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Citation: Roda, E.; De Luca, F.; Ratto,
D.; Priori, E.C.; Savino, E.; Bottone,
M.G.; Rossi, P. Cognitive Healthy
Aging in Mice: Boosting Memory by
an Ergothioneine-Rich Hericium
erinaceus Primordium Extract. Biology
2023,12, 196. https://doi.org/
10.3390/biology12020196
Academic Editors: Serena Dato,
Giuseppina Rose and Paolina
Crocco
Received: 2 January 2023
Revised: 21 January 2023
Accepted: 25 January 2023
Published: 28 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
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4.0/).
biology
Article
Cognitive Healthy Aging in Mice: Boosting Memory by an
Ergothioneine-Rich Hericium erinaceus Primordium Extract
Elisa Roda 1, †, Fabrizio De Luca 2 ,† , Daniela Ratto 2, Erica Cecilia Priori 2, Elena Savino 3,
Maria Grazia Bottone 2and Paola Rossi 2, *
1Laboratory of Clinical & Experimental Toxicology, Pavia Poison Centre, National Toxicology Information
Centre, Toxicology Unit, Istituti Clinici Scientifici Maugeri IRCCS, 27100 Pavia, Italy
2Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy
3Department of Earth and Environmental Science, University of Pavia, 27100 Pavia, Italy
*Correspondence: paola.rossi@unipv.it; Tel.: +39-038-298-6076 or +39-038-259-2414
† These authors contributed equally to this work.
Simple Summary:
With the increase in the geriatric population worldwide, the promotion of healthy
aging arose as a key issue and, hence, the scientific community dedicates huge effort to counteract
age-related impairments. Brain aging is a crucial risk factor for several neurodegenerative disorders
and dementia. One of the most affected cognitive functions is recognition memory. Inflammation and
oxidative stress play a key role in pathogenesis of cognitive impairments, and a link exists between
frailty, oxidative stress, and inflammaging. Medicinal mushrooms represent a source to develop
new therapeutic strategy, and among them Hericium erinaceus (He) displays several actions ranging
from boosting immune system to fighting senescence, due to its active ingredients/metabolites.
Among these, Ergothioneine (ERGO) is known as the longevity vitamin. Currently, when monitoring
physiological aging in mice, we demonstrated the selective, preventive and neuroprotective effect
of an ERGO-rich He primordium extract in hippocampus, preventing recognition memory decline
during aging, decreasing key markers of inflammation and oxidative stress, and increasing the
expression of glutamate receptors, which are crucially involved in glutamatergic neurotransmission.
Abstract:
Brain aging is a crucial risk factor for several neurodegenerative disorders and dementia.
The most affected cognitive function is memory, worsening early during aging. Inflammation and
oxidative stress are known to have a role in pathogenesis of cognitive impairments, and a link
exists between aging/frailty and immunosenescence/inflammaging. Based on anti-aging properties,
medicinal mushrooms represent a source to develop medicines and functional foods. In particular,
Hericium erinaceus (He) displays several actions ranging from boosting the immune system to fighting
senescence, due to its active ingredients/metabolites. Among these, Ergothioneine (ERGO) is known
as the longevity vitamin. Currently, we demonstrated the efficacy of an ERGO-rich He primordium
extract (He2) in preventing cognitive decline in a murine model of aging. We focused on recognition
memory deterioration during aging, monitored through spontaneous behavioral tests assessing
both memory components and frailty index. A parallel significant decrease in key markers of
inflammation and oxidative stress, i.e., IL6, TGF
β
1, GFAP, Nrf2, SOD1, COX2, NOS2, was revealed
in the hippocampus by immunohistochemistry, accompanied by an enhancement of NMDAR1and
mGluR2, crucially involved in glutamatergic neurotransmission. In summary, we disclosed a selective,
preventive and neuroprotective effect of He2 on aged hippocampus, both on recognition memory as
well on inflammation/oxidative stress/glutamate receptors expression.
Keywords:
aging; frailty; memory; medicinal mushroom supplementation; Hericium erinaceus
primordium; ergothioneine; inflammation; oxidative stress; hippocampus; neuroprotection
Biology 2023,12, 196. https://doi.org/10.3390/biology12020196 https://www.mdpi.com/journal/biology
Biology 2023,12, 196 2 of 28
1. Introduction
Aging is a process characterized by progressive frailty development in animals and
humans. With the increase in the geriatric population worldwide, the promotion of healthy
aging arose as a key issue. In particular, novel studies focus on therapeutic strategies with
the goal to identify natural substances that may prevent, or even revert, aging-induced
adverse effects.
Among cognitive functions known to worsen during brain aging, the most affected
cognitive ability is episodic memory, declining early over time and displaying various
severity extent [
1
–
3
]. Among episodic memory, recognition memory refers to the capa-
bility to discriminate a stimulus or an environment as familiar or novel. Recognizing
objects, people, or environments previously encountered is a vital cognitive function in
animals. Indeed, the recognition memory is considered one of the essential features of
human and mammalian personalities [
4
]. Brain aging could be associated with cognitive
impairment, motor disorders and emotive disturbances, being these alterations provoked
by different morphological and functional changes involving the brain. Specifically, cellular,
biochemical and molecular studies showed the imperative role played by inflammation and
oxidative stress in the pathogenesis of cognitive impairments and age-associated neuronal
diseases [
5
,
6
]. Undeniable evidence linked aging and frailty to both immunosenescence
and chronic systemic inflammation, the alleged inflammaging, this latter mechanism being a
distinctive feature of accelerated aging [
7
]. Chief molecules are involved in this process such
as cytokines Interleukin-6 (IL6) and Transforming Growth Factor-beta1 (TGF
β
1), inflamma-
tory mediators known to participate in clinical and pathologic events during inflammatory
diseases that are typically involved in the onset of chronic inflammation-induced fibrotic
phenomena [8,9].
It is well established that glial cells are essentially involved during injury and neu-
rodegenerative processes in the CNS. In particular, glial fibrillary acidic protein (GFAP)
is usually employed as a marker of reactive gliosis associated with brain injury and CNS
fibrotic events [
10
,
11
]. Experimental and epidemiological evidence demonstrated that
aging brain is characterized by extensive gliosis in specific brain areas [
12
]. Therefore,
the association between activated astrocytes/microglial cells with the release of soluble
cytokines strongly suggests that inflammatory processes may play a critical task in the
complicated pathophysiological interactions that occur during aging [13].
Other than inflammation and gliosis, mounting evidence emphasized the oxidative
damage role in aging and age-associated cognitive and locomotor decline as the result
of boosted reactive oxygen species (ROS) production and/or reduction in antioxidant
scavengers [
6
,
14
–
16
]. Furthermore, in a loop manner, inflammaging onset enhances the
vulnerability and responsiveness to stress-related molecules.
Concerning chief oxidative stress mediating factors, nuclear factor erythroid 2–related
factor 2 (Nrf2) regulates the expression levels of several enzymes involved in oxidative
stress and maintains cellular resistance to oxidants and the onset of the aging process [
17
].
Moreover, superoxide dismutase 1 (SOD1) is another principal protein involved in the
enzymatic defenses against ROS production, participating in the maintenance of an appro-
priate free radicals’ level needed to preserve the physiological CNS functioning [
18
]. The
overexpression of the inducible cyclooxygenase 2 (COX2) appears to play a dual role in the
brain, acting as marker and effector of neural damage after brain injuries, in physiological
or pathological aging [19,20].
The inducible form NOS2 is responsible for the generation of NO in different cells.
Diverse studies confirmed that NO and pro-oxidants surplus are capable of triggering
neuronal functional impairment and structural injury in some brain areas [21,22].
Glutamatergic pathway is involved in learning, memory formation/storage and
synaptic plasticity [
23
]. It is also well known that aging is associated with cognitive
deficits/decline via diverse mechanisms, including the change/impairment of glutamater-
gic pathway [
5
,
24
] and also affecting receptors’ binding and density [
25
–
27
]. Changes in the
expression of the ionotropic receptors N-methyl-D-aspartate receptor (NMDARs) have been
Biology 2023,12, 196 3 of 28
previously described in rat and murine hippocampus during aging or disease [
28
,
29
]. The G
protein-coupled metabotropic glutamate receptors (mGluRs) are enriched in the hippocam-
pal formation and interact physically with other proteins, including glutamate ionotropic
receptors, to ensure the maintenance of cognitive performance. Changes in hippocampal
mGluR expression levels were described in pathological cognitive aging [26,30,31].
