Testosterone Reduces Myelin Abnormalities in the Wobbler Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1+ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1+ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR1 and P2Y12R) vs. controls or WR + T. IBA1+ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors' mRNA remained low. A reduction of GS+ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation.

Effects of the mineralocorticoid receptor antagonist eplerenone in experimental autoimmune encephalomyelitis.

There is growing evidence indicating that mineralocorticoid receptor (MR) expression influences a wide variety of functions in metabolic and immune response. The present study explored if antagonism of the MR reduces neuroinflammation in the spinal cord of mice with experimental autoimmune encephalomyelitis (EAE). Eplerenone (EPLE) (100 mg/kg dissolved in 30% 2-hydroxypropyl-ß-cyclodextrin) was administered intraperitoneally (i.p.) daily from EAE induction (day 0) until sacrificed on day 17 post-induction. The MR blocker (a) significantly decreased the inflammatory parameters TLR4, MYD88, IL-1ß, and iNOS mRNAs; (b) attenuated HMGB1, NLRP3, TGF-ß mRNAs, microglia, and aquaporin4 immunoreaction without modifying GFAP. Serum IL-1ß was also decreased in the EAE+EPLE group. Moreover, EPLE treatment prevented demyelination and improved clinical signs of EAE mice. Interestingly, MR was decreased and GR remained unchanged in EAE mice while EPLE treatment restored MR expression, suggesting that a dysbalanced MR/GR was associated with the development of neuroinflammation. Our results indicated that MR blockage with EPLE attenuated inflammation-related spinal cord pathology in the EAE mouse model of Multiple Sclerosis, supporting a novel therapeutic approach for immune-related diseases.

Beneficial effects of the phytoestrogen genistein on hippocampal impairments of spontaneously hypertensive rats (SHR).

Hippocampal neuropathology is a recognized feature of the spontaneously hypertensive rat (SHR). The hippocampal alterations associate with cognitive impairment. We have shown that hippocampal abnormalities are reversed by 17ß-estradiol, a steroid binding to intracellular receptors (estrogen receptor α and ß subtypes) or the membrane-located G-protein coupled estradiol receptor. Genistein (GEN) is a neuroprotective phytoestrogen which binds to estrogen receptor ß and G-protein coupled estradiol receptor. Here, we investigated whether GEN neuroprotection extends to SHR. For this purpose, we treated 5-month-old SHR for 2 weeks with 10 mg kg-1 daily s.c injections of GEN. We analyzed the expression of doublecortin+ neuronal progenitors, glial fibrillary acidic protein+ astrocytes and ionized calcium-binding adapter molecule 1+ microglia in the CA1 region and dentate gyrus of the hippocampus using immunocytochemistry, whereas a quantitative real-time polymerase chain reaction was used to measure the expression of pro- and anti-inflammatory factors tumor necrosis factor α, cyclooxygenase-2 and transforming growth factor ß. We also evaluated hippocampal dependent memory using the novel object recognition test. The results showed a decreased number of doublecortin+ neural progenitors in the dentate gyrus of SHR that was reversed with GEN. The number of glial fibrillary acidic protein+ astrocytes in the dentate gyrus and CA1 was increased in SHR but significantly decreased by GEN treatment. Additionally, GEN shifted microglial morphology from the predominantly activated phenotype present in SHR, to the more surveillance phenotype found in normotensive rats. Furthermore, treatment with GEN decreased the mRNA of the pro-inflammatory factors tumor necrosis factor α and cyclooxygenase-2 and increased the mRNA of the anti-inflammatory factor transforming growth factor ß. Discrimination index in the novel object recognition test was decreased in SHR and treatment with GEN increased this parameter. Our results indicate important neuroprotective effects of GEN at the neurochemical and behavioral level in SHR. Our data open an interesting possibility for proposing this phytoestrogen as an alternative therapy in hypertensive encephalopathy.

