Activation of constitutive nitric oxide synthase(s) and absence of inducible isoform in aged rat brain

In this study, the effect of aging on nitric oxide synthases (NOS) was investigated in homogenates and cytosolic fractions from hippocampus, brain cortex and cerebellum of adult, old adult and old Wistar rats (3-4, 14, and 24 months old, respectively). Our results indicate the enhancement of Ca(2+) and calmoduline-dependent NOS activity in all investigated aged brain parts. Significantly higher NOS activity was found in the cerebellum.In the absence of Ca(2+) or in the presence of N-nitro-L-arginine (NNLA) the activity of NOS was absent. Inhibitor of constitutive NOS isoforms which preferentially inhibits neuronal NOS (nNOS), 7-nitroindazole, decreased NOS activity by 60 and 75% in adult and aged brain, respectively. However, using RT-PCR a significantly lower amount of mRNA for nNOS was detected in hippocampus. The ratio of NOS activity to nNOS mRNA was significantly higher in hippocampus and cerebellum of aged brain. No expression of the gene for inducible NOS was observed in adult and aged brain.These results indicate that probably nNOS is responsible for higher NOS activity in aged brain. Our data suggest that alteration of nNOS phosphorylation state may be responsible for the activation of NOS in aged brain. The down-regulation of nNOS mRNA expression may be an adaptive mechanism that protects the brain against excessive NO release.     

Age-related alteration of poly(ADP-ribose) polymerase activity in different parts of the brain

Poly(ADP-ribose) polymerase (PARP) is a conserved enzyme involved in the regulation of DNA repair and genome stability. The role of PARP during aging is not well known. In this study PARP activity was investigated in nuclear fractions from hippocampus, cerebellum, and cerebral cortex of adult (4 months), old adult (14 months) and aged (24-27 months) rats. Concomitantly, the free radical evoked lipid peroxidation was estimated as thiobarbituric acid reactive substances (TBARS). The specific activity of PARP in adult brain was about 25, 21 and 16 pmol/mg protein per min in hippocampus, cerebellum and cerebral cortex, respectively. The enzyme activity was higher in all investigated parts of the brain of old adults. In aged animals PARP activity was lower in hippocampus by about 50%, and was unchanged in cerebral cortex and in cerebellum comparing to adult rats. The concentration of TBARS was the same in all parts of the brain and remained unchanged during aging. There is no direct correlation between PARP activity and free radical evoked lipid peroxidation during brain aging. The lowered enzyme activity in aged hippocampus may decrease DNA repair capacity which subsequently may be responsible for the higher vulnerability of hippocampal neurons to different toxic insults.     

Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53

PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed double-strand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARα breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40-based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganuclease-mediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADP-ribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity.     

Effect of a Novel Prolyl Endopeptidase Inhibitor, JTP-4819, on Prolyl Endopeptidase Activity and Substance P- and Arginine-Vasopressin-Like Immunoreactivity in the Brains of Aged Rats

Abstract: The effects of a novel prolyl endopeptidase (PEP) inhibitor, ( S )-2-[[( S )-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]- N -(phenylmethyl)-1-pyrrolidinecarboxamide (JTP-4819), on the PEP activity in the brain and on the contents of substance P (SP)- and arginine-vasopressin (AVP)-like immunoreactivity (LI) in the cerebral cortex and hippocampus of young and aged rats were investigated using enzyme immunoassay. JTP-4819 exhibited a concentration-dependent in vitro inhibitory action on PEP activity in the brains of both young and aged rats, with IC₅₀ values of ∼0.7 and 0.8 n M , respectively. A single dose of JTP-4819 (3 mg/kg, p.o.) increased the SPLI content in the cerebral cortex but not the hippocampus of aged rats (23–24 months old). In addition, repeated administration of JTP-4819 (1 mg/kg, p.o., for 21 days) increased the SPLI content in the cerebral cortex and restored the SPLI content in the hippocampus, which had decreased with aging. In contrast, single (1 mg/kg, p.o.) and repeated (1 mg/kg, p.o., for 21 days) administration of JTP-4819 only tended to increase the AVPLI content of the hippocampus and cerebral cortex in aged rats, respectively. These results indicate that JTP-4819 increases the cerebral and hippocampal SPLI content in aged rats by inhibiting the action of PEP.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

