Sergio Veiga

Glial expression of estrogen and androgen receptors after rat brain injury

Estrogens and androgens can protect neurons from death caused by injury to the central nervous system. Astrocytes and microglia are major players in events triggered by neural lesions. To determine whether glia are direct targets of estrogens or androgens after neural insults, steroid receptor expression in glial cells was assessed in two different lesion models. An excitotoxic injury to the hippocampus or a stab wound to the parietal cortex and hippocampus was performed in male rats, and the resultant expression of steroid receptors in glial cells was assessed using double‐label immunohistochemistry. Both lesions induced the expression of estrogen receptors (ERs) and androgen receptors (ARs) in glial cells. ERα was expressed in astrocytes immunoreactive (ERα‐ir) for glial fibrillary acidic protein or vimentin. AR immunoreactivity colocalized with microglial markers, such as Griffonia simplicifolia lectin‐1 or OX‐6. The time course of ER and AR expression in glia was studied in the stab wound model. ERα‐ir astrocytes and AR‐ir microglia were observed 3 days after lesion. The number of ERα‐ir and AR‐ir glial cells reached a maximum 7 days after lesion and returned to low levels by 28 days postinjury. The studies of ERβ expression in glia were inconclusive; different results were obtained with different antibodies. In sum, these results suggest that reactive astrocytes and reactive microglia are a direct target for estrogens and androgens, respectively. J. Comp. Neurol. 450:256–271, 2002.

Progesterone and its derivatives dihydroprogesterone and tetrahydroprogesterone reduce myelin fiber morphological abnormalities and myelin fiber loss in the sciatic nerve of aged rats

Previous studies indicate that steroid hormones may be protective for Schwann cells and promote the expression of myelin proteins in the sciatic nerve of adult rats. In this study, we have evaluated the effect of progesterone (P), dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), dihydrotestosterone (DHT) and 5alpha-androstan-3alpha, 17beta-diol (3alpha-diol) on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24-month-old male rats. The sciatic nerves of untreated old male rats, showed a general disorganization and a significant reduction in the density of myelinated fibers, compared to nerves from 3-month-old male rats. The effect of aging was particularly evident in myelinated fibers of small caliber (<5 microm in diameter). In addition, the sciatic nerves of old rats showed a significant increase in the number of fibers with myelin infoldings in the axoplasm and in the number of fibers with irregular shapes. Treatments of old rats with P, DHP and THP resulted in a significant increase in the number of myelinated fibers of small caliber, a significant reduction in the frequency of myelin abnormalities and a significant increase in the g ratio of small myelinated fibers. Furthermore, P treatment significantly reduced the frequency of myelinated fibers with irregular shapes. In contrast, treatments with T, DHT or 3alpha-diol did not significantly affect any of the morphological parameters examined. In conclusion, our data indicate that P, and its derivatives DHP and THP, are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve. These data suggest that P, DHP and THP may represent useful therapeutic alternatives to maintain peripheral nerve integrity in aged animals.

Aromatase expression by reactive astroglia is neuroprotective.

Abstract: The enzyme aromatase catalyzes the conversion of testosterone and other C19 steroids to estradiol. Under normal circumstances, the expression of aromatase in the central nervous system of mammals is restricted to neurons. However, the expression of the enzyme is induced in astrocytes in vitro by stressful stimuli. Furthermore, different types of brain injury induce in vivo the expression of aromatase in reactive astrocytes. The expression of aromatase by reactive astrocytes is neuroprotective, because the pharmacological inhibition of the enzyme in the brain exacerbates neuronal death after different forms of mild neurodegenerative stimuli that do not significantly affect neuronal survival under control conditions. These findings indicate that the induction of aromatase in reactive astrocytes, and the consecutive increase in the local production of estradiol in the brain at injured sites, may be an endogenous neural response to reduce the extent of neurodegenerative damage.