Hericium erinaceus (He), also called Lion’s mane and Monkey Head Mushroom, is
a palatable medicinal mushroom possessing many health properties including antioxi-
dant, anti-inflammatory, antisenescence, neuroprotective and nootropic effects [
32
–
35
].
Ergothioneine (ERGO), a potent antioxidant molecule which gained the name of “longevity
vitamin” [36,37], is often de novo synthesized in several fungi species.
In frail elderly people a negative correlation exists between plasma ERGO levels and
cognitive decline. In particular, patients suffering from dementia have impaired cognitive
ability correlated with declining plasma ERGO levels [
38
]. Scientists hypothesized that
plasma ERGO levels could be a valuable tool, not only to early diagnose but also to provide
effective therapeutic interventions able to mitigate cognitive decline. Recently, a significant
enhancement of object recognition memory, determined by both NOR and OL tasks, was
reported after oral administration of the food-derived ERGO in mice where an increased
ERGO concentration was measured both in plasma and hippocampus level [39].
In previous recent papers we reported the presence of ERGO and other nootropic
metabolites, in different strains of Italian Hericium erinaceus, highlighting their advanta-
geous effects on both cognitive and locomotor performances in a murine model of physio-
logical aging [
7
,
40
,
41
]. Lately, we encountered an H. erinaceus primordium that showed
a peculiar high content of ergo but lacked both hericenones and erinacines [
6
,
40
]. There-
fore, we seized the opportunity to explore the ergo-rich primordium preventive action on
hippocampus during aging. In the present study a multi-tiered methodology allowed exam-
ination of potential neuroprotective effects with power to prevent cognitive decline during
aging in mice. In particular, the experiments focused on: (i) monitoring spontaneous behav-
ioral tests to gauge both components of recognition memory (i.e., knowledge/familiarity
and remember/recollection), and the frailty index; and then (ii) investigating inflammag-
ing, oxidative stress, gliosis and glutamatergic receptors expression in the hippocampus,
appraising specific biomarkers. This study will give important information about the
role of those mechanisms in this CNS area and on the selective protective effect of the
ergothioneine rich primordium extract.
2. Materials and Methods
2.1. Animals
Fifteen pathogen-free C57BL-6J wild-type male mice were purchased from Charles
River Italia, Calco, Italy. Mice were maintained in 12 h light/dark cycle in the Animal
Facility of the University of Pavia at controlled temperature (21
±
2
◦
C) and humidity
(50 ±10%).
Water and food were provided ad libitum. All the mice were acclimatized for at
least one month before starting the study.
All experiments were carried out in compliance with the guidelines laid out by the
Ethics Committee of Pavia University for animal welfare (Ministry of Health, License
number 774/2016-PR), and in accordance with the European Council Directive 2010/63/EU
on the care and use of laboratory animals.
2.2. Experimental Plan Schedule
In vivo
experiments were performed at four different animal ages (corresponding to
specifically reported experimental times): 11- and 14-months old (T0 and T1, respectively,
occurring during adulthood phase); 20- and 23-months old (T2 and T3, respectively, taking
place during senescence). At T3, tissue sampling and immunohistochemistry experiments
were carried out. An 8-month lasting H. erinaceus oral supplementation was performed.
Specifically, starting from 15 months of age, nine randomized mice were oral supplemented
with a drink made with H. erinaceus (strain 2) primordium (called He2) ethanol extract
Biology 2023,12, 196 4 of 28
solubilized in water (group I, namely P mice). The remaining six mice did not receive
any supplementation (group II, namely C animals). The selected final dose of 1 mg of
primordium/day was chosen to mimic the human oral supplementation (about 1 g/day).
Hence, He2 primordium supplementation began during adulthood and continued until
sacrifice at T3, during senescence. For experimental details see Figure 1.
Biology 2023, 12, x 5 of 29
Figure 1. Physiological decline of the “Knowledge” component of recognition memory during aging
in control mice and neuroprotection by He2 primordium extract in supplemented mice. For each
panel, control (C) animals are represented with red bars, dots, and histograms, whereas supple-
mented (P) mice are symbolized by green bars, dots, and histograms. Panel (A−C) refer to Emer-
gence test: (A) exits number, (B) exploring time, (C) 1st latency to firs exit. Panel (D, E) describe
NOR test: (D) discrimination index (DI) of number of approaches, (E) DI of time of approaches.
Panel (F): scatter plot showing integrated FIs for individual C and P mice (left upright); linear least-
square regression of experimental points averaged data (left downright). Separated tables on the
Figure 1.
Physiological decline of the “Knowledge” component of recognition memory during aging
in control mice and neuroprotection by He2 primordium extract in supplemented mice. For each
panel, control (C) animals are represented with red bars, dots, and histograms, whereas supplemented
Biology 2023,12, 196 5 of 28
(P) mice are symbolized by green bars, dots, and histograms. Panel (
A−C
) refer to Emergence test:
(
A
) exits number, (
B
) exploring time, (
C
) 1st latency to firs exit. Panel (
D
,
E
) describe NOR test:
(
D
) discrimination index (DI) of number of approaches, (
E
) DI of time of approaches. Panel (
F
):
scatter plot showing integrated FIs for individual C and P mice (
left upright
); linear least-square
regression of experimental points averaged data (
left downright
). Separated tables on the right
display statistical results about aging effect in C (
upright
) and P (
downright
) animals. Statistically
significant data: p< 0.05 (£, #, *); p< 0.01 (££, ##, **); p< 0.001 (£££, ###, ***).
Scientists conducting experimental procedures, i.e., spontaneous behavioral tests,
immunohistochemistry, and statistical analyses, were blinded to the experimental condition.
2.3. Behavioral Tests and Cognitive Frailty Index
Spontaneous behavioral tests were performed to explore cognitive performances dur-
ing aging. The choice of the behavioral test has been based on two main needs: (i) avoiding
excessive animal handling; and (ii) translating the experimental results to human clinical
settings. The emergence test and NOR tasks used in mice for studying the “Knowledge
component” of the recognition memory are similar to the “Stenberg Item Recognition
paradigm” conventionally utilized in clinical practice. Furthermore, OL and Y maze tasks
used in mice to investigate the Remember component of the recognition memory are
like the “Four mountains test and Image-location memory task” employed by hospital
clinicians.
Animals’ performances were measured by using SMART video tracking system (2 Bi-
ological Instruments, Besozzo, Varese, Italy) and Sony CCD color video camera (PAL).
At different selected experimental times all mice performed different spontaneous be-
havioral tests for investigating both components of spontaneous recognition memory:
knowledge/familiarity and remember/recollection.
Emergence and Novel Object Recognition tasks (NOR) evaluated the knowledge com-
ponent, whereas Y maze and Object location tasks (OL) assessed the remember component.
All these tests were conducted as previously described [
42
,
43
]. For each test we
selected specific parameters that decline during aging (Table 1).
Table 1.
Selected parameters for studying cognitive (Emergence, NOR, OL, Y maze) and locomotor
(Open arena) performances of mice at four selected experimental time.
Spontaneous Behavioral Test Selected Cognitive Parameters
Emergence
Exit Number (n)
Latency of First Exit (s)
Time of Exploration (s)
NOR Number of Approaches: DI
Time of Approaches: DI
OL Number of Approaches: DI
Time of Approaches: DI
Y maze Alternation %
For each selected parameter we obtained the corresponding cognitive frailty index
(FI), by using the following formula [41]:
FI = (Value-Mean Value at T0)/(SD at T0) ×0.25
For each test we averaged the FIs for each parameter and obtained the Fi score for the
specific spontaneous test. Then, we obtained the averaged cognitive FI for all tests and the
FI for each component of the recognition memory. Finally, by averaging the knowledge
and remember recognition memory FIs we obtained a global cognitive FI.
