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Brown Norway F1 Hybrid Rats
Brain Research Bulletin, Vol. 43, No. 2, pp. 229–233, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
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Nerve Growth Factor, Central Nervous System Apoptosis, and Adrenocortical Activity in Aged Fischer-344/Brown Norway F1 Hybrid Rats GIULIO TAGLIALATELA, 1 ROBERT ROBINSON, MATTHEW GEGG AND J. REGINO PEREZ–POLO Department of Human Biological Chemistry & Genetics, the University of Texas Medical Branch at Galveston, Galveston, TX 77555-0652, USA [Received 27 August 1996; Accepted 16 December 1996] ABSTRACT: During aging there is a progressive loss of neuronal function in the basal forebrain that results in cognitive impairment and cholinergic deficits. While altered neurotrophin (NT)mediated signal transduction may account for some age-associated deficits, there are differences in the extent of NT responsiveness among different laboratory rat strains. Here we measured nerve growth factor (NGF) protein levels and fragmented DNA in the CNS, and basal and NGF-stimulated activity levels of the hypothalamus–pituitary–adrenocortical axis (HPAA) in 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. Our results show that while there is no age-associated differences in NGF protein levels, in aged Fischer-344/Brown Norway rats, there are increases in levels of immunoreactive fragmented DNA in the CNS and in adrenocortical responses to the peripheral administration of NGF. These data contribute to the characterization of the Fischer-344/Brown Norway F1 hybrid rat and provide baseline values useful for future studies on aged CNS. Q 1997 Elsevier Science Inc.
cholinergic neuron survival and cognitive function [10]. Thus, NT treatment has been proposed to rescue neurons from ageassociated neurodegeneration [13,27,30]. In addition to its trophic effects on cholinergic neurons of the basal forebrain, NGF may also play a role as a cytokine modulating the neuroendocrine stress response. The intravenous injection of NGF stimulates the activity of the hypothalamus–pituitary–adrenocortical axis (HPAA) in the rat [29,37], the main neuroendocrine axis activated in response to stressful stimuli [9]. In mice, NGF is released from the submaxillary glands into peripheral circulation in response to stressful stimuli [18] and, in rats, treatment with antibodies to NGF significantly reduces stress-induced increases in plasma corticosteroids [37], consistent with the hypothesis that NGF has a role in HPAA modulation. The observation that glucocorticoids and stress stimuli affect NGF and NGF receptor expression in the basal forebrain, hypothalamus, and hippocampus suggests that there is a physiological relationship between the stress-related release of corticosteroids and NGF action in the CNS [34]. There is evidence that there is involvement of stress-induced corticosteroids in the processes of age-associated neuronal cell death [33,36]. Thus, the role of NGF in both the modulation of the stress-induced activity of the HPAA and the rescue of apoptotic aged neurons suggests that NGF provides protection to neurons against glucocorticoid-induced cytotoxicity. The use of a common animal model for aging studies is particularly useful. The Fischer-344/Brown Norway F1 hybrid rats has been proposed as an elective animal model in aging studies [35]. Here, we report on NGF, CNS apoptosis levels, and HPAA activity in aged Fischer-344/Brown Norway hybrid rats.
KEY WORDS: Nerve growth factor, Neuronal death, Fragmented DNA, Corticosterone, Adrenocortical axis.
INTRODUCTION Neuronal apoptotic degeneration in Alzheimer’s disease and extreme aging results in impaired cognitive function [6,27,39,40]. Some of the affected neurons depend on their survival on a constant supply of factors such as neurotrophins (NT) [13,15,21,30]. The NT family is made up of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT4/5, and NT-6 [11,12,16,20,24]. An obligatory step for the action of neurotrophins is their binding to specific high-affinity tyrosine kinase receptors [4,7,17] and to the common low-affinity p75NTR receptor [5,7,32]. In the central nervous system (CNS), NGF receptors synthesized in the cholinergic neurons of the basal forebrain are anterogradely transported to the axon terminals, where they bind the NGF secreted by target neurons. This NGF is internalized and retrogradely transported back to the basal forebrain [21]. The continuous flux of NGF and NGF receptors between the septum and hippocampus is essential for 1
MATERIALS AND METHODS Animals Three-, 18-, and 30-month-old Fischer-344/Brown Norway F1 hybrid male rats were purchased from NIA colonies kept at Harlan Sprague–Dawley (Indianapolis, IN). Rats were maintained in an approved animal care facility at 37 { 27C temperature, 12-h light cycle, food and water ad lib.
To whom requests for reprints should be addressed.