Tibolone restrains neuroinflammation in mouse experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an immune-mediated and degenerating disease in which myelin sheaths are damaged as a result of chronic progressive inflammation of the central nervous system. Tibolone [(7α,17α)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-in-3-one], a synthetic estrogenic compound with tissue-specific actions and used for menopausal hormone therapy, shows neuroprotective and antioxidant properties both in vivo and in vitro. In the present study, we analyzed whether tibolone plays a therapeutic role in experimental autoimmune encephalomyelitis (EAE) mice, a commonly used model of MS. Female C57BL/6 mice were induced with the myelin oligodendrocyte glycoprotein MOG35-55 and received s.c. tibolone (0.08 mg kg-1 ) injection every other day from the day of induction until death on the acute phase of the disease. Reactive gliosis, Toll like receptor 4 (TLR4), high mobility group box protein 1 (HMGB1), inflammasome parameters, activated Akt levels and myelin were assessed by a real-time polymerase chain reaction, immunohistochemistry, and western blot analysis. Our findings indicated that, in the EAE spinal cord, tibolone reversed the astrocytic and microglial reaction, and reduced the hyperexpression of TLR4 and HMGB1, as well as NLR family pyrin domain containing 3-caspase 1-interleukin-1ß inflammasome activation. At the same time, tibolone attenuated the Akt/nuclear factor kappa B pathway and limited the white matter demyelination area. Estrogen receptor expression was unaltered with tibolone treatment. Clinically, tibolone improved neurological symptoms without uterine compromise. Overall, our data suggest that tibolone may serve as a promising agent for the attenuation of MS-related inflammation.

Developmental expression of genes involved in progesterone synthesis, metabolism and action during the post-natal cerebellar myelination.

Progesterone is involved in dendritogenesis, synaptogenesis and maturation of cerebellar Purkinge cells, major sites of steroid synthesis in the brain. To study a possible time-relationship between myelination, neurosteroidogenesis and steroid receptors during development of the postnatal mouse cerebellum, we determined at postnatal days 5 (P5),18 (P18) and 35 (P35) the expression of myelin basic protein (MBP), components of the steroidogenic pathway, levels of endogenous steroids and progesterone's classical and non-classical receptors. In parallel with myelin increased expression during development, P18 and P35 mice showed higher levels of cerebellar progesterone and its reduced derivatives, higher expression of steroidogenic acute regulatory protein (StAR) mRNA, cholesterol side chain cleavage enzyme (P450scc) and 5α-reductase mRNA vs. P5 mice. Other steroids such as corticosterone and its reduced derivatives and 3ß-androstanodiol (ADIOL) showed a peak increase at P18 compared to P5. Progesterone membrane receptors and binding proteins (PGRMC1, mPRα, mPRß, mPRγ, and Sigma1 receptors) mRNAs levels increased during development while that of classical progesterone receptors (PR) remained invariable. PRKO mice showed similar MBP levels than wild type. Thus, these data suggests that progesterone and its neuroactive metabolites may play a role in postnatal cerebellar myelination.

Long-term effects of the glucocorticoid receptor modulator CORT113176 in murine motoneuron degeneration.

The Wobbler mouse spinal cord shows vacuolated motoneurons, glial reaction, inflammation and abnormal glutamatergic parameters. Wobblers also show deficits of motor performance. These conditions resemble amyotrophic lateral sclerosis (ALS). Wobbler mice also show high levels of corticosterone in blood, adrenals and brain plus adrenal hypertrophy, suggesting that chronically elevated glucocorticoids prime spinal cord neuroinflammation. Therefore, we analyzed if treatment of Wobbler mice with the glucocorticoid receptor (GR) antagonist CORT113176 mitigated the mentioned abnormalities. 30 mg/kg CORT113176 given daily for 3 weeks reduced motoneuron vacuolation, decreased astro and microgliosis, lowered the inflammatory mediators high mobility group box 1 protein (HMGB1), toll-like receptor 4, myeloid differentiation primary response 88 (MyD88), p50 subunit of nuclear factor kappa B (NFκB), tumor necrosis factor (TNF) receptor, and interleukin 18 (IL18) compared to untreated Wobblers. CORT113176 increased the survival signal pAKT (serine-threonine kinase) and decreased the death signal phosphorylated Junk-N-terminal kinase (pJNK), symptomatic of antiapoptosis. There was a moderate positive effect on glutamine synthase and astrocyte glutamate transporters, suggesting decreased glutamate excitotoxicity. In this pre-clinical study, Wobblers receiving CORT113176 showed enhanced resistance to fatigue in the rota rod test and lower forelimb atrophy at weeks 2-3. Therefore, long-term treatment with CORT113176 attenuated degeneration and inflammation, increased motor performance and decreased paw deformity. Antagonism of the GR may be of potential therapeutic value for neurodegenerative diseases.