Biology 2023,12, 196 6 of 28
2.4. H. erinaceus (He2) Primordium: Isolation/Cultivation, Extraction Procedures and
Analytical Determinations
As previously described, the He2 strain was isolated from a wildtype sporophore
collected in Italy in 2018 [44]. The sporophore was aseptically cut into small pieces (about
1 mm
3
) that were inoculated in Petri dishes containing 2% malt extract agar (MEA). The
isolated strain is preserved in the MicUNIPV, the Fungal Research Culture Collection of
University of Pavia, Italy. Through isolation in pure culture, mycelium was for obtaining
the sporophore, as previously reported [
41
]. The primordium developed at the initial stages
of growth; it was collected, analyzed, and used for the murine supplementation.
Concerning extraction procedure and analytical measurements, they were carried out
as previously reported in detail [
6
]. Briefly, 1 g of dried He2 primordium was blended
with ethanol 70% (10 mL) and then processed as described in literature [
41
,
45
]. HPLC-UV-
ESI/MS was used to identify and measure the ERGO amount in He2 primordium extract
by comparison with ERGO calibration curve (L-(+)-Ergothioneine (497-30-3, TETRAHE-
DRON, Paris, France) as standard (for details, see [
6
]). The ERGO calibration curve was
constructed by injecting five different concentrations of standard mixture solutions (10, 70,
150, 350 mg/L, analyzed in triplicate).
2.5. Necropsy, Tissue Sampling and Immunohistochemical Analyses
2.5.1. Brain Specimens’ Preparation
At T3, 23-months-old mice were deeply anesthetized by isoflurane inhalation (Aldrich,
Milwaukee, WI, USA) before sacrifice. Brains were immediately excised as previously
described [
7
], washed in 0.9% NaCl, and fixed by immersion for 48 h at room temperature
in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Tissues were then dehydrated
in absolute ethanol, followed by acetone, and finally embedded in Paraplast X-TRA (Sigma
Aldrich, Milan, Italy). Eight micrometer-thick sections of brains were cut in the coronal
plane and collected on silane-coated slides. After employing Hematoxylin and Eosin (H&E)
histological staining [46], precise sections were chosen to study the hippocampus.
2.5.2. Immunohistochemistry and Bright-Field Microscopy
To explore expression and distribution of specific molecules, immunohistochemi-
cal experiments were performed as previously reported [
7
] using commercial antibod-
ies on murine brain specimens. Interleukin-6 (IL6), Transforming Growth Factor-beta1
(TGF
β
1), glial fibrillary acidic protein (GFAP), nuclear factor erythroid 2–related factor 2
(Nrf2), superoxide dismutase 1 (SOD1), cyclooxygenase 2 (COX2), nitric oxide synthase 2
(NOS2), metabotropic glutamate receptor 2 (mGluR2) and ionotropic N-methyl-D-aspartate
(NMDA) receptor 1 (NMDAR1). Coronal brain sections from C and P mice were incubated
overnight at RT with PBS-diluted monoclonal and polyclonal primary antibodies (Table 2).
IL6, TGF
β
1 and GFAP were evaluated as specific markers of inflammation and reactive
gliosis, respectively; Nrf2, SOD1, COX2 and NOS2 were assessed being essentially involved
in oxidative stress cascade [
6
,
47
–
49
]; metabotropic glutamate receptors-2 (mGluR2) and
N-methyl-d-aspartate (NMDA) receptor-1 (NMDAR1) were considered for their crucial
role in glutamatergic pathway [
50
,
51
]. To reveal the antigen/antibody interaction sites,
proper biotinylated secondary antibodies (Table 2) and an avidin biotinylated horseradish
peroxidase complex (Vector Laboratories, Burlingame, CA, USA) were used. The 3,3
0
-
diaminobenzidine tetrahydrochloride peroxidase substrate (Sigma, St. Louis, MO, USA)
was employed as the chromogen, and the nuclear counterstaining was achieved by us-
ing Carazzi’s Hematoxylin. Then, the sections were dehydrated in ethanol, cleared in
xylene, and mounted in Eukitt (Kindler, Freiburg, Germany). As negative control some
sections were incubated with PBS only, in the absence of the primary antibodies, revealing
a complete lack of immunoreactivity.
Biology 2023,12, 196 7 of 28
Table 2. Primary and secondary antibodies.
Antigen Immunogen
Manufacturer, Species,
Mono-Polyclonal, Cat./Lot.
No., RRID
Dilution
Primary
antibodies
Anti-Interleukin-6
(M-19)
Purified antibody raised
against a peptide mapping at
the C-terminus of murine IL6
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Goat
polyclonal IgG, Cat# sc-1265,
RRID: AB_2127470
1:100
Anti-Transforming
Growth Factor β1 (V)
Purified antibody raised
against a peptide mapping at
the C-terminus of TGF-β1
of human origin
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Rabbit
polyclonal IgG, Cat# sc-146,
RRID: AB_632486
1:100
Anti-Glial fibrillary
acidic protein (C-19)
Purified antibody raised
against a peptide mapping at
the C-terminus of GFAP
of human origin
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Goat
polyclonal IgG, Cat# sc-6170,
RRID: AB_641021
1:100
Anti-Nuclear factor
erythroid 2–related
factor 2
Purified antibody raised
against a peptide within
Human Nrf2 aa 550 to the
C-terminus
Abcam (Cambridge, UK),
Rabbit polyclonal IgG, Cat#
ab31163,
RRID: AB_881705
1:100
Anti-Superoxide
Dismutase-1
(FL-154)
Purified antibody raised
against amino acids 1–154
representing full-length
human SOD-1
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Rabbit
polyclonal IgG, Cat# sc-11407,
RRID: AB_2193779
1:100
Anti-
Cyclooxygenase-2
(M-19)
Purified antibody raised
against a peptide mapping at
the C-terminus of COX2
of mouse origin
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Goat
polyclonal IgG, Cat# sc-1747,
RRID: AB_2084976
1:100
Anti-Nitric Oxide
Synthases-2 (M19)
Purified antibody raised
against a peptide mapping at
the C-terminus of NOS2
of mouse origin
Santa Cruz Biotechnology
(Santa Cruz, CA, USA), Rabbit
polyclonal IgG, Cat# sc-650,
RRID: AB_631831
1:100
Anti-
N-methyl-D-aspartate
Receptors 1
Purified antibody raised
against a peptide
corresponding to the
C-terminus of rat NMDA
receptor subunit
Millipore—Merck KGaA
(Darmstadt, Germany),
Rabbit monoclonal IgG,
Cat# AB9864, RRID:
AB_2112158
1:500
Anti-Glutamate
Receptor 2 and 3
Purified antibody raised
against a peptide mapping at
the C-terminus of rat GluR2
Millipore—Merck KGaA
(Darmstadt, Germany),
Rabbit polyclonal IgG,
Cat# AB1506, RRID: AB_ 90710
1:100
Secondary
Antibodies
Biotinylated goat
anti-rabbit IgG
Gamma
immunoglobulin
Vector Laboratories
(Burlingame, CA, USA),
Goat, lot# PK-6101,
RRID: AB_2336820
1:200
Biotinylated rabbit
anti-goat IgG
Gamma
immunoglobulin
Vector Laboratories
(Burlingame, CA, USA),
Rabbit, Cat# PK-6105, RRID:
AB_2336824
1:200
2.5.3. Immunohistochemical Evaluations
Sections were observed in brightfield microscopy using an Olympus BX51 optical
microscope (model BX51TF, Olympus Italia S.r.l, Segrate, Italy) and images were acquired
with an Olympus CAMEDIA C4040ZOOM camera. For each assessed marker, five slides
(about 20 sections) per mouse were examined. In both experimental groups’ hippocampal
Biology 2023,12, 196 8 of 28
specimens with diverse immunolabeling extents were considered. For each immunohis-
tochemical reaction the most representative figures were selected and are shown. Im-
munohistochemical labeling degree was measured on acquired digitized section images
under exposure time avoiding any pixel saturation effect. Immunoreactive cell density
(number of immunopositive cells/area in mm
2
) and labeling intensity were calculated
utilizing densitometric analysis (Image-J 1.48i; NIH, Bethesda, MA, USA), as previously
described [
7
]. Briefly, the immunocytochemical intensity, namely OD, was evaluated in
three randomized images/section per five slides/animal from each experimental group.
Data were recorded on Microsoft Office Excel Software spreadsheets and the analysis was
achieved using the ImageJ software.
