Introduction
To age in a healthy way is very important to the population that grows older. The capability of being able to recover from certain stresses and to be able to distinguish between similar memory patterns might be determined by AHN, or adult hippocampal neurogenesis. Adult hippocampal neurogenesis is said to decline with age in mice, as well as primates that are non-human. However, in humans, the dentate gyrus of the adult hippocampus is able to generate new neurons while aging even past the middle-age. Still, there is still not enough information or measurable studies on how much neurogenesis occurs in the human race, which is why the authors of this study decided to research and experiment with this topic of developmental biology.
Humans, compared to rodents, are different in their phylogenetics, since they call for altered arrangement of the neuronal maturation stages in the dentate gyrus of humans. So, for example, olfactory bulb neurogenesis is not found in humans, while striatal neurogenesis is unique to humans. There have been earlier studies on the analysis of human AHN, however, they did not take the effects of age into consideration. In addition, there were other studies that focused on AHN in the older population, but they did not take drug use or medication (which affect AHN) into consideration. These research groups also had trouble differentiating immature and mature neurons, which in turn led to most of these groups theorizing that dentate gyrus neurons did not reduce as human age. Some groups used in vivo brain-imaging observations, which gave contradictory results in regard to age-related changes of the hippocampal areas. This was due to poor spatial resolution, as well as, not being able to tell the differences between the sub-regions of the hippocampus.
For this study, the authors stated that angiogenesis and AHN are co-regulated. The blood volume in the brain is said to increase when exercised, and as a result of this, there is better cognitive performance of humans and more AHN in mice as well. However, cerebral blood volume may have a reduced effect in people who are older. So, for this study, the authors calculated dentate gyri (DG) volume, adult hippocampal neurogenesis (AHN), and angiogenesis and their association with differently-aged people.
The hypothesis of this particular study was that DG volume, AHN, and angiogenesis will simultaneously decrease as a person ages; moreover, all three are assumed to be correlated with each other. Because of AHN being so persistent throughout a human’s life, elderly people have major potential to stay emotionally and cognitively more complete than most people believe. On the contrary, the reduction in resilience of cognitive-emotion may be caused by factors such as: diminished angiogenesis, smaller quiescent neural progenitor pool (QNPs), or decreasing neuroplasticity in the anterior subsection of the dentate gyrus.
Since the rostral and caudal sections of the dentate gyrus have different functions, the authors of this study evaluated the posterior, mid, and anterior hippocampus post-mortem. The study included 28 woman and men, all from ages 14 to 79 years. For each region of the hippocampus, the researchers measured and categorized cells, volume, and angiogenesis at different maturation periods in the neurogenic niche in the dentate gyrus. And this was done using stereological methods for unbiased results. The 28 subjects that were observed in this experiment were chosen based off of them not having any neuropsychiatric treatment or disease; they did this to avoid confounders throughout the study.
Results
So, for a new generation of neurons to arise in the DG neurogenic process, it begins with QNPs, or quiescent radial-glia-like type I neural progenitor cells. These QNPs express brain lipid-binding proteins (BLBPs), glial fibrillary acid protein (GFAP), nestin, and the sex determining region Y-box 2 (Sox2). The QNPs first job would be the generation of type II intermediate neural progenitors (INPs), which is done by asymmetric divisions of the QNPs. This is to be able to express Ki-67 and nestin, which are expressed by the amplification of INPs. After this, the type II INPs go through differentiation which turns them into neuroblasts, also known as type III INPs. In addition to this, the type III INPs (neuroblasts) lose the expression of GFAP and Sox2, which are then substituted by the expression of polysialylated neural cell adhesion molecule (PSA-NCAM) and DCX as well. These two molecules are expressed by mature and immature granule neurons, or GNs. GNs would be the finished product in the process of differentiation, and in turn GN expresses calbindin, neuronal nuclear marker (NeuN), βIII-tubulin, and Prox-1.
For this study, the authors observed and measured the following in the posterior, mid, and anterior sections of the DG: QNPs expression of Sox2, GFAP, and Nestin; both type I-II INPs expression of Nestin and Ki-67; Immature GNs and Type III INPs (neuroblasts) expression in PSA-NCAM and DCX; and lastly, mature GNs and their expression of NeuN. It was also decided for this study that the anterior DG portion would be from the most rostral (towards nose) part of the DG, to the front of the lateral geniculate (LG). The mid DG area is defined as the whole LG area (front to back), and the posterior DG would stretch from the back of the LG to the most caudal part of the DG (towards the back of the head).