Comparative effects of progesterone and the synthetic progestin norethindrone on neuroprotection in a model of spontaneous motoneuron degeneration.

The Wobbler mouse has been proposed as an experimental model of the sporadic form of amyotrophic lateral sclerosis (ALS). The administration of natural progesterone (PROG) to Wobbler mice attenuates neuropathology, inhibits oxidative stress, enhances the expression of genes involved in motoneuron function, increases survival and restores axonal transport. However, current pharmacological treatments for ALS patients are still partially effective. This encouraged us to investigate if the synthetic progestin norethindrone (NOR), showing higher potency than PROG and used for birth control and hormone therapy might also afford neuroprotection. Two-month-old Wobbler mice (wr/wr) were left untreated or received either a 20 mg pellet of PROG or a 1 mg pellet of NOR for 18 days. Untreated control NFR/NFR mice (background strain for Wobbler) were also employed. Wobblers showed typical clinical and spinal cord abnormalities, while these abnormalities were normalized with PROG treatment. Surprisingly, we found that NOR did not increase immunoreactivity and gene expression for choline-acetyltransferase, drastically decreased GFAP + astrogliosis, favored proinflammatory mediators, promoted the inflammatory phenotype of IBA1+ microglia, increased the receptor for advanced glycation end products (RAGE) mRNA and protein expression and the activity of nitric oxide synthase (NOS)/NADPH diaphorase in the cervical spinal cord. Additionally, NOR treatment produced atrophy of the thymus. The combined negative effects of NOR on clinical assessments (forelimb atrophy and rotarod performance) suggest a detrimental effect on muscle trophism and motor function. These findings reinforce the evidence that the type of progestin used for contraception, endometriosis or replacement therapy, may condition the outcome of preclinical and clinical studies targeting neurodegenerative diseases.

A functional progesterone receptor is required for immunomodulation, reduction of reactive gliosis and survival of oligodendrocyte precursors in the injured spinal cord.

The anti-inflammatory effects of progesterone have been increasingly recognized in several neuropathological models, including spinal cord inflammation. In the present investigation, we explored the regulation of proinflammatory factors and enzymes by progesterone at several time points after spinal cord injury (SCI) in male rats. We also demonstrated the role of the progesterone receptor (PR) in inhibiting inflammation and reactive gliosis, and in enhancing the survival of oligodendrocyte progenitors cells (OPC) in injured PR knockout (PRKO) mice receiving progesterone. First, after SCI in rats, progesterone greatly attenuated the injury-induced hyperexpression of the mRNAs of interleukin 1ß (IL1ß), IL6, tumor necrosis factor alpha (TNFα), inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), all involved in oligodendrocyte damage. Second, the role of the PR was investigated in PRKO mice after SCI, in which progesterone failed to reduce the high expression of IL1ß, IL6, TNFα and IκB-α mRNAs, the latter being considered an index of reduced NF-κB transactivation. These effects occurred in a time framework coincident with a reduction in the astrocyte and microglial responses. In contrast to wild-type mice, progesterone did not increase the density of OPC and did not prevent apoptotic death of these cells in PRKO mice. Our results support a role of PR in: (a) the anti-inflammatory effects of progesterone; (b) the modulation of astrocyte and microglial responses and (c) the prevention of OPC apoptosis, a mechanism that would enhance the commitment of progenitors to the remyelination pathway in the injured spinal cord.