The effects of acute ethanol exposure and ageing on rat brain glutathione metabolism

Abstract

Binge alcohol consumption in adolescents is increasing, and it has been proposed that immature brain deals poorly with oxidative stress. The aim of our work was to study the effect of an acute dose of ethanol on glutathione (GSH) metabolism in frontal cortex, hippocampus and striatum of juvenile and adult rats. We have observed no change in levels of glutathione produced by acute alcohol in the three brain areas studied of juvenile and adult rats. Only in the frontal cortex the ratio of GSH/GSSG was increased in the ethanol-treated adult rats. GSH levels in the hippocampus and striatum were significantly higher in adult animals compared to young ones. Higher glutathione peroxidase (GPx) activity in adult rats was observed in frontal cortex and in striatum. Our data show an increased GSH concentration and GPx activity in different cerebral regions of the adult rat, compared to the young ones, suggesting that age-related variations of total antioxidant defences in brain may predispose young brain structures to ethanol-induced, oxidative stress-mediated tissue damage.     

The Expression of Cdk5, p35, p39, and Cdk5 Kinase Activity in Developing, Adult, and Aged Rat Brains

The expression of cyclin-dependent kinase 5 (Cdk5) and its regulatory subunits, p35 and p39, was investigated in rat brain from embryonic day 12 (E12) to postnatal 18 months (18M). The Cdk5 protein levels increased from E12 to postnatal day 7 (P7) and remained at this level until 18M. The Cdk5 kinase activity and the levels of both p35 mRNA and protein were low at E12, became prominent at E18-P14 but then decreased in the adult and aged rat brains of 3M to 18M. In comparison, the expression pattern of p39 appeared to have an inverse relationship to that of Cdk5 and p35. In regional distribution studies, p35 protein levels and Cdk5 kinase activity were significantly higher in the cerebral cortex and hippocampus, but lower in the cerebellum and striatum. These results suggested that Cdk5, p35 and p39 might have region-specific and developmental stage-specific functions in rat brain.     

Expression of alpha-synuclein in different brain parts of adult and aged rats.

The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.     

ABRA在正常成年大鼠大脑皮质和海马区的表达

目的观察ABRA(Actin binding Rho activator)在成年大鼠大脑皮质和海马中的表达。方法制备成年大鼠脑的冰冻切片,采用共聚焦免疫荧光技术和免疫荧光强度测量检测ABRA在大鼠大脑皮质和海马区的表达。结果 ABRA在神经元的胞核、胞浆、突起内可见,其中胞核着色最强。在大脑皮质,ABRA阳性的神经元胞体和突起广泛分布于皮质的分子层、外颗粒层、外锥体细胞层、内颗粒层、内锥体细胞层、多形细胞层,其免疫荧光强度分别为129.22±16.94、125.39±29.83、117.67±22.50、105.85±17.65、103.90±18.00、100.23±20.38,ABRA阳性细胞率分别为0.51±0.01、0.69±0.02、0.64±0.03、0.58±0.05、0.65±0.09、0.63±0.01。在海马,ABRA均匀分布于海马各部,阳性神经元集中于锥体细胞层,而其阳性突起弥散分布于海马分子层和多形层。海马锥体细胞层、分子层、多形层免疫荧光强度分别为141.19±35.48、53.19±10.38、43.32±9.59,ABRA阳性细胞率分别为0.62±0.04、0.27±0.07、0.25±0.03。结论 ABRA广泛表达于大鼠大脑皮质和海马各层,提示ABRA可能在大鼠这些部位的神经细胞功能活动方面起重要作用。     

Specificity and efficiency of Cre-mediated recombination in Emx1-Cre knock-in mice

Emx1 is a mouse homologue of the Drosophila homeobox gene empty spiracles and its expression is restricted to the neurons in the developing and adult cerebral cortex and hippocampus. We reported previously the creation of a line of transgenic mice in which the cre gene was placed directly downstream of the putative Emx1 promoter using ES cell technology. We showed that Cre protein was present in the cerebral cortex of the transgenic mice and was able to mediate loxP-specific recombination in vitro. In the present study, the specificity and efficiency of the cre-mediated recombination were determined using three independent lines of reporter mice and a combination of histochemical staining, neuronal culture, and Southern detection of the genomic DNA. Our results showed that the recombination was highly efficient in all three lines of reporter mice tested and confirmed that the deletion was restricted to the neurons in the cerebral cortex and hippocampus. Furthermore, we have determined that the recombination efficiency in the cerebral cortex was 91%. Our results suggest that Emx1 is not expressed in every neuron in the developing and adult cerebral cortex. This line of cre mice should contribute to the studies of cortical development and plasticity.     

