the key responder to IL-6 during both physiological and pathological conditions. In order to test the role of astrocytic IL-6 in neuroinflammation, we studied an extensively-used animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), in mice with an IL-6 deficiency in astrocytes (Ast-IL-6 KO). Results indicate that lack of astrocytic IL-6 did not cause major changes in EAE symptomatology. However, a delay in the onset of clinical signs was observed in Ast-IL-6 KO females, with fewer inflammatory infiltrates and decreased demyelination and some alterations in gliosis and vasogenesis, compared to floxed mice. These results suggest that astrocyte-secreted IL-6 has some roles in EAE pathogenesis, at least in females.
The aim of the present study was to test the hypothesis that peroxisome proliferator activated re... more The aim of the present study was to test the hypothesis that peroxisome proliferator activated receptor co-activator (PGC)-1 is required for exercise-induced adaptive gene responses in skeletal muscle. Whole body PGC-1 knock-out (KO) and littermate wild-type (WT) mice performed a single treadmill running exercise bout. Soleus and white gastrocnemius (WG) were obtained immediately, 2h or 6h after exercise. Another group of PGC-1 -KO and WT mice performed 5 weeks exercise training. Soleus, WG and quadriceps were obtained ~37 hours after the last training session. Resting muscles of the PGC-1 -KO mice had lower (~ 20%) cytochrome c (cyt c), cytochrome oxidase (COX)I and aminolevulinate synthase (ALAS)1 mRNA and protein levels than WT, but similar levels of AMP activated protein kinase (AMPK) 1, AMPK 2 and hexokinase (HK) II compared with WT mice. A single exercise bout increased phosphorylation of AMPK and acetyl-CoA carboxylase-and the level of HKII mRNA similarly in KO and WT. In contrast cyt c mRNA in soleus was up regulated in WT muscles only. Exercise training increased cyt c, COXI, ALAS1 and HKII mRNA and protein levels equally in WT and KO animals, but cyt c, COXI and ALAS1 expression remained ~20% lower in KO animals. In conclusion, lack of PGC-1 reduced resting expression of cyt c, COXI and ALAS1 and exercise-induced cyt c mRNA expression. However PGC-1 is not mandatory for training-induced increases in ALAS1, COXI and cyt c expression showing that other factors than PGC-1 can exert these adaptations.
is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cyto... more is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-␣ (TNF-␣) are considered important for induction and pathogenesis of EAE/MS disease, which is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE. However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-␣ during EAE. Zn-MT-II treatment could also significantly reduce apoptotic cell death of neurons and oligodendrocytes during EAE, as judged by using TUNEL and immunoreactivity for cytochrome c and caspases 1 and 3. In contrast, the number of apoptotic lymphocytes and macrophages was less affected by Zn-MT-II treatment. The Zn-MT-II-induced decrease in proinflammatory cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE in vivo and that MT-II might be a potentially useful factor for treatment of EAE/MS.
In addition to detrimental inflammation, widespread axon degeneration is an important feature of ... more In addition to detrimental inflammation, widespread axon degeneration is an important feature of multiple sclerosis (MS) pathology and a major correlate for permanent clinical deficits. Thus, treatments that combine immunomodulatory and neuroprotective effects are beneficial for MS. Using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG)-induced experimental autoimmune encephalomyelitis (EAE) as a model of MS, we recently showed that daily treatment with the phosphodiesterase 5 (PDE5) inhibitor sildenafil at peak disease rapidly ameliorates clinical symptoms and neuropathology . We have now investigated the immunomodulatory and neuroprotective actions of sildenafil treatment from the onset of EAE when the immune response prevails and show that early administration of the drug prevents disease progression. Ultrastructural analysis of spinal cord evidenced that sildenafil treatment preserves axons and myelin and increases the number of remyelinating axons. Immunostaining of oligodendrocytes at different stages of differentiation showed that sildenafil protects immature and mature myelinating oligodendrocytes. Brain-derived neurotrophic factor (BDNF), a recognized neuroprotectant in EAE, was up-regulated by sildenafil in immune and neural cells suggesting its implication in the beneficial effects of the drug. RNA microarray analysis of spinal cord revealed that sildenafil up-regulates YM-1, a marker of the alternative macrophage/microglial M2 phenotype that has neuroprotective and regenerative properties. Immunostaining confirmed up-regulation of YM-1 while the classical macrophage/microglial activation marker Iba-1 was down-regulated. Microarray analysis also showed a notable up-regulation of several members of the granzyme B cluster (GrBs). Immunostaining revealed expression of GrBs in Foxp3+-T regulatory cells (Tregs) suggesting a role for these proteases in sildenafil-induced suppression of T effector cells (Teffs). In vitro analysis of splenocytes from sildenafil-treated animals showed down-regulation of Th1/Th2/Th17 responses while Tregs were up-regulated. Additionally, sildenafil treatment prevented MOGspecific IgG2b accumulation in serum. Taken together these data demonstrates that daily sildenafil treatment from the initiation of EAE symptoms prevents further clinical deterioration by stimulating immunomodulatory and neuroprotective mechanisms. Importantly, we also show here that sildenafil enhances the ability of human Tregs from healthy donors to down-regulate the proliferation of Teffs in vitro, strongly supporting the potential of sildenafil for therapeutic intervention in MS.
