Our results demonstrate that bilateral injections of the A-β25-35 fragment in the HIPP of ovariectomized female rats produce marked deficits in olfactory perception and social recognition and spatial memory as shown in Figures and . Bilateral injections of the same dose of A-β25-35 into the OB did not produce any behavioral impairment. These behavioral effects of HIPP A-25–35 β injections were associated with increased LPO and 4-NE, in both HIPP and OB; although only with injections into the HIPP did actual neuronal degeneration occur in the HIPP, as shown in Figure . These behavioral and degenerative effects of A-β25-35 injection occurred at 24 h and 8 days after treatment although they had largely disappeared by 15 days post injection. It is important to highlight that two weeks pre-treatment before A-β25-35 injection, with E2 or one or two weeks after prevented the occurrence of all perceptual and memory impairments and significantly reduced associated neurodegenerative changes. Thus, E2 treatment can play a potent role in protecting the brain from the neurotoxic effects of A-β25-35.
A-β is the main constituent of senile plaques found in the aging brain and has been extensively linked with disturbances of learning and memory processing characteristics of aging-associated disorders, such as AD [1
]. It is also known that aggregation of the amyloid peptides is responsible for neurotoxicity [20
Up to date, there is no data regarding the formation of plaques in A-β25–35
injection models. Which was neither observed in our model in any of the time points being assessed (24 h, 8 and 15 days). The injections of A-β25-35
did not produce neurodegenerative changes restricted to the region of the injection. At this point, we are unsure how the A-β25-35
spread from the HIPP to OB and vice versa despite simple transport within the cerebroventricular system seems unlikely due to the absence of effects in the frontal cortex. Instead, a more likely explanation is transport along migratory routes between the two structures. Both HIPP and OB are sites of neurogenesis within the brain but also where cells migrate from the sub-ventricular zone into both regions. [44
]. Stem cells applied intranasally have also been shown to track from olfactory regions into the HIPP [45
] so our findings may suggest a mechanism where A-β formation occurring within the OB can rapidly move into the HIPP and vice versa.
Our finding that both olfactory perception and social recognition memories were impaired following A-β25-35
injection into the HIPP was also unexpected as a previous research work suggested a role for the HIPP in social recognition memory [46
] and other forms of olfactory memory [47
] but not in olfactory perception per se
. Possibly, the profound olfactory perception deficits we observed may have been caused by the spread of A-β from the HIPP to the OB, although we did not find similar deficits following direct injection of the same A-β25-35
dose into the OB despite similar levels of lipoperoxidation. However, as a result of the olfactory perception deficits, we obviously cannot conclude that social recognition memory was impaired since this is highly dependent on odor cues [50
]. Nevertheless, since deficits in a non-odor dependent spatial memory task spontaneous alternation were also found, we can conclude that the A-β25-35
injection into the HIPP impaired both olfactory perception and spatial learning. These data suggest that neurodegeneration in HIPP could explain in part, olfactory impairment found in some neurodegenerative diseases such as Alzheimer’s.
Our findings show that oxidative stress due to A-β25-35
injection failed to produce actual neurodegeneration in the OB which was expected to happen given the effects observed following HIPP injections. However, there is evidence that the pyramidal neurons of the CA1 HIPP subfield are very sensitive to oxidative stress [51
] and so perhaps this may explain why only the HIPP show actual evidence for neurodegenerative cells thus resulting in behavioral changes. Other studies have also reported that A-β25-35
can damage the HIPP and impair learning and short-term memory [15
]. Another one has reported that bilateral injection of A-β25-35
into the amygdala of rats induced histopathological changes such as the appearance of reactive astrocytes and neuronal shrinkage, but did not cause any disturbance in spatial learning or in conditioned avoidance learning [54
]. Interestingly, in agreement with our observations, spatial memory impairments following intracerebroventricular (i.c.v) injections of A-β25-35
have also been reported to be correlated with actual neuronal cell loss in HIPP [53
LPO is a reliable marker of oxidative stress because it reflects damage to membranes and produces a variety of damaging reactive oxidizing species associated with cell death [55
]. For instance, oxidative stress caused by environmental stimuli
is proposed to be involved in brain neuronal death in many neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases [56
Previous evidence from our laboratory has shown that ozone inhalation causes oxidative stress in a number of different brain regions in rats [57
] and in this paper, we show that A-β25-35
injection in the HIPP increases LPO in it as well as in the OB compared with control groups. It is well known that HIPP is one of the key sites vulnerable to neurotoxicity in vivo
and in relation to AD [52
Our experiments showed that both behavioral and neurodegenerative impairments induced by A-β25-35
injections were transient with changes either fading or disappearing by 15 days post-injection. To the best of our knowledge, this ability of the brain to largely recover from the neurotoxic effects of A-β25-35
injections has not been reported, with most studies focusing on single time points [15
For instance, in the hippocampus, there are reports that CA1 region neurons are more susceptible to oxidative stress impairment than CA2 or CA3 neurons [60
]. The aforementioned statement means that even though similar oxidative levels are produced by the A-β25-35
injection in both sites HIPP and OB, it results in a neuronal degeneration in only the CA1 region of the hippocampus but not in the that of the olfactory bulb where the olfactory behavior remains intact even after being the A-β25-35
injected directly in the bulb. In fact, in order to produce an olfactory behavior impairment injecting the A-β25-35
in the OB, we need to administer a double dosage than that in HIPP (4 μl), (data not shown), which evidences the susceptible difference to oxidative stress between hippocampus and olfactory bulb neurons.
