Age-related loss of recognition memory and its correlation with hippocampal and perirhinal cortex changes in female Sprague Dawley rats.

Ageing is associated with impaired performance in recognition memory, a process that consists of the discrimination of familiar and novel stimuli. Previous studies have shown the impact of ageing on object recognition memories. However, the early stages of memory impairment remain unknown. To fill this gap, we aimed at evaluating the ability of young (Y), middle-aged (MA), and senile (S) female Sprague-Dawley rats to retain 24 h long-term recognition memory. The MA cohort was included to characterise early memory deficits under two behavioural paradigms based on spontaneous location recognition (SLR) and spontaneous object recognition (SOR) tasks. In the SLR task, there was a markedly diminished novel discrimination capacity in the MA and S rats compared with the Y ones. In the SOR task, S rats evidenced a deterioration in novelty discrimination, while MA rats partially preserved the capacity to distinguish the new stimulus as compared with Y rats. Regarding early changes from MA to S rats, immunohistochemistry showed a marked decrease in the number and diameter of adult-born immature neurons in the Dentate Gyrus (DG) with a positive correlation with behavioural performance in the SLR task. Furthermore, we found a slight reduction in CA3 mature neurons and a decrease in the number of total microglia in the perirhinal cortex (Prh) in MA and S rats as compared with Y rats. As regards changes that were only observed in S rats, we found an increase in the number of total and reactive microglia in CA3 and a reduction in the number of total microglia in the DG. We conclude that spatial discrimination capacity could be affected earlier than feature discrimination capacity. We suggest that early depletion of neurogenesis in MA rats is involved in object location recognition deficits, whereas the disruption of microglial homeostasis in the Prh could be associated with object feature discrimination capacity.

Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells.

The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

DNA nanoparticle-mediated thymulin gene therapy prevents airway remodeling in experimental allergic asthma.

Thymulin has been shown to present anti-inflammatory and anti-fibrotic properties in experimental lung diseases. We hypothesized that a biologically active thymulin analog gene, methionine serum thymus factor, delivered by highly compacted DNA nanoparticles may prevent lung inflammation and remodeling in a mouse model of allergic asthma. The DNA nanoparticles are composed of a single molecule of plasmid DNA compacted with block copolymers of poly-L-lysine and polyethylene glycol (CK30PEG), which have been found safe in a human phase I/II clinical trial. Thymulin plasmids were detected in the lungs of ovalbumin-challenged asthmatic mice up to 27days after administration of DNA nanoparticles carrying thymulin plasmids. A single dose of DNA nanoparticles carrying thymulin plasmids prevented lung inflammation, collagen deposition and smooth muscle hypertrophy in the lungs of a murine model of ovalbumin-challenged allergic asthma, leading to improved lung mechanics. In the present model of chronic allergic asthma, highly compacted DNA nanoparticles using thymulin analog gene modulated the inflammatory and remodeling processes improving lung mechanics.

Transferencia génica asistida por campos magnéticos: Estudios en células gliales / Magnetic field-assisted gene transfer: Studies in glial cells / TransferÛncia gÛnica assistida por camposmagnéticos: Estudos em células gliais

Las nanopartículas magnéticas (MNP) complejadas con vectores génicos pueden, en presencia de un campo magnético externo, amplificar sustancialmente la eficiencia de la transferencia génica. Esta técnica, denominada magnetofección, es de gran interés en el campo de la terapia génica. En este estudio se caracterizó la mejora de transferencia génica en células gliales B92 utilizando complejos constituidos por diferentes proporciones de MNP asociadas a dos vectores adenovirales, a saber: los complejos entre las MNP denominadas PEI-Mag2 asociadas al adenovector RAd-GFP que expresa la proteína fluorescente verde GFP o al adenovector RAd-DsRed que expresa la proteína fluorescente roja DsRed2. Se demostró que para ambos vectores, a medida que la relación MNP/partícula viral física (PVF) va aumentando, la amplificación de la transfección también aumenta hasta que se llega a una relación MNP/PVF a partir de la cual el factor de amplificación alcanza un plateau. Se determinó que para el complejo PEI-Mag2/RAd-GFP la relación a partir de la cual se alcanza el plateau es de aproximadamente 0,5 fg Fe/PVF mientras que para el complejo PEI-Mag2/RAd-DsRed, esta relación corresponde a aproximadamente 71 fg Fe/PVF. Se concluye que los dos complejos magnéticos estudiados representan promisorias herramientas para mejorar la eficiencia en la terapia génica en células cerebrales.(AU)