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Experimental Procedure For sacrifice, rats were deeply anesthetized by exposure to 100% CO2 and, within 15–20 s from the beginning of CO2 exposure, decapitated according to ACUC-approved protocols. Upon sacrifice, trunk blood was collected in prechilled plastic tubes and left to clot overnight prior to centrifugation to prepare serum. Renin-free mouse b(2.5S)NGF, purified from adult mouse submaxillary gland as previously described [26] was diluted in phosphate-buffered saline and injected intraperitoneally at a dose of 10 pmol/g body weight. NGF Assay Tissue samples were disrupted by sonication in 31 vol (w:v) of extraction buffer (100 mM Tris-HCl; 400 mM NaCl; 2% BSA; 0.05% NaN3 ; 1 mM PMSF; 7 mg/ml Aprotinin; 4 mM EDTA; pH 7.0) and centrifuged at 12,000 1 g for 20 min at 47C. After centrifugation, the supernatant was collected into fresh tubes and added with an equal volume of tissue solution (20 mM CaCl2 ; 0.2% Triton X-100) and thoroughly mixed. One-hundred microliters of aliquot from each sample were then added in triplicate to the wells of a 96-well plate that were previously coated with 0.2 mg/ml monoclonal antibody anti-NGF (Boehringer–Mannheim, clone 27/21) in coating buffer (50 mM Na2CO3 /NaHCO3 buffer, pH 9.6; 0.1% NaN3 ). The standard curve was prepared by adding to parallel wells increasing concentrations of NGF ranging from 7.8 to 500 pg/ml of standard buffer (50 mM TrisHCl; 200 mM NaCl; 10 mM CaCl2 ; 1% BSA; 0.2% Triton X100; 0.1% NaN3 ; pH 7.0). After incubation overnight at 47C, the wells were washed 31 with wash buffer (50 mM Tris-HCl; 200 mM NaCl; 10 mM CaCl2 ; 0.1% Triton X-100; 0.05% NaN3 ; pH 7.0) and 150 ml conjugate solution (same as sample buffer, containing 0.08 U/ml of a monoclonal antibody to NGF conjugated with b-galactosidase) was added. Following incubation for 4 h at 377C, the wells were washed with wash buffer, added with 200 ml of substrate solution (100 mM HEPES; 150 mM NaCl; 2 mM MgCl2 ; 0.1% NaN3 ; 1% BSA; pH 7.0, containing 2 mg/ml chlorophenol red-b-galactopyranoside) and incubated at 377C for 1 additional hour. The intensity of the color developed for each well was then assayed in an ELISA plate reader at 570 nm wavelength emission. The amount of NGF in the samples was calculated by comparison with the readings of the standard curve and the results expressed as pg of NGF per 100 mg of wet weight of tissue.
cubation and two washes, we add the secondary antibody (antiDNA) conjugated with horseradish peroxidase. At the end of an additional period of incubation, the wells are treated with the chromogen substrate and the intensity of the color developed is assayed with an ELISA plate reader at 405/490 nm wavelength. Statistical Analysis Statistical differences between groups were assessed by analysis of variance (ANOVA) followed by the Fisher’s least statistical difference (LSD) tests for multiple comparisons. An a level below 5% (p õ 0.05) between groups was considered statistically significant. When multiple measurements were performed on the same animal (NGF assays and DNA fragmentation assays), the data were first analyzed by multiple measurements ANOVA to assess interactions between age and the multiple measured parameter. If a significant interaction was found, significant differences among individual groups were assesses by ANOVA and LSD test as described above. RESULTS Figure 1 reports the results of a two-site ELISA specific for murine NGF performed in the hippocampus, basal forebrain, and frontal cortex of 3-, 18-, and 30-month-old Fischer-344/Brown Norway hybrid rats. The relative NGF levels in the hippocampus, basal forebrain, and frontal cortex were consistent with those previously reported [31,38]. Multiple measurement ANOVA showed no significant age-dependent differences in the levels of NGF [age 1 brain region: F (4) Å 1.77, p Å 0.1618, NS]. Upon sacrifice, the trunk blood from these animals was also collected and the serum prepared for corticosterone assays. As shown in Fig. 1, we observed a progressive age-related increase in the serum levels of corticosterone that reached a statistical significant differnce at 30 months of age. The stimulatory effect of a peripheral injection of NGF on the activity of the HPA axis [18,37] was assessed in 3- and 30month-old Fischer-344/Brown Norway rats by administering NGF intraperitoneally and assaying serum corticosterone levels (as an index of adrenocortical activation) 1 h later (Fig. 2). We observed a significant NGF-related induction of serum corticosterone release in both 3- and 30-month-old rats. Notably, the