Estrogens are neuroprotective factors for hypertensive encephalopathy.

Estrogens are neuroprotective factors for brain diseases, including hypertensive encephalopathy. In particular, the hippocampus is highly damaged by high blood pressure, with several hippocampus functions being altered in humans and animal models of hypertension. Working with a genetic model of primary hypertension, the spontaneously hypertensive rat (SHR), we have shown that SHR present decreased dentate gyrus neurogenesis, astrogliosis, low expression of brain derived neurotrophic factor (BDNF), decreased number of neurons in the hilus of the dentate gyrus, increased basal levels of the estrogen-synthesizing enzyme aromatase, and atrophic dendritic arbor with low spine density in the CA1 region compared to normotensive Wistar Kyoto (WKY) ratsl. Changes also occur in the hypothalamus of SHR, with increased expression of the hypertensinogenic peptide arginine vasopressin (AVP) and its V1b receptor. Following chronic estradiol treatment, SHR show decreased blood pressure, enhanced hippocampus neurogenesis, decreased the reactive astrogliosis, increased BDNF mRNA and protein expression in the dentate gyrus, increased neuronal number in the hilus of the dentate gyrus, further increased the hyperexpression of aromatase and replaced spine number with remodeling of the dendritic arbor of the CA1 region. We have detected by qPCR the estradiol receptors ERα and ERß in hippocampus from both SHR and WKY rats, suggesting direct effects of estradiol on brain cells. We hypothesize that a combination of exogenously given estrogens plus those locally synthesized by estradiol-stimulated aromatase may better alleviate the hippocampal and hypothalamic encephalopathy of SHR. This article is part of a Special Issue entitled "Sex steroids and brain disorders".

Progesterone prevents nerve injury-induced allodynia and spinal NMDA receptor upregulation in rats.

BACKGROUND: Peripheral nerve injury-evoked neuropathic pain still remains a therapeutic challenge. Recent studies support the notion that progesterone, a neuroactive steroid, may offer a promising perspective in pain modulation. OBJECTIVES: Evaluate the effect of progesterone administration on the development of neuropathic pain-associated allodynia and on the spinal expression of N-Methyl-D-Aspartate Receptor subunit 1 (NR1), its phosphorylated form (pNR1), and the gamma isoform of protein kinase C (PKCγ), all key players in the process of central sensitization, in animals subjected to a sciatic nerve constriction. METHODS: Male Sprague-Dawley rats were subjected to a sciatic nerve single ligature constriction and treated with daily subcutaneous injections of progesterone (16 mg/kg) or vehicle. The development of hindpaw mechanical and thermal allodynia was assessed using the von Frey and Choi tests, respectively. Twenty two days after injury, the number of neuronal profiles exhibiting NR1, pNR1, or PKCγ immunoreactivity was determined in the dorsal horn of the lumbar spinal cord. RESULTS: Injured animals receiving progesterone did not develop mechanical allodynia and showed a significantly lower number of painful responses to cold stimulation. In correlation with the observed attenuation of pain behaviors, progesterone administration significantly reduced the number of NR1, pNR1, and PKCγ immunoreactive neuronal profiles. CONCLUSIONS: Our results show that progesterone prevents allodynia in a rat model of sciatic nerve constriction and reinforce its role as a potential treatment for neuropathic pain.

Progesterone attenuates astro- and microgliosis and enhances oligodendrocyte differentiation following spinal cord injury.