Effect of swinging on EEG of rats of juvenile age in the wakefulness state

Simultaneous recording of the EEG activity of superficial cortical and deep (caudate nucleus, dorsal hippocampus, anterior hypothalamus) brain parts has been performed for the first time after a 2-h swinging of frequency of 0.2 Hz in Wistar rats of juvenile age. Swinging was produced on a 4-bar parallel swing. Using a Neuron-Spectr electroencephalograph and a Diana program, normalized power spectra of wave EEG components, synchronization coefficients, and coefficients of cross-correlation between bioelectrical potentials of various brain structures were determined. After a 2-h swinging, the mean value of normalized power of slow waves of delta-diapason in hypothalamus and hippocampus was found to increase statistically significantly, while normalized power of fast waves of alpha- and beta1-diapasons in hippocampus decreased (p < 0.05). A statistically significant increase of synchronization coefficient was observed in hypothalamus and hippocampus. Changes of coefficients of cross-correlation between hypothalamus and hippocampus and other brain strictures were of the oppositely directed, individual character. In the parietal occipital brain cortex and in caudate nucleus, the changes of the EEG spectral composition also were of individual character. The obtained results on the whole correspond to data about an enhancement of the EEG low-frequency rhythms at swinging and agree with the resonance hypothesis of motion sickness.     

Decreased Insulin-Like Growth Factor-I and Its Receptor Expression in the Hippocampus and Somatosensory Cortex of the Aged Mouse

Insulin-like growth factor-I (IGF-I) is a multifunctional polypeptide and has diverse effects on brain functions. In the present study, we compared IGF-I and IGF-I receptor (IGF-IR) immunoreactivity and their protein levels between the adult (postnatal month 6) and aged (postnatal month 24) mouse hippocampus and somatosensory cortex. In the adult hippocampus, IGF-I immunoreactivity was easily observed in the pyramidal cells of the stratum pyramidale in the hippocampus proper and in the granule cells of the granule cell layer of the dentate gyrus. In the adult somatosensory cortex, IGF-I immunoreactivity was easily found in the pyramidal cells of layer V. In the aged groups, IGF-I expression was dramatically decreased in the cells. Like the change of IGF-I immunoreactivity, IGF-IR immunoreactivity in the pyramidal and granule cells of the hippocampus and in the pyramidal cells of the somatosensory cortex was also markedly decreased in the aged group. In addition, both IGF-I and IGF-IR protein levels were significantly decreased in the aged hippocampus and somatosensory cortex. These results indicate that the apparent decrease of IGF-I and IGF-IR expression in the aged mouse hippocampus and somatosensory cortex may be related to age-related changes in the aged brain.     

Synthesis and regulation of lipoprotein lipase in the hippocampus

Lipoprotein lipase (LPL) expression was determined in adult rat hippocampus and compared to enzyme expression in other brain regions. Hippocampus LPL mRNA levels were at least 2.5-fold higher than those detected in the cerebral cortex, cerebellum, and remaining brain regions. Enzyme mass and activity levels in the hippocampus were also increased to a similar degree. De novo synthesis of LPL in the hippocampus was confirmed by [35S]methionine-labeling of the tissue and identification of a 57 kDa protein obtained by immunoprecipitation. Addition of an excess amount of bovine LPL completely prevented the immunoprecipitation of this protein. The effect of nutritional modulations on brain LPL activity was determined after a 12-h fast. While no significant changes were observed in other regions of the brain, hippocampus LPL activity in fasted rats increased by 60% compared to the fed control group. Simultaneously, fasting reduced adipose LPL activity by 60%. Intraperitoneal injection of ACTH over a 5-day period had no effect on hippocampus LPL activity, while adipose LPL levels increased 2.3-fold and heart LPL levels decreased 1.4-fold. We conclude that LPL is synthesized, active and regulated in a tissue-specific manner in the adult rat hippocampus.     