Corticosterone Assay Levels of serum corticosterone were assayed with a commercially available rat corticosterone radio immune assay (RIA) kit (ICN) employing a monoclonal antibody to corticosterone as carrier, accordingly to the manufacturer’s instructions. Fragmented DNA Detection by Enzyme-Linked Immunosorbant Assay (ELISA) The amount of fragmented DNA in CNS tissue was measured by a specific two-site ELISA employing antihistone as primary antibody and anti-DNA as secondary antibody according to the manufacturer’s instructions (Boehringer–Mannheim, Terre Haute, CA). Briefly, tissues were gently homogenized using a hand-operated glass-glass homogenizer in sample buffer. After resting 30 min at 47C, the samples were centrifugated for 20 min at 15,000 1 g. The supernatant (cytosol containing low-molecular weight, fragmented, DNA) was then diluted 1:50 (v:v) with sample buffer and 0.1 ml of the solution pipetted into the wells of a 96-well plate precoated with antihistone antibody. After in-
FIG. 1. NGF content in the hippocampus (HIPP), frontal cortex (FCX) and basal forebrain area (BFA) and basal serum corticosterone (CORT, right ordinate axis) levels in 3-, 18-, and 30-month-old Fischer-344/ Brown Norway hybrid rats. *p õ 0.001 vs. 3-month-old rats (ANOVA / Fisher’s LSD test). n Å 6 rats per group.
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CNS AGING MARKERS IN THE FISCHER/BN RAT NGF-induced increase of serum corticosterone levels in 30month-old rats was significantly higher when compared to that in 3-month-old animals. Figure 3 shows the results of an ELISA detecting nucleosomeassociated fragmented DNA in the cytosolic fraction of CNS area homogenates from 3-, 18-, and 30-month-old Fischer-344/ Brown Norway hybrid rats. Multiple measurements ANOVA revealed a significant interaction between age and DNA fragmentation levels throught the different brain areas, F (8) Å 3.73, p õ 0.005. When the data within individual brain regions were analyzed by ANOVA followed by Fisher’s LSD test, the amount of immune-reactive fragmented DNA in the hippocampus and basal forebrain of 30-month-old rats was significantly higher than in the same CNS areas of 18-month-old rats, whereas it did not differ from that in the 3-month-old animals. On the other hand, fragmented DNA levels in the frontal cortex of the 30-monthold rats were statistically higher then those in the frontal cortex of both 18-month- and 3-month-old animals. There was no agerelated difference in the amount of fragmented DNA in the striatum or the cerebellum.
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FIG. 3. Levels of nucleosome-associated fragmented DNA in the hippocampus (HIPP), basal forebrain area (BFA), frontal cortex (FCX), striatum (STR), and cerebellum (CB) of 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. *p õ 0.05 vs. 18-month-old rats; # p õ 0.05 vs. 3-month- and 18-month-old rats (ANOVA / Fisher’s LSD test). n Å 5 rats per group.
DISCUSSION We measured tissue NGF content in the CNS of 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats using a two-site ELISA specific for murine NGF. The relative amounts of NGF in the hippocampus, frontal cortex, and basal forebrain were comparable to those described for other strains [31,38]. When we compared the NGF levels present in hippocampus, frontal cortex, and basal forebrain of 3-, 18-, and 30-month-old rats, we could not detect significant age-associated differences. Previous reports on NGF levels aged CNS are not consistent. There are reports of aged-associated declines, increases, or no changes in NGF content in the CNS of aged rats [1,8,14,19,31]. These differences are not strain dependent (e.g., compare [1] to [14]). Rather, one could conclude that NGF levels in the rodent CNS do not correlate directly with biological age. It may be that factors implicit to the experimental conditions used affect the measurements. For example, NT levels in the rodent CNS are affected by stress and glucocorticoid circulating levels [18,22,34]. Thus, differences in experimental animal manipulation prior to sacrifice could alter stress levels and circulating glucocorticoid levels, which in turn, would affect CNS NT levels, and explain hetero-
FIG. 2. Serum corticosterone levels in 3-month- and 30-month-old Fischer-344/Brown Norway hybrid rats 30 min after peripheral administration of NGF (10 pmol/g b.wt.). *p õ 0.05 and *** p õ 0.001, vs. cyt.C group or vs. the 3-month-old NGF-injected group, as indicated (ANOVA / Fisher’s LSD test). n Å 5 rats per group.