2.6. Statistics
Data were reported as mean standard error of the mean (SEM). We performed Bartlett
and Shapiro–Wilk Tests to establish and confirm the normality of parameters. Concerning
behavioral tests, One-way Anova for repeated measures was used for investigating the
aging effect (£ vs. 11 months (T0), # vs. 14 months; $ vs. 20 months), whereas two-way
Anova was used to compare C and P groups. The differences were considered statistically
significant for p< 0.05 (£, #, $, *), p< 0.01 (££, ##, $$, **), and p< 0.001 (£££, ###, $$$, ***).
The statistical analysis for immunohistochemical evaluations was carried out using
an unpaired Student’s t-test. The differences were considered statistically significant for
p< 0.05 (*), p< 0.01 (**), and p< 0.001 (***).
All statistical analyses were performed with GraphPad Prism 8.0 software (GraphPad
Software Inc., La Jolla, CA, USA).
3. Results
As previously reported, He2 primordium extract, characterized by HPLC-UV-ESI/MS,
contains 1.3
±
0.57 mg/g ERGO [
6
], being this amount three times higher than that mea-
sured in both mycelium (0.58 mg/g) and sporophore (0.34 mg/g) of the strain He1 [
7
].
Furthermore, primordium extract does not contain the nootropic metabolites hericenones
or erinacine [6].
3.1. Cognitive Outcomes
3.1.1. He2 Preventive Effect on Aging Decline of the “Knowledge” Memory Component
For a longitudinal study of the “knowledge” component during aging, we measured
selected parameters (see Table 1in Section 2) in the emergence and NOR tests at chosen
experimental times: 11 (T0), 14 (T1), 20 (T2), and 23 (T3) months (Figure 1) in both C and
He2 supplemented (namely P) mice.
In C animals, all parameters recorded during Emergence task, such as exits number,
exploring time, and latency to first exit (Figure 1A–C, see red bars and dots) worsened
over time, from T0 to T3. Notably, after T1 the oral supplementation with He2 primordium
extract significantly improved all recorded parameters until T3. Indeed, P group showed
similar values for all recorded parameters in adulthood (T0) and in senescence phases (T2
and T3) (Figure 1A–C, see green bars and dots).
In NOR test the discrimination index related to both number and time of approaches
(Figure 1D,E, respectively), deteriorated after T2 in C mice, reaching negative values at
T2 and T3. Remarkably, after T1 the He2 supplementation significantly inhibited the
cognitive deterioration in wild-type mice both at T2 and T3. Indeed, P mice displayed
similar values for all recorded parameters during the entire investigated life span (from
adulthood to senescence phases) and, further, these values were significantly different from
those measured in C animals during senescence, i.e., T3 and T4.
Biology 2023,12, 196 9 of 28
Finally, we calculated the cognitive FI for each parameter in the emergence and in
the NOR tests and we separately averaged the FIs for the two tests. Then, averaging the
two obtained FIs, we calculated the global, integrated cognitive FIs of the knowledge
component of the recognition memory (Figure 1F).
The integrated knowledge cognitive FI significantly increased during aging in C
animals (C, red dots) in a straight way, as demonstrated by linear least-square regression
analysis (R
2
= 0.99). Outstandingly, P mice (P, green dots) did not develop frailty hallmarks
during aging, thus demonstrating the preventive effect of the oral supplementation with
He2 primordium extract on this specific component of the recognition memory.
3.1.2. He2 Preventive Effect on Aging Decline of the “Remember” Memory Component
For a longitudinal study of the “remember” component during aging we gauged
selected parameters (see Table 1in Section 2) in OL and Y maze tests at specific chosen
experimental times: 11 (T0), 14 (T1), 20 (T2), and 23 (T3) months in C and He2 supplemented
P mice.
During physiological aging, in OL test the discrimination index related to both number
and time of approaches worsened after T2 in C mice (Figure 2A,B, red bars). Differently,
after T1 the He2 primordium extract oral supplementation prevented the cognitive decline
hindering the deterioration of the discrimination index during the senescence phases
(Figure 2A,B, green bars). Furthermore, in P mice the discrimination index of both selected
parameters, measured at T2 and T3, was significantly higher compared to that gauged in C
animals, suggesting not only a prevention of cognitive decline during aging but also an
He2-induced “gain of function” of this memory component.
The cognitive parameters evaluated during Y maze task, the alternation triplets’ per-
centage, appreciably decreased during the senescence phases, both at T2 and T3 (Figure 2C).
Remarkably, after T1 the He2 primordium extract oral supplementation substantially pre-
vented this cognitive deterioration, and, furthermore, increased this cognitive parameter
in a significant manner compared to C mice. In accordance with previously described
data, even in this case we assumed a gain of function in the remember memory component
adjuvated by He2 primordium oral supplementation.
Next, we calculated the cognitive FI for each parameter and the obtained value for
each test was averaged. Finally, we averaged the two-frailty index for OL and Y maze test
in an integrated, global FI of the remember component of recognition declarative memory
(Figure 2D). Similar to the situation above described for knowledge memory component,
remember cognitive FI significantly increased during aging in C animals (C, red dots)
in a straight way, as demonstrated by linear least-square regression analysis (R
2
= 0.98).
Notably, in P mice (P, green dots) after supplementation, the remember component reached
a value higher than that measured in young animals suggesting a boosting effect or a “gain
of function” of this component despite animal age.
Biology 2023,12, 196 10 of 28
Biology 2023, 12, x 11 of 29
Figure 2. Physiological decline of the “Remember” component of recognition memory during aging
in control mice and neuroprotection by He2 primordium extract in supplemented mice. For each
panel, control (C) animals are represented with red bars, dots, and histograms, while supplemented
(P) mice are symbolized by green bars, dots, and histograms. Panel (A, B) describe OL test: discrim-
ination index (DI) of the number (A) and time of approaches (B). Panel (C) is related to Y maze task
(alternation triplets %). Panel (D) display Remember cognitive Frailty Index (FI): scatter plot show-
ing integrated FIs for individual C and P mice (left upright); linear least-square regression of exper-
imental points averaged data (left downright). On the right, separated tables report statistical data
Figure 2.
Physiological decline of the “Remember” component of recognition memory during aging in
control mice and neuroprotection by He2 primordium extract in supplemented mice. For each panel,
control (C) animals are represented with red bars, dots, and histograms, while supplemented (P) mice
are symbolized by green bars, dots, and histograms. Panel (
A
,
B
) describe OL test: discrimination
index (DI) of the number (
A
) and time of approaches (
B
). Panel (
C
) is related to Y maze task
(alternation triplets %). Panel (
D
) display Remember cognitive Frailty Index (FI): scatter plot showing
integrated FIs for individual C and P mice (
left upright
); linear least-square regression of experimental
points averaged data (
left downright
). On the right, separated tables report statistical data concerning
age-related effect in C (
upright
) and P (
downright
) mice. Statistically significant data: p< 0.05 (£, #,
*); p< 0.01 (££, ##, **); p< 0.001 (***).
Biology 2023,12, 196 11 of 28
3.2. Histological and Immunohistochemical Data
All reactions were conducted on coronal brain sections from both aged non-supple-
mented, i.e., controls (C animals), and aged He2 primordium-treated mice (P group) at
T3 (23-month-old animals), focusing on the hippocampus being crucial in recognition and
spatial memory [52,53].
All examinations focused on the dentate gyrus (DG), and the Ammon’s horn region
(including CA subdivisions) of the hippocampus where oxidative stress and inflammation
were predominantly localized.
3.2.1. He2 Supplementation Preserves Hippocampus Healthy Cytoarchitecture
H&E staining was employed to estimate the potential occurrence of age- and/or He2-
related changes in hippocampus cytoarchitecture in aged C and P mice. Representative
pictures obtained by H&E in C (Figure 3a–c,f) and P (Figure 3d,e,g) mice are illustrated in
Figure 3. The physiological gross morphology of the whole hippocampus was preserved in
both experimental groups (Figure 3a–d). In particular, in C and P mice high-magnification
micrographs of the DG revealed well-defined three layers, namely molecular layer (ML),
granule cell layer (GL) and pleomorphic layer (PL). Concerning the CA1, the typical three
layered-structure was observed, characterized by the presence of the outer polymorphic
layer, namely Stratum oriens (SO), the middle pyramidal cell layer, namely Stratum pyrami-
dale (SP) and the inner molecular layer, namely Stratum radiatum (SR).
