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Ovarian steroids decrease DNA fragmentation in the serotonin neurons of non-injured rhesus macaques.

Lima FB, Bethea CL - Mol. Psychiatry (2009)

Bottom Line: Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area.A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2, and the TUNEL-positive cells were counted.In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL-positive cells (Mann-Whitney test, both treatments P=0.04) and E+P treatment reduced the number of TUNEL-positive cells per mm(3) (Mann-Whitney test, P=0.04).

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA.

ABSTRACT
We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys were implanted with silastic capsules that were empty (placebo) or containing estradiol (E), progesterone (P) or estradiol and progesterone (E+P) for 1 month. In all animals, eight levels of the dorsal raphe nucleus in a rostral-to-caudal direction were immunostained using the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) method. Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area. A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2, and the TUNEL-positive cells were counted. In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL-positive cells (Mann-Whitney test, both treatments P=0.04) and E+P treatment reduced the number of TUNEL-positive cells per mm(3) (Mann-Whitney test, P=0.04). Double immunocytochemistry for TUNEL and tryptophan hydroxylase (TPH) indicated that DNA fragmentation was prominent in serotonin neurons. These data suggest that in the absence of ovarian steroids, a cascade of gene and protein expression leads to an increase in DNA fragmentation in serotonin neurons. Conversely, ovarian steroids have a neuroprotective role in the non-injured brain and prevent DNA fragmentation and cell death in serotonin neurons of nonhuman primates.

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Histograms illustrating the results of stereological analysis of the number of TUNEL-positive neurons in the dorsal raphe nucleus of rhesus macaques treated for 28 days with placebo (OVX), estradiol (E), progesterone (P) or estradiol plus progesterone (EP). TUNEL stained neurons were counted on the montage within a defined area (μ2).(A) Average (±SEM) of the total number of TUNEL-positive cells in 8 levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in the total number of TUNEL positive neurons in the P and EP- treated groups compared with the OVX group (p=0.04 for P versus Ovx and p=0.04 for EP versus Ovx, Mann-Whitney nonparametric test).(B) Average (±SEM) of the TUNEL-positive neurons per cubic millimeter (mm3) of eight levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in TUNEL-positive cells/mm3 in the E+P- treated group (p=0.04, Mann-Whitney nonparametric test), while the P group was nearly significantly different (p=0.1, Mann-Whitney nonparametric test).(C) Comparison of the total volume of the area measured in each treatment group showed no difference.
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Figure 4: Histograms illustrating the results of stereological analysis of the number of TUNEL-positive neurons in the dorsal raphe nucleus of rhesus macaques treated for 28 days with placebo (OVX), estradiol (E), progesterone (P) or estradiol plus progesterone (EP). TUNEL stained neurons were counted on the montage within a defined area (μ2).(A) Average (±SEM) of the total number of TUNEL-positive cells in 8 levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in the total number of TUNEL positive neurons in the P and EP- treated groups compared with the OVX group (p=0.04 for P versus Ovx and p=0.04 for EP versus Ovx, Mann-Whitney nonparametric test).(B) Average (±SEM) of the TUNEL-positive neurons per cubic millimeter (mm3) of eight levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in TUNEL-positive cells/mm3 in the E+P- treated group (p=0.04, Mann-Whitney nonparametric test), while the P group was nearly significantly different (p=0.1, Mann-Whitney nonparametric test).(C) Comparison of the total volume of the area measured in each treatment group showed no difference.

Mentions: Eight levels of the dorsal raphe nucleus, which were 250μ apart in a rostral to caudal direction from all 20 animals were processed for TUNEL staining (n=5 animals/group) and the results are illustrated in Figure 4. However, there was individual variation between monkeys hindering analysis of variance across all treatment groups. Nonetheless as illustrated in Figure 4A, comparison of individual groups with the OVX-placebo group using a Mann-Whitney nonparametric test confirmed that both P treatment and E+P treatment caused a significant decrease in the total number of TUNEL-positive cells in the 8 levels of dorsal raphe nucleus that were measured (p =0.04, both treatments). When the number of TUNEL-positive cells/cubic millimeter (mm3) was computed, there was a significant reduction of TUNEL-positive cells after treatment with E+P (p=0.04), and a nearly significant reduction occurred with P-alone (p=0.1) as illustrated in Figure 4B. The total area that was measured did not differ between groups (Figure 4C).


Ovarian steroids decrease DNA fragmentation in the serotonin neurons of non-injured rhesus macaques.