It is known that certain types of magnetic nanoparticles (MNPs) complexed to gene vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells. This technique, called magnetofection, is of great relevance to gene therapy. In the present study the ability of MNP/adenovector complexes to enhance gene transfer to B92 glial cells was assessed. Two complexes were assessed, namely PEI-Mag2/RAd-GFP and PEI-Mag2/RAd-DsRed, which are constituted by the MNP PEI-Mag2 complexed to the adenovector RAd-GFP (expressing the green fluorescent protein GFP) and RAd-DsRed (expressing the red fluorescent protein DsRed2), respectively. It was shown that for both vectors, an increase in the ratio MNP/PVP (physical viral particle) is paralleled by an increase in transduction efficiency, up to a certain threshold value at which an efficiency plateau is reached. This threshold value was 0.5 fg Fe/PVP for the RAd-GFP complex and about 71 fg Fe/PVP for the RAd-DsRed complex. It can be concluded that both magnetic complexes assessed in this study represent promising tools for enhancing the efficiency of gene therapy in brain cells.(AU)

As nanopartículas magnéticas (MNPs) complexadas com vetores de genes podem, em presenþa de um campo magnético externo, aumentar consideravelmente a eficiÛncia da transferÛncia gÛnica. Esta técnica, chamada magnetofecþÒo, é de grande relevÔncia para a terapia genética. No presente estudo, foi caracterizada a melhoria de transferÛncia de genes em células gliais B92 utilizando complexos constituídos por diferentes proporþ§es de MNP associadas a dois vetores adenovirais, a saber: os complexos entre as MNP denominadas PEI-Mag2 associadas ao adenovetor RAd-GFP que expressa a proteína fluorescente verde GFP ou ao adenovetor RAd-DsRed que expressa a proteína fluorescente vermelha DsRed2. Foi demonstrado que para ambos os vetores, enquanto a relaþÒo MNP/partícula viral física (PVF) vai aumentando, a amplificaþÒo da transfecþÒo também aumenta até que se chega a uma relaþÒo MNP/PVF a partir da qual o fator de amplificaþÒo alcanþa um limiar. Determinou-se que para o complexo PEI-Mag2/RAd-GFP a relaþÒo a partir da qual se atinge o limiar é de aproximadamente 0,5 fg Fe/PVF ao passo que para o complexo PEI-Mag2/RAd-DsRed, esta relaþÒo corresponde a aproximadamente 71 fg Fe/PVF. Conclui-se que os dois complexos magnéticos estudados representam promissoras ferramentas para melhorar a eficiÛncia na terapia de genes em células cerebrais.(AU)

Transferencia génica asistida por campos magnéticos: Estudios en células gliales / Magnetic field-assisted gene transfer: Studies in glial cells / Transferência gênica assistida por camposmagnéticos: Estudos em células gliais

Las nanopartículas magnéticas (MNP) complejadas con vectores génicos pueden, en presencia de un campo magnético externo, amplificar sustancialmente la eficiencia de la transferencia génica. Esta técnica, denominada magnetofección, es de gran interés en el campo de la terapia génica. En este estudio se caracterizó la mejora de transferencia génica en células gliales B92 utilizando complejos constituidos por diferentes proporciones de MNP asociadas a dos vectores adenovirales, a saber: los complejos entre las MNP denominadas PEI-Mag2 asociadas al adenovector RAd-GFP que expresa la proteína fluorescente verde GFP o al adenovector RAd-DsRed que expresa la proteína fluorescente roja DsRed2. Se demostró que para ambos vectores, a medida que la relación MNP/partícula viral física (PVF) va aumentando, la amplificación de la transfección también aumenta hasta que se llega a una relación MNP/PVF a partir de la cual el factor de amplificación alcanza un plateau. Se determinó que para el complejo PEI-Mag2/RAd-GFP la relación a partir de la cual se alcanza el plateau es de aproximadamente 0,5 fg Fe/PVF mientras que para el complejo PEI-Mag2/RAd-DsRed, esta relación corresponde a aproximadamente 71 fg Fe/PVF. Se concluye que los dos complejos magnéticos estudiados representan promisorias herramientas para mejorar la eficiencia en la terapia génica en células cerebrales.