Nonetheless, distinct structural alteration of the choroid plexus was revealed in control
mice only (Figure 3f), with ependymal (choroid epithelial) cells displaying an evident cilia
reduction. Additionally, comparing C and P animals, a significant increase in shrunken cell
density was measured both in DG and particularly in the CA1 region of Ammon’s horn in
the C mice only (Figure 3).
3.2.2. He2 Supplement Decreases Inflammaging
In the current study we immunohistochemically assessed the presence/distribution of
specific molecules, i.e., IL6, TGF
β
1, and GFAP, as typical indicators of the inflammatory
pathway (Figures 4and 5and Table 3). For all investigated markers, in both experimental
groups, i.e., C and P mice, the immunopositivity was mainly localized in the DG and CA1
region of Ammon’s horn.
Table 3.
Quantitative measurement of inflammation markers, namely IL6, TGF
β
1 and GFAP in the
hippocampus, precisely in the DG and CA1 of C and P mice. n.c.: not comparable.
IL6 TGFbeta1 GFAP
Cell Density OD Cell Density OD Cell Density OD
DG C 310.79 ±17.86 178.50 ±9.40 728.24 ±33.81 142.20 ±2.96 298.24 ±19.37 137.69 ±17.17
P 42.26 ±6.27 120.00 ±4.21 119.01 ±35.41 121.06 ±4.62 165.69 ±12.69 86.78 ±4.49
CA1 C 289.13 ±19.61 127.80 ±6.63 n.c. 30.49 ±1.44 109.87 ±13.47 163.84 ±3.68
P 44.48 ±21.82 96.68 ±3.47 n.c. 30.97 ±0.56 89.71 ±7.53 138.49 ±1.55
Biology 2023,12, 196 12 of 28
Biology 2023, 12, x 13 of 29
Figure 3. Histological characterization by H&E staining. Representative brain sections, showing the
well-preserved physiological hippocampal cytoarchitecture both in non-supplemented controls (a–
d) and P (d–e) aged mice. (a): low magnification micrograph shows the whole hippocampus, formed
of cornu Ammonis (CA) and dentate gyrus (DG). CA is further partitioned into: CA1, CA2, CA3
and CA4. The choroid plexus (CP) of the lateral ventricle can be also observed. (b,d): higher magni-
fications of the DG area revealing well-defined three layers: molecular layer (ML), granule cell layer
(GL) and pleomorphic layers (PL). (c,e): higher magnifications of the CA1 region, showing the typ-
ical three layered-structure. Outer polymorphic layer, i.e., Stratum oriens (SO); middle pyramidal
cell layer, namely Stratum pyramidale (SP); inner molecular layer, i.e., Stratum radiatum (SR). (f,g):
choroid plexus (CP) in C and P mice, respectively. (f): evident structural alterations were observable,
with ependymal cells displaying cilia reduction. Light microscopy magnification: 40× (a), 200× (b,d),
400× (c,e–g). Scale bars: 500 µM (a); 200 µM (b,d); 100 µM (c,e–g). Lower left panels: Histograms show-
ing the quantitative assessment of shrunken cell density in DG and CA1 region of Ammon’s horn.
p values calculated by unpaired Student’s t-test: p < 0.01 (**), and p < 0.001 (***).
3.2.2. He2 Supplement Decreases Inflammaging
In the current study we immunohistochemically assessed the presence/distribution
of specific molecules, i.e., IL6, TGFβ1, and GFAP, as typical indicators of the inflammatory
pathway (Figures 4 and 5 and Table 3). For all investigated markers, in both experimental
groups, i.e., C and P mice, the immunopositivity was mainly localized in the DG and CA1
region of Ammon’s horn.
Figure 3.
Histological characterization by H&E staining. Representative brain sections, showing
the well-preserved physiological hippocampal cytoarchitecture both in non-supplemented controls
(
a
–
d
) and P (
d
,
e
) aged mice. (
a
): low magnification micrograph shows the whole hippocampus,
formed of cornu Ammonis (CA) and dentate gyrus (DG). CA is further partitioned into: CA1, CA2,
CA3 and CA4. The choroid plexus (CP) of the lateral ventricle can be also observed. (
b
,
d
): higher
magnifications of the DG area revealing well-defined three layers: molecular layer (ML), granule cell
layer (GL) and pleomorphic layers (PL). (
c
,
e
): higher magnifications of the CA1 region, showing the
typical three layered-structure. Outer polymorphic layer, i.e., Stratum oriens (SO); middle pyramidal
cell layer, namely Stratum pyramidale (SP); inner molecular layer, i.e., Stratum radiatum (SR). (
f
,
g
):
choroid plexus (CP) in C and P mice, respectively. (
f
): evident structural alterations were observable,
with ependymal cells displaying cilia reduction. Light microscopy magnification: 40
×
(
a
), 200
×
(
b
,
d
),
400
×
(
c
,
e
–
g
). Scale bars: 500
µ
M (
a
); 200
µ
M (
b
,
d
); 100
µ
M (
c
,
e
–
g
).
Lower left panels
: Histograms
showing the quantitative assessment of shrunken cell density in DG and CA1 region of Ammon’s
horn. pvalues calculated by unpaired Student’s t-test: p< 0.01 (**), and p< 0.001 (***).
Biology 2023,12, 196 13 of 28
Biology 2023, 12, x 14 of 29
Table 3. Quantitative measurement of inflammation markers, namely IL6, TGFβ1 and GFAP in the
hippocampus, precisely in the DG and CA1 of C and P mice. n.c.: not comparable.
IL6
TGFbeta1
GFAP
Cell density
OD
Cell density
OD
Cell density
OD
DG
C
310.79 ± 17.86
178.50 ± 9.40
728.24 ± 33.81
142.20 ± 2.96
298.24 ± 19.37
137.69 ± 17.17
P
42.26 ± 6.27
120.00 ± 4.21
119.01 ± 35.41
121.06 ± 4.62
165.69 ± 12.69
86.78 ± 4.49
CA1
C
289.13 ± 19.61
127.80 ± 6.63
n.c.
30.49 ± 1.44
109.87 ± 13.47
163.84 ± 3.68
P
44.48 ± 21.82
96.68 ± 3.47
n.c.
30.97 ± 0.56
89.71 ± 7.53
138.49 ± 1.55
Figure 4. Immunostaining patterns of IL6 (a–d), TGFβ1 (e–h) and GFAP (i–n) expression in C ani-
mals (a,b,e,f,i–k) and P (c,d,g,h,l–n) mice. IL6: an evident IL6 immunolabelling is observable both
in DG and CA1 of C mice (a,b, respectively), showing several markedly immunopositive neurons
(arrows). Diversely, in supplemented P mice, a pale IL6-immunopositivity is observed in both areas
(c,d for DG and CA, respectively), where rare immunolabelled cells are discernible (arrows). TGFβ1:
a strong immunoreactivity for TGFβ1 is observed mainly in DG of C mice (e), where different clus-
ters of immunopositive neurons are visible (arrows). Sporadic immunopositive cells (arrows) are
observed in the DG of P animals (g). Differently, any immunostaining is observable in CA1 region
of both C and P mice (f,h, respectively). GFAP: a widespread GFAP immunolabelling is distributed
both in DG and CA1 of C (i–k) and P (l–n) mice. In particular, in C animals, a carpet of GFAP-
Figure 4.
Immunostaining patterns of IL6 (
a
–
d
), TGF
β
1 (
e
–
h
) and GFAP (
i
–
n
) expression in C animals
(a,b,e,f,i–k) and P (c,d,g,h,l–n) mice. IL6: an evident IL6 immunolabelling is observable both in DG
and CA1 of C mice ((
a
,
b
), respectively), showing several markedly immunopositive neurons (arrows).
Diversely, in supplemented P mice, a pale IL6-immunopositivity is observed in both areas ((
c
,
d
) for
DG and CA, respectively), where rare immunolabelled cells are discernible (arrows).