Reactive gliosis, demyelination and proliferation of NG2+ oligodendrocyte precursor cells (OPC) are common responses to spinal cord injury (SCI). We previously reported that short-term progesterone treatment stimulates OPC proliferation whereas chronic treatment enhances OPC differentiation after SCI. Presently, we further studied the proliferation/differentiation of glial cells involved in inflammation and remyelination in male rats with SCI subjected to acute (3 days) or chronic (21 days) progesterone administration. Rats received several pulses of bromodeoyuridine (BrdU) 48 and 72 h post-SCI, and sacrificed 3 or 21 days post-SCI. Double colocalization of BrdU and specific cell markers showed that 3 days of SCI induced a strong proliferation of S100ß+ astrocytes, OX-42+ microglia/macrophages and NG2+ cells. At this stage, the intense GFAP+ astrogliosis was BrdU negative. Twenty one days of SCI enhanced maturation of S100ß+ cells into GFAP+ astrocytes, but decreased the number of CC1+ oligodendrocytes. Progesterone treatment inhibited astrocyte and microglia /macrophage proliferation and activation in the 3-day SCI group, and inhibited activation in the 21-day SCI group. BrdU/NG2 double labeled cells were increased by progesterone at 3 days, indicating a proliferation stimulus, but decreased them at 21 days. However, progesterone-enhancement of CC1+/BrdU+ oligodendrocyte density, suggest differentiation of OPC into mature oligondendrocytes. We conclude that progesterone effects after SCI involves: a) inhibition of astrocyte proliferation and activation; b) anti-inflammatory effects by preventing microglial activation and proliferation, and c) early proliferation of NG2+ progenitors and late remyelination. Thus, progesterone behaves as a glioactive factor favoring remyelination and inhibiting reactive gliosis.

Short-term environmental enrichment enhances adult neurogenesis, vascular network and dendritic complexity in the hippocampus of type 1 diabetic mice.

BACKGROUND: Several brain disturbances have been described in association to type 1 diabetes in humans. In animal models, hippocampal pathological changes were reported together with cognitive deficits. The exposure to a variety of environmental stimuli during a certain period of time is able to prevent brain alterations and to improve learning and memory in conditions like stress, aging and neurodegenerative processes. METHODOLOGY/PRINCIPAL FINDINGS: We explored the modulation of hippocampal alterations in streptozotocin-induced type 1 diabetic mice by environmental enrichment. In diabetic mice housed in standard conditions we found a reduction of adult neurogenesis in the dentate gyrus, decreased dendritic complexity in CA1 neurons and a smaller vascular fractional area in the dentate gyrus, compared with control animals in the same housing condition. A short exposure -10 days- to an enriched environment was able to enhance proliferation, survival and dendritic arborization of newborn neurons, to recover dendritic tree length and spine density of pyramidal CA1 neurons and to increase the vascular network of the dentate gyrus in diabetic animals. CONCLUSIONS/SIGNIFICANCE: The environmental complexity seems to constitute a strong stimulator competent to rescue the diabetic brain from neurodegenerative progression.

Hippocampal neurovascular and hypothalamic-pituitary-adrenal axis alterations in spontaneously type 2 diabetic GK rats.

Metabolic and vascular consequences of diabetes mellitus induce several CNS complications. The dentate gyrus of the hippocampus, a well-recognized target for diabetic alterations, is a neurogenic area associated with memory and learning processes. Here, we explored the hippocampal neurogenesis and its microenvironment (astrocytes, vascularisation and glucocorticoid influence) in a spontaneous model of type 2 diabetes, the Goto-Kakizaki rat. The number of proliferative Ki67(+) cells and young doublecortin(+) neurons was 2-fold higher in the hippocampus from diabetic rats than in normoglycemic control Wistar at 4 months of age. However, there was no difference in cell survival, studied 3 weeks after bromodeoxyuridine administration. Labeling of endothelial cells against von Willebrand factor, demonstrated a 50% decrease in the granular cell layer fractional area covered by blood vessels and a diminished capillary branching in diabetic rats. Finally, Goto-Kakizaki rats exhibited decreased glucocorticoid receptor immunolabeling in CA1, associated with higher corticosteronemia. In conclusion, diabetic rats showed increased cell proliferation and neuronal differentiation without concomitant survival modification. A high proliferation rate, potentially reflecting a compensatory mechanism for neuronal suffering, also exists in various pathological situations. However, endothelial alteration induced by chronic hyperglycemia, hyperleptinemia and insulin resistance and associated with deleterious glucocorticoid effects might impair effective neurogenesis in diabetic Goto-Kakizaki rats.

