CN108272787B

CN108272787B - Use of 7,8 dihydroxyflavone for treating or preventing aging-related cognitive decline

Info

Publication number: CN108272787B
Application number: CN201711169037.7A
Authority: CN
Inventors: 陈锦辉
Original assignee: Suzhou Glanke Pharmaceutical Technology Co ltd
Current assignee: Suzhou glanke Pharmaceutical Technology Co.,Ltd.
Priority date: 2016-11-21
Filing date: 2017-11-21
Publication date: 2021-05-28
Anticipated expiration: 2037-11-21
Also published as: HK1257911A1; CN108272787A

Abstract

The present application provides the use of 7, 8-dihydroxyflavone for treating or preventing an aging-related decline in cognitive ability in a subject or for improving cognitive ability in an aging subject.

Description

Use of 7,8 dihydroxyflavone for treating or preventing aging-related cognitive decline

Background

Aging is a process in which the function of the whole body slowly declines over time, and the decline in memory and cognitive ability caused by aging affects the daily quality of life of the elderly (Hedden & Gabrieli 2004). By the year 2050, the population of elderly people over the age of 65 will reach 8370 ten thousand, approaching 20% of the total population (Jennifer m. ortman 2014), according to the data of the us statistical office. There is an urgent need for a therapeutic regimen that can effectively and specifically treat the cognitive disorders associated with aging. However, the molecular cell-level rationale for the problem of cognitive disorders associated with aging is relatively limited at this stage, thus impeding the development of effective clinical treatment protocols.

Both neuroregenerative capacity and synaptic plasticity of hippocampus are associated with cognitive function, both of which are greatly affected by aging processes (Rao et al 2006). Nerve regeneration involves several key steps, including propagation of neural stem or progenitor cells (NSCs), survival of immature neural cells, dendritic development, maturation and functional integration (Ming & Song 2005). Our previous studies demonstrated that the aging process could purposely reduce the rate of division of active neural stem cells (Romine et al 2015). In addition, the survival rate of the newborn neuronal cells continues to stay at a low level, further hindering the production of the newborn neuronal cells, resulting in the inability of the existing neural circuits of aging animals to be integrally supplemented with the newborn neuronal cells (Bondolfi et al 2004; Rao et al 2005).

After the new-born neurons are produced in the hippocampus, they also need to undergo further morphological development and synaptic integration before they function normally. Cellular morphological maturation is the fundamental prerequisite for the correct passage of the granular neurons through the axon to the CA3 region of the signal received by the dendritic spines. In the adult hippocampus dentate gyrus, the nascent neurons develop a more complete axonal, dendritic and dendritic spine morphology, usually initially within 3-4 weeks, and continue to undergo structural changes for the next few months (Zhao et al 2006). Our studies found that the aging process not only hampers neuronal regeneration, but also greatly hampers dendritic development by reducing the number and total length of dendritic branches. Since dendrites provide a large surface area for synapse formation, a reduction in the number of dendrites and the total length of dendrites will eventually hinder the formation of new synapses. To date, there is no effective means to prevent or rescue the damage that aging causes to neuronal regeneration and dendritic development.

Both neuronal regeneration and morphological maturation of nascent neurons produced by adults are regulated by brain-derived neurotrophic factor (BDNF). BDNF activates receptors by binding to its specific receptor TrkB and exerts a regulatory role in this way (Tolwani et al 2002; Binder & Scharfman 2004). The effects of BDNF include promotion of neuronal growth, survival, migration, and synaptic modulation (Alderson et al.1990; Horch & Katz 2002; Gorski et al 2003; Cohen-Cory et al 2010). BDNF plays an especially important role in regulating hippocampal dendritic development and post-synaptic function and structural plasticity (Tolwani et al 2002; Vigers et al 2012; Wang et al 2015). Therefore, BDNF function and memory formation are related to memory consolidation (Vigers et al.2012). However, the BDNF-TrkB system is very susceptible to aging, which may explain why hippocampus-associated memory is impaired by aging (Calabrese et al 2013; Budni et al 2015) and provide a therapeutic target for improving memory function in the elderly. Although BDNF is a key factor in neurotrophic factors, its limited delivery, short half-life and inability to cross the blood-brain barrier reduce its effectiveness as a drug.