TGFβ1
: a strong
immunoreactivity for TGF
β
1 is observed mainly in DG of C mice (
e
), where different clusters of
immunopositive neurons are visible (arrows). Sporadic immunopositive cells (arrows) are observed
in the DG of P animals (
g
). Differently, any immunostaining is observable in CA1 region of both C and
P mice ((
f
,
h
), respectively).
GFAP
: a widespread GFAP immunolabelling is distributed both in DG
and CA1 of C (
i
–
k
) and P (
l
–
n
) mice. In particular, in C animals, a carpet of GFAP-immunopositive
astrocytes is clearly evident, showing thickened and intensely stained soma and prolongations
(arrows). Light microscopy magnification: 200
×
(
a
,
c
,
e
,
g
,
j
,
m
); 400
×
(
b
,
d
,
f
,
h
,
i
,
k
,
l
,
n
); 600
×
(Insert in
e,g). Scale bars: 200 µM (a,c,e,g,j,m); 100 µM (b,d,f,h,i,k,l,n).
Biology 2023,12, 196 14 of 28
Biology 2023, 12, x 15 of 29
immunopositive astrocytes is clearly evident, showing thickened and intensely stained soma and
prolongations (arrows). Light microscopy magnification: 200× (a,c,e,g,j,m); 400× (b,d,f,h,i,k,l,n);
600× (Insert in e,g). Scale bars: 200 µM (a,c,e,g,j,m); 100 µ M (b,d,f,h,i,k,l,n).
Figure 5. Panels (A–C): Histograms showing the quantitative analysis of IL6-, TGFβ1- and GFAP-
immunopositive cell density and OD, respectively, as determined in DG and CA1 region of C and
P mice. p values calculated by unpaired Student’s t-test: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
Concerning IL6, in the present study deeply IL6-immunostained neurons were
clearly observed in the DG (mainly located among the round to oval granule cell bodies
aggregation in the GL) of C mice only. Notably, several immunopositive neurons localized
nearby the sub-granular zone (SGZ) were also detected. Furthermore, in the same animals,
markedly IL6-immunoreactive neurons, possibly mossy cells, were revealed in the PL.
Concerning the evaluation of CA1, the SP exhibited several IL6-immunopositive py-
ramidal neurons showing closely packed cell somas regularly arranged in 4 to 5 strings,
with evidently immunomarked prolongations.
The subsequent quantitative analyses revealed an extremely significant reduction in
IL6-immunopositive cell density measured in both DG and CA1 in P mice only. A signif-
icant reduction in IL6-immunopositive cells OD was determined in both DG and CA1 of
P mice, compared to controls (Table 3 and Figure 5).
Figure 5.
Panels (
A
–
C
): Histograms showing the quantitative analysis of IL6-, TGF
β
1- and GFAP-
immunopositive cell density and OD, respectively, as determined in DG and CA1 region of C and P
mice. pvalues calculated by unpaired Student’s t-test: p< 0.05 (*), p< 0.01 (**), and p< 0.001 (***).
Concerning IL6, in the present study deeply IL6-immunostained neurons were clearly
observed in the DG (mainly located among the round to oval granule cell bodies aggregation
in the GL) of C mice only. Notably, several immunopositive neurons localized nearby the
sub-granular zone (SGZ) were also detected. Furthermore, in the same animals, markedly
IL6-immunoreactive neurons, possibly mossy cells, were revealed in the PL.
Concerning the evaluation of CA1, the SP exhibited several IL6-immunopositive
pyramidal neurons showing closely packed cell somas regularly arranged in 4 to 5 strings,
with evidently immunomarked prolongations.
The subsequent quantitative analyses revealed an extremely significant reduction
in IL6-immunopositive cell density measured in both DG and CA1 in P mice only. A
significant reduction in IL6-immunopositive cells OD was determined in both DG and CA1
of P mice, compared to controls (Table 3and Figure 5).
TGF
β
1 immunostaining was revealed in C mice only, mainly localized in GL and
PL of DG, whereas any immunostaining was detected in C and P mice in CA1 area
(Figures 4and 5and Table 3).
In detail, different clusters of immunopositive neurons were
observed, some of which were characterized by a large soma (mainly observed in PL); other
Biology 2023,12, 196 15 of 28
smaller ones assembled in orderly chains, typically arranged in the thickness of GL. The
quantitative assessment confirmed an extremely significant reduction in immunoreactive
cell density in P mice compared to C animals. Furthermore, a significant reduction in
TGF
β
1-immunopositive cells OD was determined in the DG of P mice only (Table 3). As
regards the CA1 area, any significant difference in TGF
β
1-immunoreactive cell OD was
measured comparing P and C mice (Table 3).
Herewith, the localization of GFAP showed a widespread distribution both in DG
and CA1 of P and C mice. In particular, in these latter animals a carpet of GFAP-positive
astrocytes was observed, showing thickened and intensely stained soma and prolongations
mainly located in the PL of DG and in the whole thickness of CA1.
The subsequent quantitative study confirmed a significant lessening of immunoposi-
tive glial cells for GFAP in the DG of P mice compared to C animals. Similarly, a significant
reduction in GFAP-immunopositive cells OD was measured in the DG of P mice com-
pared to controls. Likewise, a slight decrease in immunopositive glial cell density was
measured in CA1 region of P mice compared to C. Similarly, a significant decrease in GFAP
immunopositive cells’ OD was measured in the CA1 area of P animals only (Table 3).
3.2.3. He2 Supplement Diminished Age-Related ROS Levels
In the current study we assessed, by light microscopy immunohistochemistry, the
presence and distribution of crucial player of the oxidative stress pathway, i.e., Nrf2, SOD1,
COX2 and NOS2 (Figures 6and 7and Table 4). Like the above reported data concerning
inflammation, the immunoreactivity for all these oxidative stress markers was mostly
detected in the DG and CA1 region of Ammon’s horn in both experimental groups, namely
C and P mice.
Currently, immunohistochemical reaction for
Nrf2
revealed the heaviest immunopos-
itivity almost exclusively in C mice, mainly localized in the DG region, with several
markedly immunopositive cells closed to the SGZ, showing condensed nuclei, and also
in the PL, where bigger immunoreactive neurons, characterized by large somas, were
observed (Figure 6). Notably, in the same experimental group only, namely C animals, Nrf2
antigen resulted also overexpressed in pyramidal neurons of CA1 region (Figure 6).
Table 4.
Quantitative assessment of Oxidative stress key molecules, namely Nrf2, SOD1, COX2, and
NOS2, in the DG and CA1 of C and P mice. n.c.: not comparable.
Nrf2 SOD1
Cell Density OD Cell Density OD
DG C 258.84 ±14.41 169.78 ±13.00 415.97 ±32.21 140.49 ±7.22
P 79.24 ±10.42 125.38 ±9.66 98.74 ± − 12.40 111.20 ±3.67
CA1 C 341.02 ±41.94 139.38 ±1.37 n.c. 45.68 ±2.70
P 66.72 ±23.05 82.01 ±4.72 n.c. 45.19 ±2.03
COX2 NOS2
Cell Density OD Cell Density OD
DG C 241.23 ±18.33 100.20 ±2.58 30.52 ±2.32 90.91 ±12.17
P 192.81 ±14.11 100.01 ±3.23 29.63 ±2.69 89.84 ±2.97
CA1 C 404.04 ±49.32 119.39 ±9.47 253.78 ±38.06 123.79 ±7.98
P 40.77 ±16.44 82.39 ±2.65 70.78 ±25.40 67.95 ±2.18
Biology 2023,12, 196 16 of 28
Biology 2023, 12, x 17 of 29
Figure 6. Representative micrographs showing Nrf2, SOD1, COX2 and NOS2 immunohistochemical
expression in DG and CA1 area from non-supplemented C animals ((a,b,e–h,k,l,o,p), respectively)
and P ((c,d,i,j,m,n,q–r), respectively) mice. Nrf2: the heaviest immunopositivity is detectable almost
exclusively in C mice (a,b), mainly in the DG region (a), with several markedly immunopositive
cells closed to the SGZ (arrows), and also in the PL, where bigger immunoreactive neurons are ob-
servable (arrows). Nrf2 antigen is also overexpressed in pyramidal neurons of CA1 region (b). Di-
versely, immunopositive cells are rarely detectable in P mice (c,d), only in the DG (c) while the CA1
lacks immunoreactivity (d). SOD1: heavily immunopositive cells, localized both in the width of the
GL as well as nearby the SGZ, are clearly visible in the DG (e,f) of C mice (e–h). Also, strongly
immunomarked neurons appear evident in the PL (g). Few immunopositive cells are observed in
DG of P mice (i). A widespread lack of SOD1-immunoreactivity is detectable in the CA1 region both
in C and P animals (h,j, respectively). COX2: the strongest immunopositivity was detected in the
DG of both C and P animals (k,m, respectively), showing several immunoreactive neurons in the
PL (arrows). Several strongly immunomarked pyramidal neurons are observable in the CA1 area of
P mice (l), while a complete absence of immunoreactivity is evident in C animals (n). NOS2: a scarce
NOS-immunopositivity was observed in the DG of both C and P mice (o,q, respectively), where
palely immunoreactive cells are visible in the PL (arrows). An evident immunoreactivity is identifi-
able in neurons (arrows) located in the CA1 region of C mice (p), while a complete absence of im-
munopositivity is visible in P animals (r). Light microscopy magnification: 200× (a,c,e,i,k,m,o,q);
400× (b,d,h,j,l,n); 600× (f,g,p,r and insert in (a,i)). Scale bars: 200 µM (a,c,e,i,k,m,o,q); 100 µM
(b,d,f,g,h,j,l,n); 70 µM (p,r).
