RESUMO
The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.
Assuntos
Envelhecimento/patologia , Envelhecimento/psicologia , Hipocampo/patologia , Memória Espacial/fisiologia , Animais , Astrócitos/patologia , Cognição/fisiologia , Proteína Duplacortina , Feminino , Neurônios/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.
Assuntos
Envelhecimento/metabolismo , Terapia Genética/métodos , Vetores Genéticos/farmacologia , Fator de Crescimento Insulin-Like I/genética , Transtornos dos Movimentos/terapia , Fatores Etários , Envelhecimento/genética , Animais , Feminino , Vetores Genéticos/genética , Injeções Intraventriculares/métodos , Fator de Crescimento Insulin-Like I/administração & dosagem , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Progressive dysfunction of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons during normal aging is associated in the female rat with chronic hyperprolactinemia. We assessed the effectiveness of glial cell line-derived neurotrophic factor (GDNF) gene therapy to restore TIDA neuron function in senile female rats and reverse their chronic hyperprolactinemia. Young (2.5 months) and senile (29 months) rats received a bilateral intrahypothalamic injection (10(10) pfu) of either an adenoviral vector expressing the gene for beta-galactosidase; (Y-betagal and S-betagal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF, respectively). Transgenic GDNF levels in supernatants of GDNF adenovector-transduced N2a neuronal cell cultures were 25+/-4 ng/ml, as determined by bioassay. In the rats, serum prolactin (PRL) was measured at regular intervals. On day 17 animals were sacrificed and neuronal nuclear antigen (NeuN) and tyrosine hydroxylase (TH) immunoreactive cells counted in the arcuate-periventricular hypothalamic region. The S-GDNF but not the S-betagal rats, showed a significant reduction in body weight. The chronic hyperprolactinemia of the senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-betagal rats. Neither age nor GDNF induced significant changes in the number of NeuN and TH neurons. We conclude that transgenic GDNF ameliorates chronic hyperprolactinemia in aging female rats, probably by restoring TIDA neuron function.
Assuntos
Envelhecimento/metabolismo , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Hiperprolactinemia/genética , Hiperprolactinemia/terapia , Adenoviridae/genética , Animais , Antígenos Nucleares/metabolismo , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/metabolismo , Contagem de Células , Células Cultivadas , Doença Crônica/terapia , Feminino , Genes Reporter/genética , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Hiperprolactinemia/metabolismo , Lactotrofos/metabolismo , Microinjeções/métodos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Prolactina/análise , Prolactina/sangue , Prolactina/metabolismo , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica/genética , Resultado do Tratamento , Túber Cinéreo/metabolismo , Túber Cinéreo/fisiopatologia , Tirosina 3-Mono-Oxigenase/metabolismo , beta-Galactosidase/genéticaRESUMO
I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.
Assuntos
Epêndima/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Fator de Crescimento Insulin-Like I/genética , Transdução Genética/métodos , Adenoviridae/genética , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Epêndima/citologia , Feminino , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Hipotálamo/citologia , Hipotálamo/metabolismo , Injeções Intraventriculares/métodos , Fator de Crescimento Insulin-Like I/líquido cefalorraquidiano , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Biologia Molecular/métodos , Regiões Promotoras Genéticas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Timidina Quinase/genética , Transgenes/genética , Vimentina/metabolismo , Proteínas Virais/genéticaRESUMO
Morphologic and functional studies describing the impact of aging on mesencephalic dopaminergic (DA) neurons in laboratory animals are rather scanty and inconclusive. In rats, stereological studies characterizing age changes in the mesencephalic DA neurons have not been documented. In order to fill this information gap and to determine whether the very old rat may serve as a suitable animal model of Parkinson's disease, we performed a stereological assessment of the mesencephalic tyrosine hydroxylase immunoreactive (TH-ir) neurons in young-adult (4-6 months), old (22-24 months) and senile (30-32 months) Sprague-Dawley female rats. Morphometric analysis of the TH-ir neurons of the substantia nigra (SN) and ventral tegmental area (VTA) was performed using an appropriate image analysis system. Age changes in motor performance were assessed measuring the endurance of rats to hang from a wire mesh pole or to remain on a ramp set at different angles to the floor. Age changes in locomotion and exploratory activity were evaluated by the open field test. We observed a significant age-related reduction in TH-ir neuron numbers in the SN (17 and 33% reduction in old and senile rats, respectively compared with young counterparts) but not in the VTA. The size of the TH-ir cells increased significantly in both the SN and VTA of the senescent animals but TH labeling intensity fell. Motor, locomotor and exploratory performance deteriorated markedly in the old and senile rats as compared with young animals. These findings reveal the existence of a moderate but significant vulnerability of mesencephalic DA neurons to aging in rats. This phenomenon, which is particularly marked in the SN of very old rats, may contribute to the age-related decline in motor and exploratory performance recorded in this species.