Stage dependent effects of progesterone on motoneurons and glial cells of wobbler mouse spinal cord degeneration.

In the Wobbler mouse, a mutation in the Vps54 gene is accompanied by motoneuron degeneration and astrogliosis in the cervical spinal cord. Previous work has shown that these abnormalities are greatly attenuated by progesterone treatment of clinically afflicted Wobblers. However, whether progesterone is effective at all disease stages has not yet been tested. The present work used genotyped (wr/wr) Wobbler mice at three periods of the disease: early progressive (1-2 months), established (5-8 months) or late stages (12 months) and age-matched wildtype controls (NFR/NFR), half of which were implanted with a progesterone pellet (20 mg) for 18 days. In untreated Wobblers, degenerating vacuolated motoneurons were initially abundant, experienced a slight reduction at the established stage and dramatically diminished during the late period. In motoneurons, the cholinergic marker choline acetyltransferase (ChAT) was reduced at all stages of the Wobbler disease, whereas hyperexpression of the growth-associated protein (GAP43) mRNA preferentially occurred at the early progressive and established stages. Progesterone therapy significantly reduced motoneuron vacuolation, enhanced ChAT immunoreactive perikarya and reduced the hyperexpression of GAP43 during the early progressive and established stages. At all stage periods, untreated Wobblers showed high density of glial fibrillary acidic protein (GFAP)+ astrocytes and decreased number of glutamine synthase (GS) immunostained cells. Progesterone treatment down-regulated GFAP+ astrocytes and up-regulated GS+ cell number. These data reinforced the usefulness of progesterone to improve motoneuron and glial cell abnormalities of Wobbler mice and further showed that therapeutic benefit seems more effective at the early progressive and established periods, rather than on advance stages of spinal cord neurodegeneration.

Pharmacotherapy with 17ß-estradiol and progesterone prevents development of mouse experimental autoimmune encephalomyelitis.

BACKGROUND: Pregnant women with multiple sclerosis (MS) show disease remission in the third trimester concomitant with high circulating levels of sex steroids. Rodent experimental autoimmune encephalomyelitis (EAE) is an accepted model for MS. Previous studies have shown that monotherapy with estrogens or progesterone exert beneficial effects on EAE. The aim of the present study was to determine if estrogen and progesterone cotherapy of C57BL/6 female mice provided substantial protection from EAE. METHODS: A group of mice received single pellets of progesterone (100 mg) and 17 ß-estradiol (2.5 mg) subcutaneously 1 week before EAE induction, whereas another group were untreated before EAE induction. On day 16 we compared the two EAE groups and control mice in terms of clinical scores, spinal cord demyelination, expression of myelin basic protein and proteolipid protein, macrophage cell infiltration, neuronal expression of brain-derived neurotrophic factor mRNA and protein, and the number of glial fribrillary acidic protein (GFAP)-immunopositive astrocytes. RESULTS: Clinical signs of EAE were substantially attenuated by estrogen and progesterone treatment. Steroid cotherapy prevented spinal cord demyelination, infiltration of inflammatory cells and GFAP+ astrogliocytes to a great extent. In motoneurons, expression of BDNF mRNA and protein was highly stimulated, indicating concomitant beneficial effects of the steroid on neuronal and glial cells. CONCLUSIONS: Cotherapy with estrogen and progesterone inhibits the development of major neurochemical abnormalities and clinical signs of EAE. We suggest that a combination of neuroprotective, promyelinating and immuno-suppressive mechanisms are involved in these beneficial effects.