Figure 6.
Representative micrographs showing Nrf2, SOD1, COX2 and NOS2 immunohistochemical
expression in DG and CA1 area from non-supplemented C animals ((
a
,
b
,
e
–
h
,
k
,
l
,
o
,
p
), respectively)
and P ((
c
,
d
,
i
,
j
,
m
,
n
,
q
,
r
), respectively) mice.
Nrf2:
the heaviest immunopositivity is detectable almost
exclusively in C mice (
a
,
b
), mainly in the DG region (
a
), with several markedly immunopositive
cells closed to the SGZ (arrows), and also in the PL, where bigger immunoreactive neurons are
observable (arrows). Nrf2 antigen is also overexpressed in pyramidal neurons of CA1 region (
b
).
Diversely, immunopositive cells are rarely detectable in P mice (
c
,
d
), only in the DG (
c
) while the
CA1 lacks immunoreactivity (
d
).
SOD1
: heavily immunopositive cells, localized both in the width of
the GL as well as nearby the SGZ, are clearly visible in the DG (
e
,
f
) of C mice (
e
–
h
). Also, strongly
immunomarked neurons appear evident in the PL (
g
). Few immunopositive cells are observed in
DG of P mice (
i
). A widespread lack of SOD1-immunoreactivity is detectable in the CA1 region
both in C and P animals ((
h
,
j
), respectively).
COX2
: the strongest immunopositivity was detected
in the DG of both C and P animals ((
k
,
m
), respectively), showing several immunoreactive neurons
in the PL (arrows). Several strongly immunomarked pyramidal neurons are observable in the CA1
area of P mice (l), while a complete absence of immunoreactivity is evident in C animals (n). NOS2:
a scarce NOS-immunopositivity was observed in the DG of both C and P mice (
o
,
q
, respectively),
where palely immunoreactive cells are visible in the PL (arrows). An evident immunoreactivity is
identifiable in neurons (arrows) located in the CA1 region of C mice (
p
), while a complete absence of
immunopositivity is visible in P animals (
r
). Light microscopy magnification: 200
×
(
a
,
c
,
e
,
i
,
k
,
m
,
o
,
q
);
400
×
(
b
,
d
,
h
,
j
,
l
,
n
); 600
×
((
f
,
g
,
p
,
r
) and insert in (
a
,
i
)). Scale bars: 200
µ
M (
a
,
c
,
e
,
i
,
k
,
m
,
o
,
q
); 100
µ
M
(b,d,f,g,h,j,l,n); 70 µM (p,r).
Biology 2023,12, 196 17 of 28
Biology 2023, 12, x 18 of 29
Figure 7. Panels (A–D): Histograms showing the quantitative assessment of Nrf2-, SOD1-, COX2-
and NOS2-immunopositive cell density and OD, respectively, measured in DG and CA1 region of
C and P mice. p values calculated by unpaired Student’s t-test: p < 0.05 (*), p < 0.01 (**), and p < 0.001
(***).
Currently, immunohistochemical reaction for Nrf2 revealed the heaviest immuno-
positivity almost exclusively in C mice, mainly localized in the DG region, with several
markedly immunopositive cells closed to the SGZ, showing condensed nuclei, and also in
the PL, where bigger immunoreactive neurons, characterized by large somas, were ob-
served (Figure 6). Notably, in the same experimental group only, namely C animals, Nrf2
antigen resulted also overexpressed in pyramidal neurons of CA1 region (Figure 6).
Accordingly, the quantitative analysis documented an extremely significant reduc-
tion in immunolabeled cell density in the DG of P mice compared to controls. Further-
more, a significant parallel decrease in immunopositive cells OD was observed in the
same area comparing P and C mice (Figure 7 and Table 4).
With a similar trend, a very significant reduction in Nrf2-immunopositive cell den-
sity was detected in the CA1 area comparing P and C mice. Concerning Nrf2-immunore-
active cells OD, an extremely significant lessening was measured in the CA1 area of P
mice compared to C animals (Figure 7 and Table 4).
Figure 7.
Panels (
A
–
D
): Histograms showing the quantitative assessment of Nrf2-, SOD1-, COX2- and
NOS2-immunopositive cell density and OD, respectively, measured in DG and CA1 region of C and
P mice. pvalues calculated by unpaired Student’s t-test: p< 0.05 (*), p< 0.01 (**), and p< 0.001 (***).
Accordingly, the quantitative analysis documented an extremely significant reduction
in immunolabeled cell density in the DG of P mice compared to controls. Furthermore, a
significant parallel decrease in immunopositive cells OD was observed in the same area
comparing P and C mice (Figure 7and Table 4).
With a similar trend, a very significant reduction in Nrf2-immunopositive cell density
was detected in the CA1 area comparing P and C mice. Concerning Nrf2-immunoreactive
cells OD, an extremely significant lessening was measured in the CA1 area of P mice
compared to C animals (Figure 7and Table 4).
Regarding SOD1, the expression pattern differed strikingly between the two exper-
imental groups, namely C and P mice. Specifically, in C animals only, the DG was char-
acterized by clusters of heavily immunopositive cells localized both in the width of the
GL as well as nearby the SGZ. Moreover, in the same area, strongly immunomarked
neurons appeared evident in the PL. Concerning the CA1 region, a widespread lack of
immunoreactivity for SOD1 was revealed both in C and P animals (Figure 6).
Hence, the subsequent quantitative evaluation determined an extremely significant
decrease in SOD1-immunolabeled cell density in the DG comparing P and C mice. Likewise,
Biology 2023,12, 196 18 of 28
a significant reduction in SOD1-immunopositive cells OD was observed in the DG of P
animals only (Figure 7and Table 4). As regards the CA1 area, in line with the qualitative
observation noticed in light microscopy any significant change in SOD1-immunoreactive
cell OD was measured comparing P and C mice (Figure 7and Table 4).
Presently, the cellular localization and distribution of COX2 revealed a similar im-
munostaining pattern in the DG of both experimental groups, namely C and P mice. In
detail, the stronger immunopositivity was detected at cytoplasmic level in the neurons
of PL (Figure 6). Concerning the CA1 area, the COX2-immunoreactivity greatly diverged
between P and C mice. Specifically, several strongly immunomarked pyramidal neurons
were observed in P mice only, while a complete absence of immunoreactivity was revealed
in C animals (Figure 6).
The quantitative analysis documented only a slight reduction in COX2-immunopositive
cell density in the DG comparing P and C mice (Figures 6and 7and Table 4). Likewise,
focusing on immunolabeled cells OD, any significant difference was measured in the DG
comparing the two experimental groups (Table 4). Diversely, with regard to the CA1 area,
an extremely significant reduction in COX2-immunolabeled cell density was detected in P
mice compared to controls. Moreover, in the same supplemented animals, a very significant
lessening of immunopositive cell OD was determined (Figure 7and Table 4).