Accordingly, there is a need in the art for a medicament for treating or preventing cognitive decline in an aging subject.

Disclosure of Invention

The inventor finds in studies that aging impairs the development of new neuronal dendrites. These impairments are all responsible for the decline of cognitive abilities in the elderly. There is currently no effective method to promote nerve regeneration or the development of neogenetic neuron dendrites in the aging brain.

The inventors further surprisingly found that systemic injection of 7,8 Dihydroxyflavone (DHF) into aged mice can significantly increase the length of neonatal neuronal dendrites in aged mice, thereby saving the damage of aging on the development of neonatal neuronal dendrites. Therefore, DHF can slow down the decline of brain cognitive ability due to aging by promoting the development of hippocampal neogenic neuron dendrites.

The invention includes methods and uses for the treatment or prevention of cognitive decline caused by aging. In one embodiment, the invention relates to the use of 7, 8-Dihydroxyflavone (DHF) or a derivative thereof, or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof, in the manufacture of a composition for treating or preventing an aging-related decline in cognitive ability in a subject, or for improving cognitive ability in an aging subject.

In one embodiment, the invention relates to the use of 7, 8-Dihydroxyflavone (DHF) or a derivative thereof, or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof, in the manufacture of a composition for extending the dendrite length of a nascent neuron in an aging subject.

The present invention includes administering DHF or a derivative thereof to an aging subject. DHF or derivatives thereof may also be administered in a clinical setting. In one embodiment, DHF or a derivative thereof is administered by intraperitoneal or intravenous injection.

In one embodiment, the present invention comprises prophylactic administration of DHF or a derivative thereof, thereby preventing cognitive decline from aging.

In one embodiment, the composition of the invention is a medicament or pharmaceutical composition.

In one embodiment, DHF or a derivative thereof, or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof, of the invention is also administered in combination with at least one therapeutic agent.

In one embodiment, the subject is a mammal, preferably a human.

Drawings

Figure 1 shows that aging reduces the number of immature neonatal neurons in the hippocampus of aging mice and impairs their dendritic morphological development. FIGS. 1A-B: immature neurons in the Hippocampal Dentate Gyrus (HDG) of 3-month old mice (A) and 12-month old mice (B) were shown by labeling microtubule-associated protein (deubelocin [ Dcx ] red) using an antibody. HDG structures were stained with 4', 6-diamidino-2-phenylindole (DAPI, blue). FIGS. 1C-D: the high magnification image shows the structure of the Dcx positive immature neurons in fig. 1A and B (indicated by white boxes). FIG. 1E: the number of Dcx-positive neonatal immature neurons in HDG was quantified in 3-month old mice (n ═ 3) and 12-month old mice (n ═ 4). FIGS. 1F-G: dendrites of Dcx-positive neonatal immature neurons in HDG of 3-month old mice (F) and 12-month old mice (G) were reconstituted. FIGS. 1H-I: quantification of average dendrite branch number (H) and total dendrite length (I) of neonatal immature neurons showing Dcx positivity in HDG of 3-month old mice (n ═ 3) and 12-month old mice (n ═ 4) (. p <0.05,. p <0.01)

Figure 2 shows that DHF treatment did not significantly increase the number of nascent immature neurons in the hippocampus of aged mice. FIG. 2A: schematic diagram of experimental method. FIGS. 2B-D: treatment of microtubule-associated proteins (Dcx, red) using immunostaining showed immature neurons in the Hippocampus Dentate Gyrus (HDG) of 12-month-old mice receiving different injection treatments, respectively: phosphate buffered saline injection group (PBS; n-4, fig. 2B), dimethyl sulfoxide injection group (DMSO; n-4, fig. 2C) and 7,8 dihydroxyflavone injection group (DHF; n-4, fig. 2D). HDG structures were stained with 4', 6-diamidino-2-phenylindole (DAPI, blue). FIGS. 2E-G: the high magnification picture of fig. 2B, C, D (indicated by white boxes) shows that individual immature neurons showing Dcx positivity in 12-month-old mice HDG received PBS injection (fig. 2E), DMSO injection (fig. 2F) and DHF injection (fig. 2G), respectively. FIG. 2H: the number of immature neurons showing Dcx positivity in HDG of 12-month-old mice receiving PSB, DMSO and DHF treatment, respectively, was quantified.