It is known that certain types of magnetic nanoparticles (MNPs) complexed to gene vectors can, in the presence of an external magnetic field, greatly enhance gene transfer into cells. This technique, called magnetofection, is of great relevance to gene therapy. In the present study the ability of MNP/adenovector complexes to enhance gene transfer to B92 glial cells was assessed. Two complexes were assessed, namely PEI-Mag2/RAd-GFP and PEI-Mag2/RAd-DsRed, which are constituted by the MNP PEI-Mag2 complexed to the adenovector RAd-GFP (expressing the green fluorescent protein GFP) and RAd-DsRed (expressing the red fluorescent protein DsRed2), respectively. It was shown that for both vectors, an increase in the ratio MNP/PVP (physical viral particle) is paralleled by an increase in transduction efficiency, up to a certain threshold value at which an efficiency plateau is reached. This threshold value was 0.5 fg Fe/PVP for the RAd-GFP complex and about 71 fg Fe/PVP for the RAd-DsRed complex. It can be concluded that both magnetic complexes assessed in this study represent promising tools for enhancing the efficiency of gene therapy in brain cells.

As nanopartículas magnéticas (MNPs) complexadas com vetores de genes podem, em presença de um campo magnético externo, aumentar consideravelmente a eficiência da transferência gênica. Esta técnica, chamada magnetofecção, é de grande relevância para a terapia genética. No presente estudo, foi caracterizada a melhoria de transferência de genes em células gliais B92 utilizando complexos constituídos por diferentes proporções de MNP associadas a dois vetores adenovirais, a saber: os complexos entre as MNP denominadas PEI-Mag2 associadas ao adenovetor RAd-GFP que expressa a proteína fluorescente verde GFP ou ao adenovetor RAd-DsRed que expressa a proteína fluorescente vermelha DsRed2. Foi demonstrado que para ambos os vetores, enquanto a relação MNP/partícula viral física (PVF) vai aumentando, a amplificação da transfecção também aumenta até que se chega a uma relação MNP/PVF a partir da qual o fator de amplificação alcança um limiar. Determinou-se que para o complexo PEI-Mag2/RAd-GFP a relação a partir da qual se atinge o limiar é de aproximadamente 0,5 fg Fe/PVF ao passo que para o complexo PEI-Mag2/RAd-DsRed, esta relação corresponde a aproximadamente 71 fg Fe/PVF. Conclui-se que os dois complexos magnéticos estudados representam promissoras ferramentas para melhorar a eficiência na terapia de genes em células cerebrais.

Decrease in PTEN and increase in Akt expression and neuron size in aged rat spinal cord.

PTEN is a tumor suppressor gene known to play an important role in the regulation of cell size. In this study we compared PTEN expression in the spinal cord of young (5 months old) vs. aged (32 months old) female rats and correlated them with alterations in neuron size and morphology in the same animals. Total and phosphorylated PTEN (pPTEN) as well as its downstream target phosphorylated Akt (pAkt) were assessed by Western blotting. Spinal cord neurons were morphometrically characterized. Total PTEN, pPTEN and total Akt expression were significantly higher in young rats than in aged animals. Expression of pAkt was stronger in aged animals. A significant increase in neuronal size was observed in large motoneurons of aged as compared with young rats. Our data show that in the spinal cord of rats, neuronal PTEN expression diminishes with advanced age while neuronal size increases. These results suggest that in the spinal cord, an age-related reduction in PTEN and increase of pAkt expression may be involved in the progressive enlargement of neurons.

Impact of very old age on the expression of cervical spinal cord cell markers in rats.

Aging is a process associated with both anatomical changes and loss of expression of some cell markers. Intermediate filaments are known to impart mechanical stability to cells and tissues. Some of them are present in different cell populations of the central nervous system. In order to explore the impact of extreme age we immunohistochemically characterized the changes in intermediate filaments and other cellular markers present in cells populating the gray matter cervical spinal cord of very old rats (28 months) taking young (5 months) counterparts as a reference. The spinal cord weight of the senile animals (12.6+/-1.1 g) was significantly higher (P<0.001) than that of the young animals (8.4+/-1.1 g). Spinal cord length also increased significantly (P<0.05) with age (7.9+/-0.3 cm vs. 8.28+/-0.1 cm for young and senile, respectively). An increase in both neurofilament staining area and density was observed in senile rats in comparison to young animals. A significant (P<0.05) age-related increment in the mean area of the cervical segments was observed. Vimentin expression in the ependymal zone decreased in area and intensity during aging. Our data show that there are some significant changes in the morphological and histochemical patterns of the cervical spinal cord in senile rats. However, they do not necessarily represent a pathologic situation and may rather reflect plastic reorganization.