Journal of Neuropathology and Experimental Neurology, May 1, 1999
The balance between trophic factors and inhibitory molecules is likely to determine the outcome o... more The balance between trophic factors and inhibitory molecules is likely to determine the outcome of neural tissue damage. The growth inhibitory factor (GIF), a member of the metallothionein family of proteins named metallothionein-III (MT-III), has been suggested to play an important role in tissue repair after adult brain injury. Because no information is available on this factor in relation to immature brain damage, we examined the chronological changes of GIF (MT-III) mRNA and protein following excitotoxic lesions to the postnatal day 9 brain using in situ hybridization and immunocytochemical techniques. We observed a significant decrease of neuronal GIF (MT-III) mRNA and protein levels between 4 and 24 hours postinjury and an increase in glial GIF (MT-III) levels. Double immunocytochemical techniques showed GIF (MT-III) and GFAP positive astrocytes from 2-4 hours postinjury. From 3 days postinjury strongly reactive astrocytes expressed strong levels of both GIF (MT-III) mRNA and protein, which were maintained in the glial scar formed at longer times. These results show the expression of an inhibitory molecule by postnatal reactive astrocytes. Glial GIF (MT-III) expression may play an important role in the tissue reconstruction after immature brain damage.
Background and Purpose-Exposure of animals for a few hours to moderate hypoxia confers relative p... more Background and Purpose-Exposure of animals for a few hours to moderate hypoxia confers relative protection against subsequent ischemic brain damage. This phenomenon, known as hypoxic preconditioning, depends on new RNA and protein synthesis, but its molecular mechanisms are poorly understood. Increased expression of IL-6 is evident, particularly in the lungs of animals subjected to hypoxic preconditioning. Stanniocalcin-1 (STC-1) is a 56-kDa homodimeric glycoprotein originally discovered in bony fish, where it regulates calcium/phosphate homeostasis and protects against toxic hypercalcemia. We originally reported expression of mammalian STC-1 in brain neurons and showed that STC-1 guards neurons against hypercalcemic and hypoxic damage. Methods-We treated neural Paju cells with IL-6 and measured the induction of STC-1 mRNA. In addition, we quantified the effect of hypoxic preconditioning on Stc-1 mRNA levels in brains of wild-type and IL-6 deficient mice. Furthermore, we monitored the Stc-1 response in brains of wild-type and transgenic mice, overexpressing IL-6 in the astroglia, before and after induced brain injury. Results-Hypoxic preconditioning induced an upregulated expression of Stc-1 in brains of wild-type but not of IL-6 -deficient mice. Induced brain injury elicited a stronger STC-1 response in brains of transgenic mice, with targeted astroglial IL-6 expression, than in brains of wild-type mice. Moreover, IL-6 induced STC-1 expression via MAPK signaling in neural Paju cells. Conclusion-These findings indicate that IL-6 -mediated expression of STC-1 is one molecular mechanism of hypoxic preconditioning-induced tolerance to brain ischemia.
The metallothionein (MT) gene family consists of four members (MT-I through -IV) that are tightly... more The metallothionein (MT) gene family consists of four members (MT-I through -IV) that are tightly regulated during development. Whereas MT-I and MT-II are widely expressed isoforms, MT-III has been found to be mainly expressed in the central nervous system in adult animals, and is the only isoform that inhibits survival and neurite formation of cortical neurons in vitro. A number of models of brain injury have been shown to affect MT-III mRNA levels, which has been suggested to be related to the putative neurotrophic role of this protein. However, a stress response will presumably be associated to the brain injury which could, in turn, drive MT-III regulation. In the present report the effect of a classical stress model, immobilization stress, on brain MT regulation has been studied in rats. MT-I+II protein levels were measured by radioimmunoassay in up to eight brain areas and, as expected, it was found that stress increased selectively MT-I+II levels. Adrenalectomy (ADX) had a general decreasing effect on basal MT-I+II levels; however, ADX blunted the MT-I+II response to stress in cerebellum and presumably in frontal cortex and medulla plus pons but not in the hypothalamus. MT-I mRNA measurements were in accordance with the MT-I+II protein levels in the brain areas studied. In contrast to MT-I mRNA, MT-III mRNA levels of brain cortex tended to decrease during stress, although this effect was not statistically significant. ADX also tended to decrease basal MT-III mRNA levels. Northern blot assays of pooled mRNAs suggested similar differential regulation of these two brain MT isoforms in the cerebellum. These results indicate that glucocorticoids mediate brain MT-I+II response to stress in some but not all brain areas, that a role of these hormones is likely also for MT-III, and that the regulation of MT isoforms differs substantially in the brain.
Uploads
Papers by Juan Hidalgo