injection in the hippocampus produces a fluctuation in the spatial behavior [15
]. In our model, we found that there are also fluctuations in the rat’s olfactory behavior; these are observed in the first few days after A-β25-35
injection as Figures and show. However, a recovery of the olfactory behavior is observed afterwards.
It has been reported that cell neurogenesis in the subventricular area and its migration to the lesion area may partly explain this recovery [62
]. Our injection model shows that the affected neurons are those found in an adjacent A-β25-35
injected area, no bigger than 600 microns, thus the impairment does not invade other areas of the hippocampus keeping the rest of the structure’s functions intact.
Some studies have reported memory impairments following i.c.v A-β25-35
administration after periods around or in excess of 15 days [15
]. It is possible, therefore, that the brain’s capacity to compensate following A-β treatment may be increased when localized injections in the HIPP or OB are used as opposed to more global i.c.v administration. There is continuous cell migration from the subventricular and subgranular zones of the HIPP to the OB and to the HIPP itself following damage [59
]. Thus, possibly, cell migration from the subventricular zone to the OB together with neurogenesis within the OB contributed to both functional and neurodegenerative recovery by 15 days after HIPP A-β25-35
injections and E2
induced neurodegeneration is traceable by means of a Fluoro-Jade C technique which is positive from 24 hours after injection. This technique mainly stains the neurons in degeneration process [63
]. This degeneration will result in cell death and the neuronal remains will eventually vanish together with the astrogliosis and inflammatory reaction. As the Fluoro-Jade C is mainly used to signal the cells in degeneration process, the intensity of the signal gathered at day 15 is lesser than that obtained at 24 hours or 8 days later, there are scarcely left few neuronal remains, thus, less fluorescence. When we assess hippocampus cuts stained with eosin and hematoxylin after 15 days, we can observe the absence of pyramidal neurons in the injected area.
Neuroprotective actions of estradiol have been shown in a number of different contexts [29
]. The 17 β-estradiol dosage used in this research work has shown to have antioxidant effects in other models such as the exposure to ozone [57
]. In the current study, the protective effects we observed following a two week pre-treatment and a one or two weeks after E2
in ovariectomized rats were clearly very strong, with a complete absence of any olfactory perception or olfactory learning or spatial learning deficits. While, following the E2
treatment, there was still some evidence for increased lipoperoxidation and neurodegenerative changes at 24 h after A-β25-35
treatment in either HIPP or OB; this was significantly lower compared with that of A-β25-35
treatment alone. There is a significant decrease in the lipoperoxidation levels after A-β25–35
injection in the group with estradiol supplement, while in the groups without it the oxidative stress levels were higher. It can be observed that the dosage used (25 mg/kg) has an antioxidant effect which is reflected in a lower neuronal degeneration which is related to a lesser intensity of the Fluoro-Jade stain.
We have previously shown that similar E2
treatment to ovariectomized rats protects against ozone-induced olfactory memory deficits and lipoperoxidation in the olfactory system [58
]. Here, we have extended these findings to include protection against the neurodegenerative and behavioral effects of A-β.
We deliberately chose to use an ovariectomy model in order to demonstrate potential neuroprotective effects of E2
treatment since it reflects similar hormonal changes that occur in women following menopause. While the incidence of AD is significantly higher in women than in men, clear evidence that post-menopausal reductions in estrogens contribute to this as opposed to greater longevity has yet to be produced [64
], despite early influential studies suggesting otherwise [30
]. It does, however, seem that there may be a particular period of vulnerability in the early stages of menopause and there is still considerable interest in establishing potential therapeutic efficacy of estrogen treatment [64
]. At this stage, studies in rodents have reported that brain estrogens deficiency can accelerate A-β plaque formation in a transgenic mouse model of AD [67
]. It also seems to be that both estrogen α and β-receptors may contribute to increases and decreases respectively in hippocampal apolipo protein E expression [68
]. Furthermore, the potential neuroprotective mechanism whereby estrogen is acting to reduce A-β may be due to reductions in oxidative stress via the mitochondria. Clearly, we still need further evidence to support both estrogen interactions with A-β injection as well as its potential for therapeutic use in AD.