Appearance of parvalbumin-specific immunoreactivity in the cerebral cortex and hippocampus of the developing rat and gerbil brain

Summary Developmental changes in the distribution of parvalbumin-specific immunoreactivity in the brain, in particular in the cerebral cortex and hippocampus, were followed immunohistochemically in two different species, the rat and the Mongolian gerbil (Meriones unguiculatus) using an antibody raised against for rat parvalbumin. The gerbil is known to develop its auditory and visual capacity later than rat. In both the rat and gerbil, parvalbumin-specific immunoreactivity appeared after birth in both the cerebral cortex and hippocampus. The timing of the development of expression of parvalbumin varied among different parts of the cerebral cortex. The parietal cortex showed evidence of the earliest expression of parvalbumin whilst the occipital and temporal cortices expressed parvalbumin at a later stage of a development. This feature was common to both the rat and gerbil but occurred at a relatively later stage in the gerbil. The profile of the distribution of parvalbumin in the brain of the developing and adult gerbil was similar to that of the rat, but there were some differences. The frequency of bead-like structures on the dendrites of the parvalbumin-positive cells in the CA1 region of the hippocampus was markedly lower in the gerbil; instead, straight non-beaded fibers which ran vertically into the pyramidal layer were stained. Parvalbumin-positive fibers were also found in the cerebral cortex of the gerbil.     

Development of Prolonged Focal Cerebral Edema and Regional Cation Changes Following Experimental Brain Injury in the Rat

The present study examined the formation of regional cerebral edema in adult rats subjected to lateral (parasagittal) experimental fluid-percussion brain injury. Animals receiving fluid-percussion brain injury of moderate severity over the left parietal cortex were assayed for brain water content at 6 h, 24 h, and 2, 3, 5, and 7 days post injury. Regional sodium and potassium concentrations were measured in a separate group of animals at 10 min, 1 h, 6 h, and 24 h following fluid-percussion injury. Injured parietal cortex demonstrated significant edema, beginning at 6 h post injury (p less than 0.05) and persisting up to 5 days post injury. In the hippocampus ipsilateral to the site of cortical injury, significant edema occurred as early as 1 h post injury (p less than 0.05), with resolution of water accumulation beginning at 3 days. Sodium concentrations significantly increased in both injured cortex (1 h post injury, p less than 0.05) and injured hippocampus (10 min post injury, p less than 0.05). Potassium concentrations fell significantly 1 h post injury within the injured cortex (p less than 0.05), whereas significant decreases were not observed until 24 h post injury within the injured hippocampus. Cation alterations persisted throughout the 24-h post injury period. These results demonstrate that regional brain edema and cation deregulation occur in rats subjected to lateral fluid-percussion brain injury and that these changes may persist for a prolonged period after brain injury.     

Aging Differentially Affects the Loss of Neuronal Dendritic Spine,Neuroinflammation and Memory Impairment at Rats after Surgery

It is known that age is an important factor for postoperative cognitive dysfunction (POCD) and the patients with POCD suffer from the impairment of multiple brain regions and multiple brain functions. However currently animal studies of POCD mainly focus on hippocampus region, therefore in this study we performed partial hepatectomy in young adult and aged rats to test the questions (1) whether POCD in animals involves other brain areas besides hippocampus; (2) how age influences POCD of young adult and aged animals. We found that (1) in young adult rats, the memory was not significantly affected (P>0.05) 1d, 3d and 7d after partial hepatectomy, but was significantly impaired (p<0.001) in aged rats 1d and 3d post-surgery; (2) in young adult rats, the surgery did not significantly affect the densities of dendritic spines of neurons at CA1, dentate gyrus (DG) and cingulate cortex (P>0.05, respectively) 1d and 3d post-surgery, but the spine densities at CA1 and DG of aged rats were significant reduced 1d and 3d post-surgery (p<0.001, respectively), however this didn’t happen at cingulate cortex (P>0.05); (3) In young adult rats, surgery didn’t affect the activation of microglia and levels of TNF-α and IL-1β at hippocampus (P>0.05), but significantly activated microglia and increased levels of TNF-α and IL-1β at hippocampus of aged rats (P<0.05). Our data suggest that (1) partial hepatectomy-induced POCD mainly involves hippocampus impairments, and (2) differential loss of neuronal dendritic spines and neuroinflammation at hippocampus are most likely the mechanism for the formation of POCD in aged rats.