Figure 3 shows that DHF treatment improved dendritic morphological development of neonatal immature neurons in the hippocampus of aged mice. FIGS. 3A-C: immunostaining with antibodies to label microtubule-associated proteins (deuterotoxin [ Dcx ] red) revealed individual immature neuronal structures in the Hippocampus Dentate Gyrus (HDG) of 12-month-old mice that received different injection treatments: phosphate buffered saline injection group (PBS; n-4, fig. 3A), dimethyl sulfoxide injection group (DMSO; n-4, fig. 3B) and 7,8 dihydroxyflavone injection group (DHF; n-4, fig. 3C). Granular cell layers were stained with 4', 6-diamidino-2-phenylindole (DAPI, blue) to reveal structure. FIG. 3D: the dendrites of immature neurons showing Dcx positivity in HDG of 12-month-old mice receiving PSB, DMSO and DHF treatment, respectively, were reconstituted. FIGS. 3E-G: the average number of dendritic branches (fig. 3E), average dendritic length (fig. 3F), and total dendritic length (fig. 3G) of immature neurons showing Dcx positivity in HDG of 12-month-old mice receiving PSB, DMSO, and DHF treatment, respectively, were quantified. FIG. 3H: the total dendrite length of Dcx positive immature neurons in hippocampus of 12-month-old mice receiving PSB, DMSO and DHF treatment, respectively, was a proportion of each group ([ p ] p <0.05, [ p ] p <0.01, [ p ] p < 0.001).

Detailed Description

Definition of

The term "combination" refers to the administration of one or more therapeutic agents to DHF or a derivative thereof, including simultaneous administration or sequential administration in any order.

The term "7, 8-dihydroxyflavone" or "DHF" refers to 7, 8-dihydroxy-2-phenyl-4H-1-benzopyran-4-one, which is a compound of the formula:

DHF is a tyrosine kinase receptor b (trkb) agonist that binds to the extracellular domain of the receptor (Kd 320nM), promoting receptor dimerization and autophosphorylation.

For the purposes of treatment or therapy, the term "mammal" refers to any animal defined as a mammal, including humans, domestic animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

The term "treatment" or "treating" is a means for obtaining a beneficial or desired clinical result. In this context, beneficial or desired clinical results include, but are not limited to, alleviation of clinical symptoms, diminishment of extent of disease, stabilized (not worsening) state of disease, delay or slowing of progression of disease, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. As used herein, "treatment" does not include "preventing", "avoiding" or "preventing".

The term "compound" refers to DHF or a derivative, or a pharmaceutically acceptable salt, ester, solvate, tautomer, optical isomer, clathrate, hydrate, polymorph, or prodrug thereof, and also includes protective derivatives thereof. The compounds may contain one or more chiral centers and/or double bonds and, therefore, exist in the following forms: stereoisomers such as double bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. According to the present invention, the chemical structures referred to herein include the compounds of the present invention, including all enantiomers, diastereomers and geometric isomers of the corresponding compounds, that is, whether in stereochemically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure form) and isomeric mixtures (e.g., geometric isomers, enantiomeric and diastereomeric mixtures). In some cases, one enantiomer, diastereomer, or geometric isomer may have better activity or improved toxicity or kinetic characteristics than the other isomer. In such cases, these enantiomers, diastereomers and geometric isomers of the compounds of the invention become preferred.