Protective effects of progesterone administration on axonal pathology in mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE), an induced model of Multiple Sclerosis presents spinal cord demyelination, axonal pathology and neuronal dysfunction. Previous work has shown that progesterone attenuated the clinical severity, demyelination and neuronal dysfunction of EAE mice (Garay et al., J. Steroid Biochem. Mol. Biol., 2008). Here we studied if progesterone also prevented axonal damage, a main cause of neurological disability. To this end, some axonal parameters were compared in EAE mice pretreated with progesterone a week before immunization with MOG(40-54) and in a group of steroid-free EAE mice. On day 16th after EAE induction, we determined in both groups and in control mice: a) axonal density in semithin sections of the spinal cord ventral funiculus; b) appearance of amyloid precursor protein (APP) immunopositive spheroids as an index of damaged axons; c) levels of the growth associated protein GAP43 mRNA and immunopositive cell bodies, as an index of aberrant axonal sprouting. Steroid-naive EAE mice showed decreased axonal density, shrunken axons, abundance of irregular vesicular structures, degenerating APP+ axons, increased expression of GAP43 mRNA and immunoreactive protein in motoneurons. Instead, EAE mice receiving progesterone treatment showed increased axonal counts, high proportion of small diameter axons, reduced APP+ profiles, and decreased GAP43 expression. In conclusion, progesterone enhanced axonal density, decreased axonal damage and prevented GAP43 hyperexpression in the spinal cord of EAE mice. Thus, progesterone also exerts protective effects on the axonal pathology developing in EAE mice.

Neuronal plasticity and antidepressants in the diabetic brain.

The hippocampus, a limbic structure linked to higher brain functions, appears vulnerable in diabetic subjects that have a higher risk of stroke, dementia, and cognitive decline. The dentate gyrus (DG) of the hippocampus is one of the limited neurogenic brain areas during adulthood; neurons born in the DG are involved in some types of learning and memory processes. We found a decrease in the ability for proliferation and neuronal differentiation of newborn cells, measured by bromodeoxyuridine incorporation in the DG, from streptozotocin-induced diabetic mice. The hilar region, formed by mature neurons presenting higher sensitivity to brain damage, showed a reduced neuronal density in diabetic mice with respect to vehicle-treated mice. Interestingly, in a spontaneous model of type 1 diabetes, we corroborated a decrease in the rate of neurogenesis in the nonobese diabetic mice compared to control strains, and this reduction was also found during the prediabetic stage. The antidepressant fluoxetine administered over a period of 10 days to diabetic mice was effective in preventing changes in proliferation and differentiation of new neurons. Confocal microscope studies, including using neuronal and glial markers, suggested that differentiation toward a neuronal phenotype was decreased in diabetic animals and was reversed by the antidepressant treatment. In addition, the loss of hilar neurons was avoided by fluoxetine treatment. Several reports have demonstrated that high susceptibility to stress and elevated corticosterone levels are detrimental to neurogenesis and contribute to neuronal loss. These features are common in some types of depression, diabetes, and aging processes, suggesting they participate in the reported hippocampal abnormalities present in these conditions.

Cognitive dysfunction and hippocampal changes in experimental type 1 diabetes.

Type 1 diabetes (T1D) is accompanied by a "diabetic encephalopathy" including hypersensitivity to stress, increased risk of stroke, dementia and cognitive impairment. In previous works we reported several brain alterations including a strong decrease in hippocampal proliferation and survival in both spontaneous and streptozotocin-induced models of experimental T1D. The aim of this study was to explore in streptozotocin-treated mice and other parameters associated to mild neurodegeneration in the dentate gyrus and the potential correlation with behavioural changes. The neurogenic status, measured by doublecortin (DCX) expression, showed an important decline in the number of positive cells in the subgranular zone (SGZ). However, neuronal migration was not affected. We found a marked enhancement of intracellular lipofuscin deposits, characteristic of increased oxidative stress and aging in both, the hilus and the SGZ and granular cell layer (GCL). Diabetic mice showed a significant impairment in learning and memory tests, exhibiting a higher latency to show an escape response and a poorer learning efficiency of an active avoiding response compared with control mice. Both, exploratory and non-exploratory activities in a conflictive environment in the asymmetric elevated plus maze were not affected by the diabetic condition. In conclusion, experimental diabetes showed clear signs of changes in the dentate gyrus, changes similar to those present in the aging process. Correlatively, these alterations were in line with a reduced performance in learning and memory tests. The mechanism that could potentially link neural and behavioural disturbances is not yet fully comprehended.