geneity of observations. This is particularly likely given that aged rats have an exaggerated stress response and exhibit a general disinhibition of the HPAA activity [9,25]. Consistent with reports describing a dishinibition of the HPAA in aged rats [9,25], we observed a significant age-related increase in basal (not stress-activated) serum corticosterone levels in 18- and 30-month-old Fischer-344/Brown Norway rats as compared to young (3-month-old) animals. According to the hypothesis stated above, one possibility could be that manipulations prior to animal sacrifice could have induced a stress response in our experimental rats, thereby leading to significantly increased plasma corticosterone levels, which would be more elevated in the aged animals [9,25]. This possibility, however, seems unlikely because the rats have been sacrificed in a random age order within 1 min from entering the sacrifice room and within 15–20 s from the beginning of the CO2 exposure that, according to ACUC-approved procedures for laboratory animal handling, we use before decapitation, a time insufficient to lead to measureable increases in plasma corticosterone levels due to a handling-related stress response. We have previously shown that intravenously delivered NGF stimulates the HPAA centrally [37]. Intravenously injected 125INGF is taken up into the CNS where it accumulates in the hippocampus after 1 h but is cleared by 6 h [23]. We hypothesized that peripheral NGF stimulates the HPAA by acting via NGF receptors in the hippocampus, a limbic structure that plays an important role in the modulation of the HPAA activity [9], or in structures projecting to the hippocampus. Injection of 30-monthold Fischer-344/Brown Norway rats with NGF resulted in increased corticosterone serum levels compared to control aged rats or injected 3-month-old rats. The one observation of reduced HPAA responses to NGF injections in aged rats is in the Sprague–Dawley rats [2], consistent with the observation that aged Sprague–Dawley rats have lower NGF receptor levels in the hippocampus [3]. On the other hand, we have preliminary data suggesting that NGF receptor levels in the hippocampus of aged Fischer-344/Brown Norway hybrid rats are not reduced as compared to young animals (not shown), which would explain the difference of HPAA responses to exogenously administered NGF between these two rat strains. Further ongoing studies will address this issue. The significantly higher HPAA response observed in 30-month-old Fischer- 344/Brown Norway hybrid rats
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would then be consistent with the documented higher responses of aged rats to inducers of HPAA activity [9], of which NGF may be one more example [18,37]. We have reported that there is DNA fragmentation, as measured by both DNA gel electrophoresis and by ELISA, in the CNS of 24month-old Fischer-344 rats, consistent with the idea that apoptosis may be responsible for age-associated neurodegeneration [6,39,40]. Here we expand these observations to the Fischer-344/Brown Norway hybrid rat at 3, 18, and 30 months of age. While there was clearly a significant increase in fragmented DNA in the CNS of 30month-old rats as compared to the 18-month-old rats, comparisons to 3-month-old rats showed no significant differences, thus indicating that in both 3- and 30-month-old rat CNS there is the presence of fragmented DNA. Presently, we cannot explain the presence of fragmented DNA in the CNS of 3-month-old Fischer-344/Brown Norway rats. In the nervous system, developmentally regulated apoptotic neuron death due to limiting supplies of trophic factors (neuronal pruning) occurs during the perinatal age [28]. Therefore, it seems unlikely that the fragmented DNA observed here in young rats may reflect ongoing developmental neuronal pruning. It is puzzling, however, that elevated DNA fragmentation is observed only in the hippocampus and the basal forebrain, a cholinergic circuitry known to depend upon hippocampal-derived NTs [11,23]. Further studies that will focus on ages between birth and 3 months are needed to clarify the nature of the fragmented DNA present in the CNS of young Fischer-344/Brown Norway hybrid rats. In conclusion, our results show that there are both similarities and differences between Fischer-344/Brown Norway hybrid rats and other rat strains in terms of adrenocortical activity, NGF, and apoptosis in the aged CNS.
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21. ACKNOWLEDGEMENTS
22. This work was supported by grants from the American Federation for Aging Research (AFAR), the UTMB Center on Aging and the UTMB Small Grant Program awarded to G.T. This is publication no. 75 of the USPHS Grant P01 AG10514 awarded by NIA (J.R.P.-P.).
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233 the hypothalamo–pituitary–adrenocortical axis during the stress response. Endocrinology 129:2212–2218; 1991. 38. Taglialatela, G.; Navarra, D.; Cruciani, R.; Ramacci, M. T.; Angelucci, L. Acetyl-L-carnitine increases nerve growth factor levels and choline acetyltransferase activity in the CNS of aged rats. Exp. Gerontol. 29:55–66; 1994. 39. Taglialatela, G.; Gegg, M.; Perez–Polo, J. R.; Williams, L. R.; Rose, G. M. Evidence for DNA fragmentation in the CNS of aged Fischer344 rats. Neuroreport 7:977–980; 1996. 40. Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science 267:1456–1462; 1995.