The term "polymorph" refers to a solid crystalline form of a compound disclosed herein or a complex thereof. Different polymorphs of the same compound may exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to, stability (e.g., stability to heat or light), compressibility and density (important in formulation and product manufacture), and dissolution rate (which can affect bioavailability). Differences in stability may be due to changes in chemical reactivity (e.g., differential oxidation, such that dosage forms formed from one polymorph discolor more rapidly than dosage forms formed from another polymorph) or mechanical properties (e.g., tablets break upon storage as kinetically favored polymorphs transform to thermodynamically more stable polymorphs), or both (e.g., tablets of a single polymorph break more easily at high humidity). The different physical properties of polycrystalline bodies can affect their processing. For example, one polymorph may be more likely to solvate than another polymorph, or it may be more difficult to filter, wash away impurities because of their different particle shapes or particle size distributions.

The term "hydrate" refers to a compound disclosed herein or a salt thereof, which further comprises a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.

The term "clathrate" refers to a compound disclosed herein or a salt thereof in a crystal lattice comprising spaces (e.g., channels) with guest molecules (e.g., solvent or water) trapped therein.

The term "prodrug" refers to a drug molecule of a compound of the invention that is biologically inactive until activated by a metabolic process. Prodrugs include derivatives of compounds that may be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compounds of the present invention. Prodrugs may be active after the reaction is carried out under biological conditions, or they may be active in their unreacted form. Examples of prodrugs useful in the present invention include, but are not limited to, analogs or derivatives of the compounds disclosed herein, which include a prodrugHydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogs. Other examples of prodrugs include compounds disclosed herein containing- -NO, - -NO₂- -ONO or- -ONO₂Derivatives of moieties. Prodrugs can generally be prepared using well known methods.

Pharmaceutically acceptable salts

Where the compound has sufficient ph to form a stable, non-toxic acidic or basic salt, the compound may be suitable for administration as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, and other acids well known in the pharmaceutical art. In particular, examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids to form physiologically acceptable anions such as: tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including sulfates, nitrates, bicarbonates, and carbonates.

Pharmaceutically acceptable salts can be obtained by standard, well-known preparative procedures, for example by reacting a sufficiently basic compound (e.g., an amine) with a suitable acid to form a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.

Composition comprising a metal oxide and a metal oxide

DHF or pharmaceutically acceptable salt based compositions may be prepared in combination with pharmaceutically acceptable additives, in any carrier or excipient in a therapeutically effective amount for any indication described herein. The therapeutically effective amount may vary depending on the condition to be treated, its severity, the treatment regimen being administered, the pharmacokinetics of the agent, and the individual condition of the patient.

In one aspect, DHF is preferably in admixture with a pharmaceutically acceptable carrier. In general, it is preferred to administer the pharmaceutical composition in an orally administrable form, but the formulation may also be administered by parenteral, intravenous, intramuscular, transdermal, buccal, subcutaneous, suppository or other routes. Intravenous and intramuscular formulations are preferably administered in sterile saline. One of ordinary skill in the art, given the teachings of this specification, can adjust the formulation to provide a variety of formulations for a particular route of administration without destabilizing or affecting the therapeutic activity of the compositions of the present invention. More specifically, for example, the desired compound is adjusted to be more stable in water or other excipients, and can be simply achieved by conventional adjustments (salt formulation, esterification, etc.).

In general, a therapeutically effective amount of a compound of the invention in a pharmaceutical dosage form is generally from about 0.1mg/kg to about 100mg/kg or more, depending on the compound used, the degree of cognitive decline, and the route of administration. For the purposes of the present invention, a prophylactically or prophylactically effective amount of a composition according to the invention falls within the same ranges as described above for a therapeutically effective amount and is generally the same as the effective amount described above.

Administration of DHF or its derivatives may be continuous (intravenous drip) to several times daily oral administrations (e.g., QID, BID, etc.), and may include oral, topical, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), buccal and suppository administrations, as well as other routes of administration. Enteric coated oral tablets may also be used to enhance the bioavailability and stability of compounds administered by the oral route. The most effective dosage form will depend on the pharmacokinetics of the particular agent chosen, as well as the severity of traumatic brain injury in the patient. Oral dosage forms are particularly preferred because of ease of administration and patient compliance.