INPs Are Stable during Human Aging but QNPs Decline
To start, they focused on the abundance of type I and II INP and QNP cells in the posterior, mid, and anterior DG of a total of 28 females (11) and males (17), all age 14 to 79 years. Sox2, and expressed transcription factor, marked QNPs and resulted in a decline as aging occurs, more specifically in the anterior to mid DG, although gender did not play a role. Furthermore, Nestin+ type I-II INPs and Sox2/nestin+ presented a steadiness and did not decline with age in the posterior, mid, and anterior DG, regardless of gender as well. In addition to this, the amount of Ki-67+ in the posterior, mid, and anterior DG were also stable in both sexes as aging progressed. So, all in all, it was found that the pool of Sox2+ QNP was less in the anterior and mid dentate gyrus of the older population and was measured to be around 1,000 cells per region of the DG (posterior, mid, anterior). On the other hand, Sox2/nestin+ INP type I and II cells were observed and calculated to have a steady amount in all sections of the DG of the older test subjects; and these cells came in the thousands per DG section. Lastly, Ki-67+ cells also had a steady amount as age progressed, and these were observed to have about 10,000 cells in each DG section. In addition, it was mentioned that these cells also have non-neuronal lineage cells that are dividing as well, which give them a lot more density per section of the DG.
Neuroplasticity May Decline but Immature GNs Are Preserved in Older Humans
In this specific part of the study, in order to find the changes of immature GNs and type II-III INPs based on age progression, or to find activity of neuroplasticity, there are certain markers that are used in this step. A main marker used is PSA-NCAM, which, when coupled with DCX, is able to work as a marker for immature GNs and neuroblasts as well. On the other hand, when PSA-NCAM is found on its own, it works as a marker for neuroplasticity. This study found that PSA-NCAM+ cells in the human sub-granular zone (INPs & immature GNs), as well as PSA-NCAM+ mature GNs were observed to be less in the anterior region of the DG as age progressed; moreover, the volume was constant as well for these prokaryon cells. This evidence was shown in both the male and females test subjects, so gender had no affect either. On the contrary, DCX/PSA-NCAM+ and DCX+ cells were measured and resulted in a steady number of cells in the span of the 14-79 years old for both genders. These results signify that there are preserved immature GNs and type III INPs.
As a result, there was no age-related observation showing a decline of the DCX/PSA-NCAM+ and DCX immature neurons and INPs. This means that in the older subjects, no matter what the gender, they have a stable neurogenesis process. In addition to this, it was also observed that there were less PSA-NCAM+ cells, which were cells that vary from mature neurons to the type II INPs. This is most likely evidence from the deterioration of neuroplasticity as a person gets older. Neuroplasticity deterioration in humans involves dendrite sprouting, activity-dependent plasticity, blunted migration, and long-term potentiation.
GN, Glia, and DG Volume Remain Unaffected while Reduced Angiogenesis and Capillary Density Correlates with Decreased Neuroplasticity
In this part of the study, the authors went ahead and measured the results in regard to angiogenesis. This involves capillary measures which includes the count of new capillaries within a specific area, the amount of bifurcations for each capillary, and the length and area of the new capillaries as well. In addition to this, the posterior, mid, and anterior regions of the hippocampus were measured for the estimated total volume of the DG, total number of glia and GNs.
Angiogenesis in the posterior, mid, and anterior sections of the DG was evaluated by measuring the area, length, and the bifurcation number of nestin+ capillaries; also, the amount of new capillaries per cubic mm was measured using Stereo Investigator and Neurolucida software (MBF) across the section of tissue thickness. The volume of the posterior, mid, and anterior DG were roughly calculated; this included the molecular layer (ML), granule cell layer (GCL), and sub-granular zone (SGZ), which was all done using the Cavalieri method. In order for the authors to be able to calculate the posterior, mid, and anterior DG and SGZ-GCL, they had to associate outlines of the DG. This included ML, GCL, and the SGZ and their measures from the most rostral end to the most caudal hippocampal end of the DG.
The results showed that there were smaller capillary length and area, as well as, capillaries that were less-branched. It is stated that the smaller sizes of the capillaries are due to less PSA-NCAM+ cells present in the anterior to mid dentate gyrus. However, the declining capillary sizes had no correlation to the amount of DG volume, Nissl glia, NeuN cells, DCX/PSA-NCAM, DCX, Ki-67, Sox2/nestin, nestin, and Sox2; these results had no differences between the male and female subjects as well. Furthermore, no age-related differences were observed in the DG volume, GCL-SGZ volume, or in the amount of Nissl+ glia and NeuN+ GNs; additionally, gender made no difference for these results. To conclude, the fact that there were PSA-NCAM+ neuronal cells indicates that there is less neuroplasticity, which ultimately, is correlated with the age-related decline in angiogenesis in both genders. Moreover, the glia, DG volume, and mature GNs were around the same in the range of subjects 14 to 79 years old, and this was for both genders as well.
Discussion
Prior to this study, the effects of aging on DG volume, angiogenesis, and AHN are yet to be studied in the hippocampus, as a whole, from subjects without the presence of neuropsychiatric treatment or disease, clean toxicology, or clean neuropathology. So, for this study, the subjects chosen were medication-free, had no cognitive disability, and had no brain disease. The test subjects in this study also had “good global function”, which was measured by the Global Assessment Scale, and low stress due to recent life-related events as measured by the St. Paul-Ramsey Life Experience Scale. The results found were of stable DG volume and constant AHN as a person aged over the 65-year span, from 14-79 years old. On the other hand, results of this study also showed decline in angiogenesis and neuroplasticity as a human gets older; additionally, possible deterioration of multipotent QNP pools, more specifically in the mid-anterior DG. In contrast, the posterior showed no QNP pool differences, which pretty much explains why there is less and less emotional-cognitive recuperation as a person becomes older.