Assuntos
Envelhecimento/patologia , Envelhecimento/fisiologia , Dopamina/metabolismo , Neurônios/patologia , Neurônios/fisiologia , Animais , Comportamento Animal/fisiologia , Feminino , Imuno-Histoquímica , Mesencéfalo/patologia , Mesencéfalo/fisiologia , Atividade Motora/fisiologia , Ratos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.
Assuntos
Envelhecimento/metabolismo , Dopamina/metabolismo , Terapia Genética/métodos , Hipotálamo/metabolismo , Fator de Crescimento Insulin-Like I/genética , Adenoviridae/genética , Animais , Contagem de Células , Feminino , Vetores Genéticos/administração & dosagem , Hipotálamo/citologia , Injeções , Camundongos , Neurônios/citologia , Prolactina/sangue , Ratos , Ratos Sprague-Dawley , Transdução Genética/métodosRESUMO
The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.
Assuntos
Doenças do Sistema Endócrino/terapia , Terapia Genética , Sistemas Neurossecretores , Envelhecimento/genética , Animais , Animais Geneticamente Modificados , Genes Transgênicos Suicidas , Hipotálamo/metabolismo , Camundongos , Proteínas Mutantes/genética , Hipófise/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/terapia , Ratos , Ratos Brattleboro , Receptores de Superfície Celular/genética , Receptores para Leptina , Retinoblastoma/genéticaRESUMO
Thymulin is a thymic peptide possessing hypophysiotropic activity and antiinflammatory effects in the brain. We constructed a synthetic DNA sequence encoding met-FTS, a biologically active analog of thymulin, and subsequently cloned it into different expression vectors. A sequence optimized for expression of met-FTS in rodents, 5'-ATGCAGGCCAAGTCGCAGGGGGGGTCGAACTAGTAG-3', was cloned in the mammalian expression vectors pCDNA3.1(+) and phMGFP (which expresses the Monster Green Fluorescent Protein), thus obtaining pcDNA3.1-metFTS and p-metFTS-hMGFP, which express met-FTS and the fluorescent fusion protein metFTS-hMGFP, respectively. The synthetic sequence was also used to construct the adenoviral vector RAd-metFTS, which expresses met-FTS. Transfection of HEK293 and BHK cells with pcDNA3.1-metFTS (experimental groups) or pcDNA3.1 (control), led to high levels of thymulin bioactivity (>600 versus <0.1 pg/ml in experimental and control supernatants, respectively). Transfection of HEK293 and BHK cells with pmetFTS-hMGFP revealed a cytoplasmic and nuclear distribution of the fluorescent fusion protein. A single intramuscular (i.m.) injection (10(7) plaque forming units (PFU)/mouse or 10(8) PFU/rat) of RAd-metFTS in thymectomized animals (nondetectable serum thymulin) restored serum thymulin levels for at least 110 and 130 days post-injection in mice and rats, respectively. We conclude that RAd-metFTS constitutes a suitable biotechnological tool for the implementation of thymulin gene therapy in animal models of chronic brain inflammation.