To treat a pharmaceutical composition according to the present invention, a therapeutically effective amount of one or more compounds of the present invention is preferably mixed with a pharmaceutically acceptable carrier according to conventional quantitative pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral. In preparing the pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations such as suspensions, elixirs and solutions, suitable carriers and additives including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like may be used. For solid oral preparations such as powders, tablets, capsules, and solid dosage forms such as suppositories, suitable carriers and additives include starches, sugar carriers such as glucose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents and the like may be used. Tablets or capsules may be enteric coated for sustained release by standard techniques, if desired. The bioavailability of the compounds in the patient can be significantly affected by the use of these dosage forms.

For parenteral formulations, the carrier will usually comprise sterile water or aqueous sodium chloride solution, although other ingredients, including those dispersion aids, may also be included. When sterile water is to be used or the sterility of sterile water is to be maintained, the compositions and carriers must also be sterilized. Can also be prepared as suspensions for injection, in which case suitable liquid carriers, suspending agents and the like can also be used.

In one embodiment, the compounds and compositions are useful for treating, preventing or delaying aging-related cognitive decline. Preferably, for the treatment, prevention or delay of cognitive decline associated with aging, the composition is administered in an oral dosage form in the following amounts: about 250 micrograms to about 1 gram or more, at least once per day, or up to four times a day. The compounds are preferably administered orally, but may also be administered parenterally, topically, or in the form of suppositories.

In one embodiment, DHF or a derivative thereof may be formulated as part of a beverage, including a sports beverage (e.g.,

) Energy drink (e.g., Red ox Red)

) A nutritional beverage (e.g.,

) Or any type of beverage supplemented or supplemented with vitamins, electrolytes and the like.

Examples

Example 1 aging greatly reduced the number of nascent immature neurons in the hippocampus and impaired dendritic morphology.

Decline in cognitive ability is a sign of aging progression, but the specific principle is not yet clear (Bishopet al.2010). Among the many possible causes, the decrease in hippocampal neuroplasticity, represented by a decrease in neuronal regeneration rate and synapse loss, is a major factor (Burke & Barnes 2006). Neuronal regeneration, including neural stem cell proliferation, immature neuronal survival, metastasis, maturation, dendritic development and functional integration (Ming & Song 2005), is affected by aging at various levels. Our previous studies showed that aging affects neuronal regeneration primarily because aging impairs neural stem cell proliferation, especially proliferation of active neural progenitor cells in the hippocampus of mice (Romine et al.2015). In this study, we further evaluated the number of neonatal immature neurons and their dendritic development in 3-month old mice (n ═ 3) and 12-month old mice (n ═ 4). Every sixth brain section was treated with immunostaining and labeled with an antibody against microtubule-associated protein (Dcx), a marker for neonatal immature neurons (Gleeson et al 1999). In 3-month-old mice, Dcx-positive neonatal immature neurons were shown to be distributed predominantly uniformly within one third of the Hippocampus Dentate Gyrus (HDG) Granular Cell Layer (GCL) (fig. 1A). In contrast to 3-month-old mice, we observed a significant decrease in the number of neonatal immature neurons in 12-month-old mice, indicating that Dcx-positive cells are less abundant and sporadically distributed in the inner third of the Hippocampus Dentate Gyrus (HDG) Granular Cell Layer (GCL) in 12-month-old mice (fig. 1B). Under high magnification, we can clearly observe each Dcx-positive cell on the Granular Cell Layer (GCL) (fig. 1C, fig. 1D). Each Hippocampus Dentate Gyrus (HDG) contained about 11,098 ± 2,841 neonatal immature neurons in 3-month-old mice, a number that dropped to 341 ± 54 in 12-month-old mice, indicating a large reduction in the number of neonatal immature neurons in aging animals (p ═ 0.022, fig. 1E).