Effects of progesterone on oligodendrocyte progenitors, oligodendrocyte transcription factors, and myelin proteins following spinal cord injury.

Progesterone is emerging as a myelinizing factor for central nervous system injury. Successful remyelination requires proliferation and differentiation of oligodendrocyte precursor cells (OPC) into myelinating oligodendrocytes, but this process is incomplete following injury. To study progesterone actions on remyelination, we administered progesterone (16 mg/kg/day) to rats with complete spinal cord injury. Rats were euthanized 3 or 21 days after steroid treatment. Short progesterone treatment (a) increased the number of OPC without effect on the injury-induced reduction of mature oligodendrocytes, (b) increased mRNA and protein expression for the myelin basic protein (MBP) without effects on proteolipid protein (PLP) or myelin oligodendrocyte glycoprotein (MOG), and (c) increased the mRNA for Olig2 and Nkx2.2 transcription factors involved in specification and differentiation of the oligodendrocyte lineage. Furthermore, long progesterone treatment (a) reduced OPC with a concomitant increase of oligodendrocytes; (b) promoted differentiation of cells that incorporated bromodeoxyuridine, early after injury, into mature oligodendrocytes; (c) increased mRNA and protein expression of PLP without effects on MBP or MOG; and (d) increased mRNA for the Olig1 transcription factor involved in myelin repair. These results suggest that early progesterone treatment enhanced the density of OPC and induced their differentiation into mature oligodendrocytes by increasing the expression of Olig2 and Nkx2.2. Twenty-one days after injury, progesterone favors remyelination by increasing Olig1 (involved in repair of demyelinated lesions), PLP expression, and enhancing oligodendrocytes maturation. Thus, progesterone effects on oligodendrogenesis and myelin proteins may constitute fundamental steps for repairing traumatic injury inflicted to the spinal cord.

Steroid protection in the experimental autoimmune encephalomyelitis model of multiple sclerosis.

OBJECTIVES: Based on evidence that pregnant women with multiple sclerosis (MS) show a decline in the relapse rate during the third trimester and an increase during the first 3 months postpartum, the suggestion was made that high levels of circulating sex steroids are responsible for pregnancy-mediated neuroprotection. As both estradiol (E(2)) and progesterone exert neuroprotective and myelinating effects on the nervous system, the effects of sex steroids were studied in the experimental autoimmune encephalomyelitis (EAE) model of MS. METHODS: EAE was induced in female C57BL/6 mice by administration of a myelin oligodendrocyte protein (MOG(40-45)) peptide. Clinical signs of EAE, myelin protein expression and neuronal parameters were determined in mice with or without hormonal treatment. RESULTS: Progesterone given prior to EAE induction attenuated the clinical scores of the disease, slightly delayed disease onset and decreased demyelination foci, according to luxol fast blue staining (LFB), myelin basic protein (MBP) and proteolipid protein (PLP) and mRNA expression. Motoneuron expression of Na,K-ATPase mRNA was also enhanced by progesterone. In turn, combined E(2) plus progesterone therapy more effectively prevented neurological deficits, fully restored LFB staining, MBP and PLP immunoreactivity and avoided inflammatory cell infiltration. On the neuronal side, steroid biotherapy increased brain-derived neurotrophic factor (BDNF) mRNA. CONCLUSION: Early treatment with progesterone alone or more evidently in combination with E(2) showed a clinical benefit and produced myelinating and neuroprotective effects in mice with MOG(40-45)-induced EAE. Therefore, sex steroids should be considered as potential novel therapeutic strategies for MS.