Assuntos
Adenoviridae/genética , Encefalite/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Fator Tímico Circulante/análise , Animais , Linhagem Celular , Núcleo Celular/química , Cricetinae , Citoplasma/química , Encefalite/sangue , Feminino , Expressão Gênica , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Timectomia , Fator Tímico Circulante/genética , Fatores de TempoRESUMO
Pituitary adenomas constitute the most frequent neuroendocrine pathology, comprising up to 15% of primary intracranial tumors. Current therapies for pituitary tumors include surgery and radiotherapy, as well as pharmacological approaches for some types. Although all of these approaches have shown a significant degree of success, they are not devoid of unwanted side effects, and in most cases do not offer a permanent cure. Gene therapy-the transfer of genetic material for therapeutic purposes-has undergone an explosive development in the last few years. Within this context, the development of gene therapy approaches for the treatment of pituitary tumors emerges as a promising area of research. We begin by presenting a brief account of the genesis of prolactinomas, with particular emphasis on how estradiol induces prolactinomas in animals. In so doing, we discuss the role of each of the recently discovered growth inhibitory and growth stimulatory substances and their interactions in estrogen action. We also evaluate the cell-cell communication that may govern these growth factor interactions and subsequently promote the growth and survival of prolactinomas. Current research efforts to implement gene therapy in pituitary tumors include the treatment of experimental prolactinomas or somatomammotropic tumors with adenoviral vector-mediated transfer of the suicide gene for the herpes simplex type 1 (HSV1) thymidine kinase, which converts the prodrug ganciclovir into a toxic metabolite. In some cases, the suicide transgene has been placed under the control of pituitary cell-type specific promoters, like the human prolactin or human growth hormone promoters. Also, regulatable adenoviral vector systems are being assessed in gene therapy approaches for experimental pituitary tumors. In a different type of approach, an adenoviral vector, encoding the human retinoblastoma suppressor oncogene, has been successfully used to rescue the phenotype of spontaneous pituitary tumors of the pars intermedia in mice. We close the article by discussing the future of molecular therapies. We point out that although, gene therapy represents a key step in the development of molecular medicine, it has inherent limitations. As a consequence, it is our view that at some point, genetic therapies will have to move from exogenous gene transfer (i.e. gene therapy) to endogenous gene repair. This approach will call for radically new technologies, such as nanotechnology, whose present state of development is outlined.
Assuntos
Terapia Genética/métodos , Neoplasias Hipofisárias/terapia , Animais , Genes Transgênicos Suicidas , Vetores Genéticos/administração & dosagem , Vetores Genéticos/efeitos adversos , Humanos , Camundongos , NanotecnologiaRESUMO
Pathological processes like cancer, chronic inflammation and autoimmune phenomena, all of which involve massive cell death, are associated with significant increases in circulating DNA. In order to clarify whether massive apoptosis occurring under physiological circumstances also causes DNA release into the circulation, we correlated the time-course of dexamethasone-induced intra thymic cell apoptosis with plasma DNA dynamics in rats. Animals were given 10 mg/l dexamethasone in their drinking water for up to 7 days. Sequential plasma samples were obtained during the treatment and DNA was quantitated by a micro fluorometric assay. Thymus and spleen weight as well as apoptotic cell levels were assessed at different times. Seven days of glucocorticoid treatment reduced thymic and spleen mass by 82 and 31%, respectively. Intra thymic apoptosis was maximal 24 h after the beginning of glucocorticoid treatment, declining markedly by 48 h. Very little apoptosis was observed in the spleen. Plasma DNA increased steadily during the first 4 days of glucocorticoid treatment (11.8 +/- 1.2 microg/ml on day 0; 24.2 +/- 1.6 microg/ml on day 4) beginning to decline afterward. Thymectomy but not splenectomy, drastically reduced the glucocorticoid-induced increase in plasma DNA. It is concluded that hormone-induced massive intra thymic cell death is followed by a delayed release of nucleosomal DNA into the circulation.