It can be clearly seen under high magnification that dendrites of immature neurons are growing on molecular layer (fig. 1C, fig. 1D). Dcx positive cells from 12-month old mice exhibited poorer dendritic morphology compared to 3-month old mice. We quantitatively analyzed the dendritic morphology of nascent immature neurons in HDG in adult mice (46 neuronal cells in 3 mice) and aged mice (91 neuronal cells in 4 mice) by dendritic reconstitution (fig. 1F, 1G). In 3-month-old mice, each neonatal neuron typically has 4.1 ± 0.4 dendritic branches with a total length of 297.0 ± 65.4 microns (fig. 1H, 1I). In 12-month-old mice, the number of de novo neuronal dendrites decreased by 58% with an average of 1.7 ± 0.2 dendrite branches per neuron (p ═ 0.004, fig. 1H), and the total length of dendrites decreased by 76% to an average of 70.6 ± 18.2 microns per neuron (p ═ 0.022, fig. 1I). In summary, we observed a large decrease in the number of neonatal immature neurons in HDG in aged mice, with damage to the neonatal neuron dendritic morphology.

Example 2 Effect of DHF on survival of neonatal neurons in aged mice

Until now, there has been no effective drug that can slow down or prevent the decline of cognitive ability caused by aging, so that it is necessary to develop a method that can promote nerve regeneration and/or dendritic morphological development.

First, we focused on whether DHF could increase neonatal neuron survival in HDG in aged mice. 12-month-old mice were divided into three groups and received DHF injections (5mg/kg dissolved in dimethylsulfoxide [ DMSO ], n ═ 4), 70% dimethylsulfoxide injections (n ═ 4), or phosphate buffered saline injections (PBS, n ═ 4), respectively, once daily for two weeks. All mice were sacrificed 24 hours after the last injection and brains were removed (fig. 2A) to assess the number of immature neurons and dendritic morphogenesis. One of 6 brain sections was selected in fixed order for immunostaining by detecting neonatal neurons in the hippocampus using antibodies to microtubule-associated protein (Dcx). In mouse brain sections receiving PBD and DMSO injections, we observed very small amounts of Dcx positive cells (fig. 1B, 2C). There was no drastic change in the number of Dcx-positive cells after receiving DHF injection (fig. 2D). Under high magnification, we can count out every Dcx positive cell (FIGS. 2E-G). In the DHF-injected group, the average number of Dcx-positive cells per HDG was 330 ± 111 on average; the number of DMSO injection groups is 293 +/-191; the number of PBS control groups was 341 ± 54 (fig. 2H), and there were no significant statistical differences between the three groups of data. Thus, DHF injection treatment over a period of two weeks did not improve neonatal immature neuronal survival in aged mice. In contrast, our previous studies showed that treatment with DHF injections at the same dose for two weeks can increase the number of neonatal immature neurons produced in young animals after adulthood (Zhao et al 2015). This suggests the difficulty and complexity of treating cognitive decline associated with aging.