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Nerve Growth Factor, Central Nervous System Apoptosis, and Adrenocortical Activity in Aged Fischer-344/Brown Norway F1 Hybrid Rats GIULIO TAGLIALATELA, 1 ROBERT ROBINSON, MATTHEW GEGG AND J. REGINO PEREZ–POLO Department of Human Biological Chemistry & Genetics, the University of Texas Medical Branch at Galveston, Galveston, TX 77555-0652, USA [Received 27 August 1996; Accepted 16 December 1996] ABSTRACT: During aging there is a progressive loss of neuronal function in the basal forebrain that results in cognitive impairment and cholinergic deficits. While altered neurotrophin (NT)mediated signal transduction may account for some age-associated deficits, there are differences in the extent of NT responsiveness among different laboratory rat strains. Here we measured nerve growth factor (NGF) protein levels and fragmented DNA in the CNS, and basal and NGF-stimulated activity levels of the hypothalamus–pituitary–adrenocortical axis (HPAA) in 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. Our results show that while there is no age-associated differences in NGF protein levels, in aged Fischer-344/Brown Norway rats, there are increases in levels of immunoreactive fragmented DNA in the CNS and in adrenocortical responses to the peripheral administration of NGF. These data contribute to the characterization of the Fischer-344/Brown Norway F1 hybrid rat and provide baseline values useful for future studies on aged CNS. Q 1997 Elsevier Science Inc.
cholinergic neuron survival and cognitive function [10]. Thus, NT treatment has been proposed to rescue neurons from ageassociated neurodegeneration [13,27,30]. In addition to its trophic effects on cholinergic neurons of the basal forebrain, NGF may also play a role as a cytokine modulating the neuroendocrine stress response. The intravenous injection of NGF stimulates the activity of the hypothalamus–pituitary–adrenocortical axis (HPAA) in the rat [29,37], the main neuroendocrine axis activated in response to stressful stimuli [9]. In mice, NGF is released from the submaxillary glands into peripheral circulation in response to stressful stimuli [18] and, in rats, treatment with antibodies to NGF significantly reduces stress-induced increases in plasma corticosteroids [37], consistent with the hypothesis that NGF has a role in HPAA modulation. The observation that glucocorticoids and stress stimuli affect NGF and NGF receptor expression in the basal forebrain, hypothalamus, and hippocampus suggests that there is a physiological relationship between the stress-related release of corticosteroids and NGF action in the CNS [34]. There is evidence that there is involvement of stress-induced corticosteroids in the processes of age-associated neuronal cell death [33,36]. Thus, the role of NGF in both the modulation of the stress-induced activity of the HPAA and the rescue of apoptotic aged neurons suggests that NGF provides protection to neurons against glucocorticoid-induced cytotoxicity. The use of a common animal model for aging studies is particularly useful. The Fischer-344/Brown Norway F1 hybrid rats has been proposed as an elective animal model in aging studies [35]. Here, we report on NGF, CNS apoptosis levels, and HPAA activity in aged Fischer-344/Brown Norway hybrid rats.
KEY WORDS: Nerve growth factor, Neuronal death, Fragmented DNA, Corticosterone, Adrenocortical axis.
INTRODUCTION Neuronal apoptotic degeneration in Alzheimer’s disease and extreme aging results in impaired cognitive function [6,27,39,40]. Some of the affected neurons depend on their survival on a constant supply of factors such as neurotrophins (NT) [13,15,21,30]. The NT family is made up of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT-3, NT4/5, and NT-6 [11,12,16,20,24]. An obligatory step for the action of neurotrophins is their binding to specific high-affinity tyrosine kinase receptors [4,7,17] and to the common low-affinity p75NTR receptor [5,7,32]. In the central nervous system (CNS), NGF receptors synthesized in the cholinergic neurons of the basal forebrain are anterogradely transported to the axon terminals, where they bind the NGF secreted by target neurons. This NGF is internalized and retrogradely transported back to the basal forebrain [21]. The continuous flux of NGF and NGF receptors between the septum and hippocampus is essential for 1
MATERIALS AND METHODS Animals Three-, 18-, and 30-month-old Fischer-344/Brown Norway F1 hybrid male rats were purchased from NIA colonies kept at Harlan Sprague–Dawley (Indianapolis, IN). Rats were maintained in an approved animal care facility at 37 { 27C temperature, 12-h light cycle, food and water ad lib.
