Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

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.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

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.

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats.

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.

The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

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.

Gene therapy in the neuroendocrine system.

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.