Example 3 improvement of dendritic morphology in aged mice by DHF

While DHF did not improve survival of neonatal neurons in aged mice, microscopic examination revealed changes in dendritic morphology in the mice (fig. 2E-G, fig. 3A-C). Images of each hippocampus Dcx positive cells were taken at 40 x magnification using a Zeiss (Zeiss) microscope system. Cell bodies and dendrites were traced and reconstructed using neurouca software (fig. 3D) and analyzed using NeuroExplorer (fig. 3E-H). We found that the average number of dendritic branches per neuron increased slightly, from 1.7 ± 0.2 in the PBS group (91 neurons in 4 mice) and 1.6 ± 0.04 in the DMSO group (94 neurons in 4 mice) to 2.1 ± 0.4 in the DHF group (109 neurons in 4 mice), but this difference did not reach a significant statistical difference (fig. 3E). Nevertheless, the average length of each dendritic branch increased significantly from 32.1 ± 3.4 microns for the PBS group and 38.3 ± 5.5 microns for the DMSO group to 47.6 ± 3.4 microns for the DHF group (fig. 3F). Thus, the total dendrite length of each neuron increased significantly from 70.6 ± 18.2 microns for the PBS group and 67.0 ± 9.8 microns for the DMSO group to 110.3 ± 23.7 microns for the DHF group (fig. 3G). By further grouping the neonatal immature neurons according to the difference in total length of dendrites, we found that in the PBS and DMSO control groups, the neonatal immature neurons developed predominantly very short dendrites within 50 microns from the cell body (PBS group mice 67.8 ± 8.8%, DMSO group mice 61.4 ± 2.4%, fig. 3H). In the DHF group, the proportion of neonatal neurons with short dendrites with a total length of less than 50 μm was greatly reduced to 45.9 ± 5.7% (fig. 3H). As a result, the mice in the DHF group had more neonatal neurons developing relatively longer dendrites (more than 200 microns from the cell body), accounting for 18.7 ± 2.6% of all neonatal neurons measured, compared to only 8.4 ± 3.3% in the PBS group and 5.1 ± 2.0% in the DMSO group (fig. 3H). In summary, while DHF does not promote the development of a greater number of dendritic branches from nascent immature neurons, DHF promotes dendritic development by lengthening dendrites, which is manifested by DHF extending the average and overall length of dendrites in aged mice, while also increasing the proportion of nascent immature neurons with longer overall dendritic lengths on an overall level.

Discussion of the related Art

Given the current rapid growth of the aging population, the decline in cognitive abilities associated with aging will place an increasing burden on our society (Jennifer m. Despite this severe situation, the molecular mechanisms by which aging brings about cognitive decline remain unclear, impeding the development of therapies that can delay or prevent such cognitive decline. Neuroplasticity, which is represented by hippocampus' ability to regenerate nerves and synaptic plasticity, is an important part of learning and memory. There are studies that demonstrate that neuroplasticity in both murine and human is affected by aging (Burke & Barnes 2006; Rao et al 2006). In the present invention, we used 12-month old mice and observed some similar features in them as in the older animals studied before, the most significant of which were a reduction in the number of neonatal immature neurons and dendritic morphological damage compared to the younger mice at 3 months of age (fig. 1). These observations represent a lack of neuroregenerative capacity and prominent plasticity in the aged experimental group. Approaches aimed at enhancing nerve regeneration and/or synaptic plasticity may be potential therapeutic targets.

To date, there is no effective treatment regimen that can enhance the neuroregenerative capacity or synaptic plasticity in older animals.

In this experiment, we injected mice with two weeks of DHF, which unexpectedly did not significantly increase neonatal neuron survival in aged mice (fig. 2). Nevertheless, we also unexpectedly found that there were significant differences in dendritic morphology between the experimental and control groups. Our data show that by daily injection of DHF for two weeks, the mean dendrite length of the nascent neurons was increased by a large 48% compared to the PBS group and 24% compared to the DMSO group, although the number of dendrite branches of the nascent neurons was not significantly changed (fig. 3F). Likewise, total dendrite length of the nascent neurons was increased by 53% compared to the PBS group and 64% compared to the DMSO group (fig. 3G). In addition, we observed an increased proportion of nascent neurons possessing longer dendrites, with a 2.2-fold increase in the number of neurons with a total length of dendrites exceeding 200 microns (fig. 3H).

BDNF has been shown to control the shape and number of dendritic spines, affecting the development of synaptic loops, especially in the hippocampus (Ji et al 2005; Cohen-Cory et al 2010). Our previous studies directly demonstrated that loss of BDNF impairs postnatal formation of synapses in hippocampal dentate gyrus granular cells (Gao et al 2009). In the present invention, we have found that the total dendrite length of each nascent neuron decreases dramatically with the progression of aging.

From 2-month-old mice to 12-month-old mice, the total dendrite length per neuron dropped to 23.7% of the original length. In this experiment, DHF treatment significantly increased the number of dendritic branches and total length of dendrites per de novo neuron in aged mice. Nevertheless, the dendrite length of the aged mice that received DHF injection was much shorter than that of the young adult mice at 3 months of age, which was only 37% of that of the young adult mice at 3 months of age.