To whom requests for reprints should be addressed.
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Experimental Procedure For sacrifice, rats were deeply anesthetized by exposure to 100% CO2 and, within 15–20 s from the beginning of CO2 exposure, decapitated according to ACUC-approved protocols. Upon sacrifice, trunk blood was collected in prechilled plastic tubes and left to clot overnight prior to centrifugation to prepare serum. Renin-free mouse b(2.5S)NGF, purified from adult mouse submaxillary gland as previously described [26] was diluted in phosphate-buffered saline and injected intraperitoneally at a dose of 10 pmol/g body weight. NGF Assay Tissue samples were disrupted by sonication in 31 vol (w:v) of extraction buffer (100 mM Tris-HCl; 400 mM NaCl; 2% BSA; 0.05% NaN3 ; 1 mM PMSF; 7 mg/ml Aprotinin; 4 mM EDTA; pH 7.0) and centrifuged at 12,000 1 g for 20 min at 47C. After centrifugation, the supernatant was collected into fresh tubes and added with an equal volume of tissue solution (20 mM CaCl2 ; 0.2% Triton X-100) and thoroughly mixed. One-hundred microliters of aliquot from each sample were then added in triplicate to the wells of a 96-well plate that were previously coated with 0.2 mg/ml monoclonal antibody anti-NGF (Boehringer–Mannheim, clone 27/21) in coating buffer (50 mM Na2CO3 /NaHCO3 buffer, pH 9.6; 0.1% NaN3 ). The standard curve was prepared by adding to parallel wells increasing concentrations of NGF ranging from 7.8 to 500 pg/ml of standard buffer (50 mM TrisHCl; 200 mM NaCl; 10 mM CaCl2 ; 1% BSA; 0.2% Triton X100; 0.1% NaN3 ; pH 7.0). After incubation overnight at 47C, the wells were washed 31 with wash buffer (50 mM Tris-HCl; 200 mM NaCl; 10 mM CaCl2 ; 0.1% Triton X-100; 0.05% NaN3 ; pH 7.0) and 150 ml conjugate solution (same as sample buffer, containing 0.08 U/ml of a monoclonal antibody to NGF conjugated with b-galactosidase) was added. Following incubation for 4 h at 377C, the wells were washed with wash buffer, added with 200 ml of substrate solution (100 mM HEPES; 150 mM NaCl; 2 mM MgCl2 ; 0.1% NaN3 ; 1% BSA; pH 7.0, containing 2 mg/ml chlorophenol red-b-galactopyranoside) and incubated at 377C for 1 additional hour. The intensity of the color developed for each well was then assayed in an ELISA plate reader at 570 nm wavelength emission. The amount of NGF in the samples was calculated by comparison with the readings of the standard curve and the results expressed as pg of NGF per 100 mg of wet weight of tissue.
cubation and two washes, we add the secondary antibody (antiDNA) conjugated with horseradish peroxidase. At the end of an additional period of incubation, the wells are treated with the chromogen substrate and the intensity of the color developed is assayed with an ELISA plate reader at 405/490 nm wavelength. Statistical Analysis Statistical differences between groups were assessed by analysis of variance (ANOVA) followed by the Fisher’s least statistical difference (LSD) tests for multiple comparisons. An a level below 5% (p õ 0.05) between groups was considered statistically significant. When multiple measurements were performed on the same animal (NGF assays and DNA fragmentation assays), the data were first analyzed by multiple measurements ANOVA to assess interactions between age and the multiple measured parameter. If a significant interaction was found, significant differences among individual groups were assesses by ANOVA and LSD test as described above. RESULTS Figure 1 reports the results of a two-site ELISA specific for murine NGF performed in the hippocampus, basal forebrain, and frontal cortex of 3-, 18-, and 30-month-old Fischer-344/Brown Norway hybrid rats. The relative NGF levels in the hippocampus, basal forebrain, and frontal cortex were consistent with those previously reported [31,38]. Multiple measurement ANOVA showed no significant age-dependent differences in the levels of NGF [age 1 brain region: F (4) Å 1.77, p Å 0.1618, NS]. Upon sacrifice, the trunk blood from these animals was also collected and the serum prepared for corticosterone assays. As shown in Fig. 1, we observed a progressive age-related increase in the serum levels of corticosterone that reached a statistical significant differnce at 30 months of age. The stimulatory effect of a peripheral injection of NGF on the activity of the HPA axis [18,37] was assessed in 3- and 30month-old Fischer-344/Brown Norway rats by administering NGF intraperitoneally and assaying serum corticosterone levels (as an index of adrenocortical activation) 1 h later (Fig. 2). We observed a significant NGF-related induction of serum corticosterone release in both 3- and 30-month-old rats. Notably, the