Presence of binucleate neurons in the spinal cord of young and senile rats.

The presence of binucleate cells constitutes a normal feature of some animal tissues but is rare in the normal brain and has not been documented in the spinal cord. We assessed different segments of the rat spinal cord in order to determine the frequency and distribution of binucleate neurons in this structure as well as the impact of aging on this neuronal population. Young (4-5 months) and senile (32 months) female Sprague-Dawley rats were used. Sections from cervical, thoracic and lumbar segments were histochemically and immunohistochemically (NeuN) stained and the frequency and distribution of binucleate neurons was determined by manual counting. The frequency of binucleate neurons in all of the analysed segments was comparable between young and senile animals. Binucleate neurons were particularly frequent in the C5 and C6 segments. The overall distribution of binucleate neurons in the different laminae assessed was, Lm-III = 19%; Lm-VI = 17%; Lm-VII = 39%; LmVIII = 8%; Lm-IX = 11%; Lm-X = 6%, and was comparable between young and senile rats. We conclude that binucleate neurons occur as a normal feature of the rat spinal cord and that their frequency and distribution does not change with aging.

Loss of NeuN immunoreactivity in rat spinal cord neurons during aging.

A morphologic study of the impact of aging on neuron marker expression was performed in different segments of the rat spinal cord. Spinal cord specimens from young (5 months), middle-aged (12 months) and senile (32 months) female rats were assessed. We found a complete loss of neuron-specific nuclear protein (NeuN) immunoreactivity in cervical, thoracic and lumbar segments of the senile animals whereas neuron-specific enolase (NSE) immunoreactivity was comparable in young and senile rats. These findings in otherwise morphologically well preserved spinal cord neurons are of interest and reveal that NeuN may not be a reliable marker to identify neurons in the spinal cord of aging rats.

Morphometry of cervical segments grey matter in the male rat spinal cord.

The cervical portion of the spinal cord is an area frequently affected by alterations of medical and veterinary importance. Since there is scarce quantitative anatomical data on this region, we undertook a morphometric study of the grey matter of all segments of the rat cervical spinal cord of male rats in order to generate reference patterns to be used in future experimental studies. Using image analysis software, the total spinal cord length and grey and white matter area of each segment was recorded. The morphometric characteristics of the neurones populating the laminae of the grey matter of the cervical segments was also recorded. Neurones were classified into small, medium and large sizes for each lamina and statistically compared. The present data fill an anatomical information gap by providing quantitative data about the normal anatomical features of the rat cervical cord. The anatomical data found could be used to better understand the physiological relevance of that region in the rat.

Impact of very old age on hypothalamic dopaminergic neurons in the female rat: a morphometric study.

Dopaminergic neurons of the A(12) (tuberoinfundibular dopaminergic system) and A(14) (periventricular dopaminergic system) hypothalamic areas exert a tonic inhibitory control of prolactin secretion. Tuberoinfundibular dopaminergic system neuron function is known to decline during aging in rats, but little is known about the impact of extreme age on neuron number and morphology in the two systems. We morphometrically assessed the neurons of the tuberoinfundibular dopaminergic system and the periventricular dopaminergic system in female rats 6 (young, Y), 24 (old, O), and 30-32 (senescent, S) months old. Serial coronal sections of fixed hypothalami were immunohistochemically labeled for tyrosine hydroxylase, and immunoreactive perikarya from the A(12) and A(14) areas were quantitatively characterized and compared among the three age groups. Radioimmunoassay was used to measure serum prolactin. The number of A(12) tyrosine hydroxylase-immunoreactive perikarya showed a steady decline with age, whereas the number of A(14) tyrosine hydroxylase-immunoreactive perikarya remained stable from young to old age but showed a sharp drop in the senescent rats. In the old rats, tyrosine hydroxylase-immunoreactive neuronal area (A(12) = 135.37 and A(14) = 158.79 microm(2)) was significantly higher than that of young (A(12) = 72.56 and A(14) = 99.7 microm(2)) and senescent animals (A(12) = 95.5 and A(14) = 106.5 microm(2)). Densitometric assessment of median eminence tyrosine hydroxylase immunoreactivity revealed a steady age-related reduction of tyrosine hydroxylase content in the median eminence. Serum prolactin levels increased steadily with age. We conclude that, in the female rat, aging brings about a progressive loss of both tuberoinfundibular dopaminergic system and periventricular dopaminergic system neurons, which becomes more conspicuous at extreme ages.