Experimental methods

Animal treatment

Male C57BL/6 mice (Jackson Laboratories) were housed in 12/12 hours light and dark cycles and were ready to receive food and water. Mice used in the experiment were 3 months old and 12 months old. All procedures were in compliance with the guidelines approved by the indiana university committee for animal welfare and use.

DHF treatment

Mice at 12 months of age received one of three treatments, DHF injection (5mg/kg in DMSO, n-4) or 70% DMSO injection (n-4) or phosphate buffered saline injection (PBS, 50mg/kg, n-4, i.p. ]), once daily for two weeks. All mice were sacrificed 24 hours after the last injection to count the number of neonatal neurons and to evaluate the dendritic morphology of the neonatal neurons (fig. 2A).

Tissue treatment

Experimental animals were deeply anesthetized with 2.5% tribromoethanol and perfused through the heart with 0.9% cold saline. Followed by fixation with cold 4% Paraformaldehyde (PFA) fixative. After overnight fixation, brains were removed, placed in 30% sucrose solution for 48 hours, cut into 30 micron brain slices with a microtome (Leica, CM1900), and stored in a freezer at-20 ℃. These sections were then subjected to immunohistochemical analysis.

Cell counting

Immunohistochemical staining was performed simultaneously on all sections to detect Dcx positive cells. Every sixth slice (30 microns thick, 180 microns apart) in each hippocampus slice series was processed. Cell counts were performed as described in reference (Gao & Chen 2013). Briefly, neonatal neural cells positive for each Dcx in the hippocampal dentate gyrus granular cell layer are counted. The total number of cells was further corrected (Coggeshall & Lekan 1996).

Microscopic imaging technique

Sections were analyzed by an inverted microscope system (Zeiss Axiovert 200M) in combination with ApoTome using a computer controlled digital camera (Zeiss AxioCam MRc5) as an interface. Images were captured by ApoTome and combined with AxioVision v4.8 software (ZeissAxioVision, v4.8), and finally the pictures were mosaicked and labeled using Photoshop 7.0(Adobe Systems).

Dendritic tracking and analysis

40-fold magnified pictures of each Dcx positive cell were used by Neuroucida software (MBFBioscience, Williston, VT) to trace neuronal cell bodies and dendrites. The neuronal tracing results were finally analyzed using NeuroExplorer software (Next Technologies, Madison, AL).

Statistical analysis

The data collected are expressed as mean ± standard deviation. Data comparisons of the number of neonatal immature neurons, the number of mean dendrites and the total dendrite length were performed using student's t-test (fig. 1E,1H,1I) in mice at 3 and 12 months of age. The number of neonatal immature neuronal cells, the mean dendritic number, the mean dendritic length and the total dendritic length of 12-month-old mice (fig. 2H,3E,3F,3G) after receiving PBS, DMSO and DHF treatment were analyzed using one-way ANOVA and Fisher Least Significant Difference (LSD) was used as a post-hoc comparative test.

The proportion of total dendrite length of each different class of neonatal immature neuron in 12-month-old mice (fig. 3H) after PBS, DMSO and DHF treatment was analyzed using two-way ANOVA (two-way ANOVA) and single-way ANOVA (one-way ANOVA) and LSD as a post-hoc comparative test. Statistical analysis was performed using SPSS software (IBM corporation) and p-value was set to < 0.05.

Claims

Use of 7, 8-dihydroxyflavone or a pharmaceutically acceptable salt thereof in the manufacture of a composition for prophylactic administration to a subject thereby preventing cognitive decline caused by aging, wherein the cognitive ability comprises learning memory, and the prophylactic administration improves dendritic morphology of hippocampal neogenesis neurons in the subject, thereby preventing cognitive decline caused by aging, and the improving dendritic morphology of hippocampal neogenesis neurons in the subject comprises an increase in the average length of dendritic branches of neogenesis neurons.
2. The use of claim 1, wherein the composition is a medicament.
3. The use of claim 1, wherein the subject is a mammal.
4. The use of claim 3, wherein the mammal is a human.