Corticosterone Assay Levels of serum corticosterone were assayed with a commercially available rat corticosterone radio immune assay (RIA) kit (ICN) employing a monoclonal antibody to corticosterone as carrier, accordingly to the manufacturer’s instructions. Fragmented DNA Detection by Enzyme-Linked Immunosorbant Assay (ELISA) The amount of fragmented DNA in CNS tissue was measured by a specific two-site ELISA employing antihistone as primary antibody and anti-DNA as secondary antibody according to the manufacturer’s instructions (Boehringer–Mannheim, Terre Haute, CA). Briefly, tissues were gently homogenized using a hand-operated glass-glass homogenizer in sample buffer. After resting 30 min at 47C, the samples were centrifugated for 20 min at 15,000 1 g. The supernatant (cytosol containing low-molecular weight, fragmented, DNA) was then diluted 1:50 (v:v) with sample buffer and 0.1 ml of the solution pipetted into the wells of a 96-well plate precoated with antihistone antibody. After in-
FIG. 1. NGF content in the hippocampus (HIPP), frontal cortex (FCX) and basal forebrain area (BFA) and basal serum corticosterone (CORT, right ordinate axis) levels in 3-, 18-, and 30-month-old Fischer-344/ Brown Norway hybrid rats. *p õ 0.001 vs. 3-month-old rats (ANOVA / Fisher’s LSD test). n Å 6 rats per group.
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CNS AGING MARKERS IN THE FISCHER/BN RAT NGF-induced increase of serum corticosterone levels in 30month-old rats was significantly higher when compared to that in 3-month-old animals. Figure 3 shows the results of an ELISA detecting nucleosomeassociated fragmented DNA in the cytosolic fraction of CNS area homogenates from 3-, 18-, and 30-month-old Fischer-344/ Brown Norway hybrid rats. Multiple measurements ANOVA revealed a significant interaction between age and DNA fragmentation levels throught the different brain areas, F (8) Å 3.73, p õ 0.005. When the data within individual brain regions were analyzed by ANOVA followed by Fisher’s LSD test, the amount of immune-reactive fragmented DNA in the hippocampus and basal forebrain of 30-month-old rats was significantly higher than in the same CNS areas of 18-month-old rats, whereas it did not differ from that in the 3-month-old animals. On the other hand, fragmented DNA levels in the frontal cortex of the 30-monthold rats were statistically higher then those in the frontal cortex of both 18-month- and 3-month-old animals. There was no agerelated difference in the amount of fragmented DNA in the striatum or the cerebellum.
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FIG. 3. Levels of nucleosome-associated fragmented DNA in the hippocampus (HIPP), basal forebrain area (BFA), frontal cortex (FCX), striatum (STR), and cerebellum (CB) of 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats. *p õ 0.05 vs. 18-month-old rats; # p õ 0.05 vs. 3-month- and 18-month-old rats (ANOVA / Fisher’s LSD test). n Å 5 rats per group.
DISCUSSION We measured tissue NGF content in the CNS of 3-, 18-, and 30-month-old Fischer-344/Brown Norway rats using a two-site ELISA specific for murine NGF. The relative amounts of NGF in the hippocampus, frontal cortex, and basal forebrain were comparable to those described for other strains [31,38]. When we compared the NGF levels present in hippocampus, frontal cortex, and basal forebrain of 3-, 18-, and 30-month-old rats, we could not detect significant age-associated differences. Previous reports on NGF levels aged CNS are not consistent. There are reports of aged-associated declines, increases, or no changes in NGF content in the CNS of aged rats [1,8,14,19,31]. These differences are not strain dependent (e.g., compare [1] to [14]). Rather, one could conclude that NGF levels in the rodent CNS do not correlate directly with biological age. It may be that factors implicit to the experimental conditions used affect the measurements. For example, NT levels in the rodent CNS are affected by stress and glucocorticoid circulating levels [18,22,34]. Thus, differences in experimental animal manipulation prior to sacrifice could alter stress levels and circulating glucocorticoid levels, which in turn, would affect CNS NT levels, and explain hetero-
FIG. 2. Serum corticosterone levels in 3-month- and 30-month-old Fischer-344/Brown Norway hybrid rats 30 min after peripheral administration of NGF (10 pmol/g b.wt.). *p õ 0.05 and *** p õ 0.001, vs. cyt.C group or vs. the 3-month-old NGF-injected group, as indicated (ANOVA / Fisher’s LSD test). n Å 5 rats per group.