Testosterone decreases reactive astroglia and reactive microglia after brain injury in male rats: role of its metabolites, oestradiol and dihydrotestosterone

Previous studies have shown that the neuroprotective hormone, testosterone, administered immediately after neural injury, reduces reactive astrogliosis. In this study we have assessed the effect of early and late therapy with testosterone or its metabolites, oestradiol and dihydrotestosterone, on reactive astroglia and reactive microglia after a stab wound brain injury in orchidectomized Wistar rats. Animals received daily s.c. injections of testosterone, oestradiol or dihydrotestosterone on days 0–2 or on days 5–7 after injury. The number of vimentin immunoreactive astrocytes and the volume fraction of major histocompatibility complex‐II (MHC‐II) immunoreactive microglia were estimated in the hippocampus in the lateral border of the wound. Both early and delayed administration of testosterone or oestradiol, but not dihydrotestosterone, resulted in a significant decrease in the number of vimentin‐immunoreactive astrocytes. The volume fraction of MHC‐II immunoreactive microglia was significantly decreased in the animals that received testosterone or oestradiol in both early and delayed treatments and in animals that received early dihydrotestosterone administration. Thus, both early and delayed administration of testosterone reduces reactive astroglia and reactive microglia and these effects may be at least in part mediated by oestradiol, while dihydrotestosterone may mediate part of the early effects of testosterone on reactive microglia. In conclusion, testosterone controls reactive gliosis and its metabolites, oestradiol and dihydrotestosterone, may be involved in this hormonal effect. The regulation of gliosis may be part of the neuroprotective mechanism of testosterone.

Ro5-4864, a peripheral benzodiazepine receptor ligand, reduces reactive gliosis and protects hippocampal hilar neurons from kainic acid excitotoxicity.

The peripheral‐type benzodiazepine receptor (PBR) is a critical component of the mitochondrial permeability transition pore, which is involved in the regulation of cell survival. Different forms of brain injury result in induction of the expression of the PBR in the areas of neurodegeneration, mainly in reactive glial cells. The consequences of induction of PBR expression after brain injury are unknown. To test whether PBR may be involved in the regulation of neuronal survival after injury, we have assessed the effect of two PBR ligands, Ro5‐4864 and PK11195, on neuronal loss induced by kainic acid in the hippocampus. Systemic administration of kainic acid to male rats resulted in the induction of a reactive phenotype in astrocytes and microglia and in a significant loss of hilar neurons in the dentate gyrus. Administration of Ro5‐4864, before the injection of kainic acid, decreased reactive gliosis in the hilus and prevented hilar neuronal loss. In contrast, PK11195 was unable to reduce reactive gliosis and did not protect hilar neurons from kainic acid. These findings suggest that the PBR is involved in control of neuronal survival and gliosis after brain injury and identify this molecule as a potential target for neuroprotective interventions.

Sex hormones and brain aging

Sex steroids exert pleiotropic effects in the nervous system, preserving neural function and promoting neuronal survival. Therefore, the age-related decrease in sex steroids may have a negative impact on neural function. Progesterone, testosterone and estradiol prevent neuronal loss in the central nervous system in different experimental animal models of neurodegeneration. Furthermore, progesterone and its reduced derivatives dihydroprogesterone and tetrahydroprogesterone reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in rat peripheral nerves. However, the results from hormone replacement studies in humans are thus far inconclusive. A possible alternative to hormonal replacement therapy is to increase local steroidogenesis by neural tissues, which express enzymes for steroid synthesis and metabolism. Proteins involved in the intramitochondrial trafficking of cholesterol, the first step in steroidogenesis, such as the peripheral-type benzodiazepine receptor and the steroidogenic acute regulatory protein, are up-regulated in the nervous system after injury. Furthermore, steroidogenic acute regulatory protein expression is increased in the brain of 24-month-old rats compared with young adult rats. This suggests that brain steroidogenesis may be modified in adaptation to neurodegenerative conditions and to the brain aging process. Furthermore, recent studies have shown that local formation of estradiol in the brain, by the enzyme aromatase, is neuroprotective. Therefore, steroidogenic acute regulatory protein, peripheral-type benzodiazepine receptor and aromatase are attractive pharmacological targets to promote neuroprotection in the aged brain.

Neuroactive steroids prevent peripheral myelin alterations induced by diabetes

The effect of the neuroactive steroids progesterone, dihydroprogesterone and tetrahydroprogesterone on myelin abnormalities induced by diabetes was studied in the sciatic nerve of adult male rats treated with streptozotocin. Streptozotocin increased blood glucose levels and decreased body weight gain, parameters not affected by steroids. Streptozotocin increased the number of fibers with myelin infoldings in the axoplasm, 8 months after the treatment. Chronic treatment for 1 month with progesterone and dihydroprogesterone resulted in a significant reduction in the number of fibers with myelin infoldings to control levels. Treatment with tetrahydroprogesterone did not significantly affect this myelin alteration. These results suggest that neuroactive steroids such as progesterone and dihydroprogesterone may represent therapeutic alternatives to counteract peripheral myelin alterations induced by diabetes.