As part of the results, there were less QNPs found to be expressing Sox2, while not expressing nestin, which mainly occurred in the anterior-mid DG. These results agree with the results in rats that showed a limited amount of QNP pool that divides asymmetrically; a division mechanism that deteriorates overtime, as aging occurs. The young and old subjects had similar amounts of nestin and Sox2 cells in the posterior, mid, and anterior DG; about 1,000 cells per DG section, with a total of about 3,000 cells in the whole DG of the subjects. These similar amounts in both young and old test subject indicates that there is a continuous proliferation, which come from INPs.
Ki-67 cells came in 10,000 cells per DG section, which gave a full amount of 30,000 cells in each DG for each subject; these numbers were constant in both younger and older subjects of this study. These results correlate to the DG cells’ density which are seen to express proliferation markers throughout the progression of the human life.
DCX/PSA-NCAM and DCX cells were observed to be steady with aging, and these were recorded with a few thousands per DG section, with a total DG amount of 10,000-15,000 immature neurons (INP type III) per person of this study. Other studies showed results with evidence that DCX cells decline from time of birth to the age of 15, though, staying constant from as a person gets older. In addition, there were fewer PSA-NCAM cells reported, as well as a stable amount of DCX/PSA-NCAM type III INPs (immature GNs) and Sox2/nestin type II INPs as a person gets older. This is not due to a diminished INP proliferation, but rather because of a decline in neuroplasticity (or migration). Furthermore, there were persistent amounts DCX/PSA-NCAM cells and declining amounts of PSA-NCAM cells as aging progressed. This could be the result of many PSA-NCAM+ cells also being NeuN+ cells; which would lead to a larger involvement of immature GNs with a smaller amount of mature GN neuroplasticity.
Conclusion
It was shown that the amount of cerebral blood due to exercise-induced growth is much lower in older people (those 60 to 79 years of age) than in younger people. This means that blood-associated factors and vascular signaling pathways, which maintain cellular plasticity and angiogenesis, declines as a person’s age progresses. Any alteration to microangiopathy, angiogenesis mediators, or angiogenesis would result to the pathophysiology of age-associated diseases of the brain, such as Alzheimer’s; additionally, this is also associated with the amounts of amyloid-β masses. Even though they were unable to calculate exercise patterns of their subjects, which is associated with hippocampal volume, AHN, and angiogenesis, it is probable that younger individuals exercised more than the older individual subjects.
In order to avoid confounders in this study, procedures such as neuropathology exams, brain and blood toxicology exams, and clinical reports were used to exclude any individuals with chronic disease or neuropsychiatric disease, as well as, psychotropic use of drugs or microvascular variations. Other factors that were checked on was a low brain tissue pH level, as well as postmortem time of at least 26 hours to guarantee the equal dispersal of brain protein quality throughout the brain. These factors would be considered as limiting factors as they are based off of the reports of the subjects.
No actual differences were observed in regard to age-related changes or levels of AHN between the male and female subjects. Another factor that was not accounted for was the menstrual cycles of the women, as the young individual females could have been going through their cycle, while the older female subjects could have possibly been going through menopause. Many studies have shown that natural hormones of the body can regulate angiogenesis and neurogenesis in humans. Some of these hormones include testosterone, corticosteroids, and estrogen as well. Consequently, these different factors among men and women may lead to age-related changes in the multipotent progenitor pool, neuroplasticity, and angiogenesis.
It was also noted that humans have less GNs and more AHN in the older population, which is opposite of what primates and rats present. Because old GNs assist in pattern completion and new GNs help with separation of patterns; the removal of older GNs and a stable AHN supports complex memory and learning of humans, and the behaviors that are driven by emotions. It is important to have AHN that is persistent, as this preserves cognitive resilience and allows for decision-making based on memory, without having to worry about the interruption of unrelated information from the memory. Age-associated cognitive-emotional fluctuations is due to less neuroplasticity, which happens when there is less vascularization of the neurogenic process, as well as a wore out QNPs (quiescent progenitor pool). As mentioned, there is a need for future studies on neuroplasticity and its association with cognitive and emotions, based on the potential benefits that diet, medications, and exercise have for aging healthily.
The methods used for this study appear to be more accurate than previous studies on the same subject. Brain selection, subject selection, matching procedure, and processing of brain tissue were all done meticulously. Moreover, more methods were put into use for this study, where as other studies missed important methods that are important to implement. Methods like stereology, which is said to be the gold standard when calculating the densities of cells. Additionally, this study made use of replication, immunocytochemistry, confocal microscopy, and immunohistofluorescence. All in all, the study found that AHN persists into the eighth decade of a person’s life. In contrast, QNPs, neuroplasticity, and angiogenesis declined as a person gets older. Lastly, from ages 14-79 years; DG volume, glia, mature & immature GNs, and proliferating neural progenitors had no change, not even between genders.