geneity of observations. This is particularly likely given that aged rats have an exaggerated stress response and exhibit a general disinhibition of the HPAA activity [9,25]. Consistent with reports describing a dishinibition of the HPAA in aged rats [9,25], we observed a significant age-related increase in basal (not stress-activated) serum corticosterone levels in 18- and 30-month-old Fischer-344/Brown Norway rats as compared to young (3-month-old) animals. According to the hypothesis stated above, one possibility could be that manipulations prior to animal sacrifice could have induced a stress response in our experimental rats, thereby leading to significantly increased plasma corticosterone levels, which would be more elevated in the aged animals [9,25]. This possibility, however, seems unlikely because the rats have been sacrificed in a random age order within 1 min from entering the sacrifice room and within 15–20 s from the beginning of the CO2 exposure that, according to ACUC-approved procedures for laboratory animal handling, we use before decapitation, a time insufficient to lead to measureable increases in plasma corticosterone levels due to a handling-related stress response. We have previously shown that intravenously delivered NGF stimulates the HPAA centrally [37]. Intravenously injected 125INGF is taken up into the CNS where it accumulates in the hippocampus after 1 h but is cleared by 6 h [23]. We hypothesized that peripheral NGF stimulates the HPAA by acting via NGF receptors in the hippocampus, a limbic structure that plays an important role in the modulation of the HPAA activity [9], or in structures projecting to the hippocampus. Injection of 30-monthold Fischer-344/Brown Norway rats with NGF resulted in increased corticosterone serum levels compared to control aged rats or injected 3-month-old rats. The one observation of reduced HPAA responses to NGF injections in aged rats is in the Sprague–Dawley rats [2], consistent with the observation that aged Sprague–Dawley rats have lower NGF receptor levels in the hippocampus [3]. On the other hand, we have preliminary data suggesting that NGF receptor levels in the hippocampus of aged Fischer-344/Brown Norway hybrid rats are not reduced as compared to young animals (not shown), which would explain the difference of HPAA responses to exogenously administered NGF between these two rat strains. Further ongoing studies will address this issue. The significantly higher HPAA response observed in 30-month-old Fischer- 344/Brown Norway hybrid rats
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would then be consistent with the documented higher responses of aged rats to inducers of HPAA activity [9], of which NGF may be one more example [18,37]. We have reported that there is DNA fragmentation, as measured by both DNA gel electrophoresis and by ELISA, in the CNS of 24month-old Fischer-344 rats, consistent with the idea that apoptosis may be responsible for age-associated neurodegeneration [6,39,40]. Here we expand these observations to the Fischer-344/Brown Norway hybrid rat at 3, 18, and 30 months of age. While there was clearly a significant increase in fragmented DNA in the CNS of 30month-old rats as compared to the 18-month-old rats, comparisons to 3-month-old rats showed no significant differences, thus indicating that in both 3- and 30-month-old rat CNS there is the presence of fragmented DNA. Presently, we cannot explain the presence of fragmented DNA in the CNS of 3-month-old Fischer-344/Brown Norway rats. In the nervous system, developmentally regulated apoptotic neuron death due to limiting supplies of trophic factors (neuronal pruning) occurs during the perinatal age [28]. Therefore, it seems unlikely that the fragmented DNA observed here in young rats may reflect ongoing developmental neuronal pruning. It is puzzling, however, that elevated DNA fragmentation is observed only in the hippocampus and the basal forebrain, a cholinergic circuitry known to depend upon hippocampal-derived NTs [11,23]. Further studies that will focus on ages between birth and 3 months are needed to clarify the nature of the fragmented DNA present in the CNS of young Fischer-344/Brown Norway hybrid rats. In conclusion, our results show that there are both similarities and differences between Fischer-344/Brown Norway hybrid rats and other rat strains in terms of adrenocortical activity, NGF, and apoptosis in the aged CNS.
11. 12. 13. 14.
15. 16.
17. 18. 19.
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21. ACKNOWLEDGEMENTS
22. This work was supported by grants from the American Federation for Aging Research (AFAR), the UTMB Center on Aging and the UTMB Small Grant Program awarded to G.T. This is publication no. 75 of the USPHS Grant P01 AG10514 awarded by NIA (J.R.P.-P.).
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