Translocator protein 18 kDa is involved in the regulation of reactive gliosis.

Translocator protein (18 kDa) (TSPO), previously known as peripheral‐type benzodiazepine receptor, is a critical component of the mitochondrial permeability transition pore. Brain inflammation results in the induction of the expression of TSPO in glial cells and some TSPO ligands decrease reactive gliosis after brain injury. However, since some TSPO ligands are neuroprotective, their effects on reactive gliosis may be the consequence of a reduced neurodegeneration. To assess whether TSPO ligands can modulate reactive gliosis in absence of neuronal death, we have tested their effects on the inflammatory response induced in the hippocampus of male rats by the intracerebroventricular infusion of lipopolysaccharide (LPS). LPS treatment did not induce neuronal death, assessed by Fluoro jade‐B staining, but increased the number of cells immunoreactive for vimentin and MHC‐II, used as markers of reactive astrocytes and reactive microglia, respectively. Furthermore, LPS produced an increase in the number of proliferating microglia. The TSPO ligand PK11195 reduced the number of MHC‐II immunoreactive cells and the proliferation of microglia in LPS treated rats. In contrast, another TSPO ligand, Ro5‐4864, did not significantly affect the response of microglia to LPS. Neither PK11195 nor Ro5‐4864 affected the LPS‐mediated increase in the number of vimentin‐immunoreactive astrocytes at the time point studied, although PK11195 reduced vimentin immunoreactivity. These findings identify TSPO as a potential target for controlling neural inflammation, showing that the TSPO ligand PK11195 may reduce microglia activation by a mechanism that is independent of the regulation of neuronal survival.

Effects of neuroactive steroids on myelin of peripheral nervous system

Peripheral nervous system (PNS) possess both classical (e.g. progesterone receptor, PR, androgen receptor, AR) and non-classical (e.g. GABA(A) receptor) steroid receptors and consequently may represent a target for the action of neuroactive steroids. Our data have indicated that neuroactive steroids, like for instance, progesterone, dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 3alpha-diol, stimulate both in vivo and in vitro (Schwann cell cultures), the expression of two important proteins of the myelin of peripheral nerves, the glycoprotein Po (Po) and the peripheral myelin protein 22 (PMP22). It is important to highlight that the mechanisms by which neuroactive steroids exert their effects on the expression of Po and PMP22 involve different kind of receptors depending on the steroid and on the myelin protein considered. In particular, at least in culture of Schwann cells, the expression of Po seems to be under the control of PR, while that of PMP22 needs the GABA(A) receptor. Because Po and PMP22 play an important physiological role for the maintenance of the multilamellar structure of the myelin of the PNS, the present observations might suggest the utilization of neuroactive steroids as new therapeutically approaches for the rebuilding of the peripheral myelin.

Sex-dimorphic effects of progesterone and its reduced metabolites on gene expression of myelin proteins by rat Schwann cells.

Abstract  Data obtained in our and other laboratories have indicated that progesterone (P) and its derivatives, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), stimulate the expression of two myelin proteins of the peripheral nervous system (PNS) [i.e., glycoprotein zero (P0) and peripheral myelin protein 22 (PMP22)]. We have now considered the effects of P and its derivatives on these and other myelin proteins [i.e., myelin‐associated glycoprotein (MAG) and myelin and lymphocyte protein (MAL)] in sex‐specific cultures of rat Schwann cells. Gene expression of myelin proteins was assessed by RNase protection assay. Treatment with P or DHP induced a stimulatory effect on P0 mRNA levels in male but not in female Schwann cells. In contrast, treatment with THP increased gene expression of P0 exclusively in female Schwann cells. A similar sex‐difference was also evident for other myelin proteins. Indeed, PMP22 expression was stimulated by treatment with P in male cultures, whereas THP induced an increase of mRNA levels in female cultures. Moreover, MAG was stimulated by THP treatment in male cultures only, whereas MAL expression was unaffected by neuroactive steroid treatment in both male and female cultures. In conclusion, the present observations indicate that the effects of neuroactive steroids on myelin proteins are sexually dimorphic. This finding might represent an important background for sex‐specific therapies of acquired and inherited peripheral neuropathies.