Lima FB, Bethea CL - Mol. Psychiatry (2009)

Histograms illustrating the results of stereological analysis of the number of TUNEL-positive neurons in the dorsal raphe nucleus of rhesus macaques treated for 28 days with placebo (OVX), estradiol (E), progesterone (P) or estradiol plus progesterone (EP). TUNEL stained neurons were counted on the montage within a defined area (μ2).(A) Average (±SEM) of the total number of TUNEL-positive cells in 8 levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in the total number of TUNEL positive neurons in the P and EP- treated groups compared with the OVX group (p=0.04 for P versus Ovx and p=0.04 for EP versus Ovx, Mann-Whitney nonparametric test).(B) Average (±SEM) of the TUNEL-positive neurons per cubic millimeter (mm3) of eight levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in TUNEL-positive cells/mm3 in the E+P- treated group (p=0.04, Mann-Whitney nonparametric test), while the P group was nearly significantly different (p=0.1, Mann-Whitney nonparametric test).(C) Comparison of the total volume of the area measured in each treatment group showed no difference.
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Related In: Results  -  Collection

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Figure 4: Histograms illustrating the results of stereological analysis of the number of TUNEL-positive neurons in the dorsal raphe nucleus of rhesus macaques treated for 28 days with placebo (OVX), estradiol (E), progesterone (P) or estradiol plus progesterone (EP). TUNEL stained neurons were counted on the montage within a defined area (μ2).(A) Average (±SEM) of the total number of TUNEL-positive cells in 8 levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in the total number of TUNEL positive neurons in the P and EP- treated groups compared with the OVX group (p=0.04 for P versus Ovx and p=0.04 for EP versus Ovx, Mann-Whitney nonparametric test).(B) Average (±SEM) of the TUNEL-positive neurons per cubic millimeter (mm3) of eight levels of the dorsal raphe nucleus in each treatment group (n=5 animals/group). There was a significant decrease in TUNEL-positive cells/mm3 in the E+P- treated group (p=0.04, Mann-Whitney nonparametric test), while the P group was nearly significantly different (p=0.1, Mann-Whitney nonparametric test).(C) Comparison of the total volume of the area measured in each treatment group showed no difference.
Mentions: Eight levels of the dorsal raphe nucleus, which were 250μ apart in a rostral to caudal direction from all 20 animals were processed for TUNEL staining (n=5 animals/group) and the results are illustrated in Figure 4. However, there was individual variation between monkeys hindering analysis of variance across all treatment groups. Nonetheless as illustrated in Figure 4A, comparison of individual groups with the OVX-placebo group using a Mann-Whitney nonparametric test confirmed that both P treatment and E+P treatment caused a significant decrease in the total number of TUNEL-positive cells in the 8 levels of dorsal raphe nucleus that were measured (p =0.04, both treatments). When the number of TUNEL-positive cells/cubic millimeter (mm3) was computed, there was a significant reduction of TUNEL-positive cells after treatment with E+P (p=0.04), and a nearly significant reduction occurred with P-alone (p=0.1) as illustrated in Figure 4B. The total area that was measured did not differ between groups (Figure 4C).

Bottom Line: Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area.A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2, and the TUNEL-positive cells were counted.In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL-positive cells (Mann-Whitney test, both treatments P=0.04) and E+P treatment reduced the number of TUNEL-positive cells per mm(3) (Mann-Whitney test, P=0.04).

View Article: PubMed Central - PubMed

Affiliation: Division of Reproductive Sciences, Oregon National Primate Research Center, Beaverton, OR 97006, USA.

ABSTRACT
We previously found that ovarian steroids promote neuroprotection in serotonin neurons by decreasing the expression of pro-apoptotic genes and proteins in the dorsal raphe nucleus of rhesus macaques, even in the absence of overt injury. In this study, we questioned whether these actions would lead to a reduction in DNA fragmentation in serotonin neurons. Ovariectomized (OVX) rhesus monkeys were implanted with silastic capsules that were empty (placebo) or containing estradiol (E), progesterone (P) or estradiol and progesterone (E+P) for 1 month. In all animals, eight levels of the dorsal raphe nucleus in a rostral-to-caudal direction were immunostained using the terminal deoxynucleotidyl transferase nick end labeling (TUNEL) method. Two staining patterns were observed, which are referred to as type I, with complete dark staining of the nucleus, and type II, with peripheral staining in the perinuclear area. A montage of the dorsal raphe was created at each level with a Marianas Stereology Microscope and Slidebook 4.2, and the TUNEL-positive cells were counted. In direct comparison with OVX animals, P treatment and E+P treatment significantly reduced the total number of TUNEL-positive cells (Mann-Whitney test, both treatments P=0.04) and E+P treatment reduced the number of TUNEL-positive cells per mm(3) (Mann-Whitney test, P=0.04). Double immunocytochemistry for TUNEL and tryptophan hydroxylase (TPH) indicated that DNA fragmentation was prominent in serotonin neurons. These data suggest that in the absence of ovarian steroids, a cascade of gene and protein expression leads to an increase in DNA fragmentation in serotonin neurons. Conversely, ovarian steroids have a neuroprotective role in the non-injured brain and prevent DNA fragmentation and cell death in serotonin neurons of nonhuman primates.

Show MeSH
Related in: MedlinePlus