Concerning NOS2, the heaviest immunoreactivity was mainly identified in the cy-
toplasm of pyramidal neurons of the CA1 region of the Ammon’s horn in C mice only
(Figure 6). Differently, concerning the DG, in both experimental groups, namely P and
C animals, a pale NOS-immunopositivity was observed in rare cells of the PL while any
immunopositivity was detected in the GL (Figure 6).
Interestingly, the quantitative evaluation revealed an immunostaining pattern trend
comparable to that above reported for COX2. Specifically, only a slight non-significant
reduction in NOS2-immunopositive cell density was calculated comparing the two experi-
mental groups (Figure 7and Table 4). Showing a similar trend, any significant difference
was measured considering NOS2-immunolabeled cell OD both in P and C mice (Figure 7
and Table 4). Considering the CA1, a very significant reduction in NOS2-immunolabeled
cell density was reported in P mice compared to controls. Likewise, an extremely significant
reduction in NOS2-immunopositive cell OD was assessed comparing P and C animals
(Figure 7and Table 4).
3.3. He2 Supplement Improved Glutamate Neurotransmission in Aging
In the current study, immunohistochemical pictures obtained assessing specific mark-
ers of glutamatergic neurotransmission, i.e., NMDAR1 and mGluR2, molecules belonging
to the two general receptors classes of glutamatergic pathway, are depicted in Figure 8. In
particular, the localization of these two key molecules showed a widespread distribution
in the DG and CA1 area of Ammon’s horn in both experimental groups, namely P and C
mice, as formerly reported for all the other investigated markers.
Interestingly, we revealed an increase in NMDAR1-immunopositivity, both in the DG
and the CA1 region of the Ammon’s horn of P mice, compared to controls
(Figures 8and 9
and Table 5). Specifically, in P animals only the heaviest immunoreactivity was clearly
detected in the DG where clusters of strikingly immunopositive cells, arranged in well-
ordered chains, were observed in the width of the GL (Figure 8). Noticeably, several
immunolabelled cells were also located in the SGZ, and many immunoreactive neurons,
characterized by large soma, were discerned in the PL (Figure 8). As regards the CA1 area,
the SP exhibited several NMDAR1-immunopositive neurons, somewhere showing palely
immunomarked tiny prolongations, deepening in the underneath SR (Figure 8).
Biology 2023,12, 196 19 of 28
Biology 2023, 12, x 20 of 29
immunolabelled cells were also located in the SGZ, and many immunoreactive neurons,
characterized by large soma, were discerned in the PL (Figure 8). As regards the CA1 area,
the SP exhibited several NMDAR1-immunopositive neurons, somewhere showing palely
immunomarked tiny prolongations, deepening in the underneath SR (Figure 8).
Table 5. Quantitative appraisal of glutamatergic neurotransmission indicators, namely NMDAR1
and mGluR2, in hippocampal areas, i.e., DG and CA1, in C and P mice.
NMDAR1
mGluR2
Cell density
OD
Cell density
OD
DG
C
127.78 ± 25.13
160.44 ± 3.59
113.08 ± 41.06
193.14 ± 12.01
P
397.74 ± 32.78
178.13 ± 5.42
753.86 ± 102.81
201.83 ± 4.28
CA1
C
64.26 ± 23.40
69.65 ± 5.44
30.15 ± 16.29
142.90 ± 8.70
P
287.26 ± 16.33
177.71 ± 7.80
275.16 ± 19.83
153.10 ± 4.18
Figure 8. Immunohistochemical labelling for NMDAR1 and mGluR2 in DG and CA1 region from C
animals (a,b,f–h, respectively) and P (c–e,i,j, respectively) mice. NMDAR1: sporadic immuno-
labelled cells (arrows) are observable both in DG (a) and CA1 region (b) of C animals. A marked
immunopositivity is clearly evident both in the DG and CA1 area of P mice (c–e, respectively). In
Figure 8.
Immunohistochemical labelling for NMDAR1 and mGluR2 in DG and CA1 region from C
animals ((
a
,
b
,
f
–
h
), respectively) and P ((
c
–
e
,
i
,
j
), respectively) mice.
NMDAR1
: sporadic immuno-
labelled cells (arrows) are observable both in DG (
a
) and CA1 region (
b
) of C animals. A marked
immunopositivity is clearly evident both in the DG and CA1 area of P mice ((
c
–
e
), respectively). In
the DG, clusters of heavy immunoreactive cells, arranged in well-ordered chains, are observable
in the width of the GL (
c
), several immunolabelled cells are in the SGZ (
d
, arrows), and many
immunoreactive neurons with large soma are detectable in the PL ((
d
), arrows). In the CA1 area
several NMDAR1-immunopositive neurons are visible localized in the SP, often showing palely
immunomarked tiny prolongations, deepening in the underneath SR ((
e
), arrows).
mGluR2
: a weak
and sporadic immunoreactivity is observable both in DG (
f
,
g
) and CA1 region (
h
) of C animals, where
rare immunolabelled cells are detected (arrows). A heavy immunopositivity is clearly evident both in
the DG and CA1 area of P mice ((
i
,
j
), respectively). Some heavily immunoreactive cells are evident
in the DG (
i
), principally localized in the width of the GL and close to the SGZ (arrows). Various
immunomarked neurons are also noticeable in the PL ((
i
), arrows). Numerous immunopositive
neurons are detectable in the SP of CA1, often characterized by immunoreactive prolongations,
deepening beneath in the underlying SR (
j
). Light microscopy magnification: 200
×
(
a
,
d
,
g
,
i
); 400
×
(b,e,h,j); 600×((c,f) and insert in (i)). Scale bars: 200 µM (a,d,g,i); 100 µM (b,c,e,f,h,j).
Biology 2023,12, 196 20 of 28
Biology 2023, 12, x 21 of 29
the DG, clusters of heavy immunoreactive cells, arranged in well-ordered chains, are observable in
the width of the GL (c), several immunolabelled cells are in the SGZ (d, arrows), and many immu-
noreactive neurons with large soma are detectable in the PL (d, arrows). In the CA1 area several
NMDAR1-immunopositive neurons are visible localized in the SP, often showing palely im-
munomarked tiny prolongations, deepening in the underneath SR (e, arrows). mGluR2: a weak and
sporadic immunoreactivity is observable both in DG (f,g) and CA1 region (h) of C animals, where
rare immunolabelled cells are detected (arrows). A heavy immunopositivity is clearly evident both
in the DG and CA1 area of P mice (i,j, respectively). Some heavily immunoreactive cells are evident
in the DG (i), principally localized in the width of the GL and close to the SGZ (arrows). Various
immunomarked neurons are also noticeable in the PL (i, arrows). Numerous immunopositive neu-
rons are detectable in the SP of CA1, often characterized by immunoreactive prolongations, deep-
ening beneath in the underlying SR (j). Light microscopy magnification: 200× (a,d,g,i); 400× (b,e,h,j);
600× (c,f and insert in (i)). Scale bars: 200 µM (a,d,g,i); 100 µ M (b,c,e,f,h,j).
Figure 9. Panels (A,B): histograms displaying the quantitative evaluation of NMDAR1- and
mGluR2-immunopositive cell density and OD, respectively, measured in DG and CA1 area of C and
P mice. p values calculated by unpaired Student’s t-test: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).
Figure 9.
Panels (
A
,
B
): histograms displaying the quantitative evaluation of NMDAR1- and mGluR2-
immunopositive cell density and OD, respectively, measured in DG and CA1 area of C and P mice. p
values calculated by unpaired Student’s t-test: p< 0.05 (*), p< 0.01 (**), and p< 0.001 (***).
Table 5.
Quantitative appraisal of glutamatergic neurotransmission indicators, namely NMDAR1
and mGluR2, in hippocampal areas, i.e., DG and CA1, in C and P mice.
NMDAR1 mGluR2
Cell Density OD Cell Density OD
DG C 127.78 ±25.13 160.44 ±3.59 113.08 ±41.06 193.14 ±12.01
P 397.74 ±32.78 178.13 ±5.42 753.86 ±102.81 201.83 ±4.28
CA1 C 64.26 ±23.40 69.65 ±5.44 30.15 ±16.29 142.90 ±8.70
P 287.26 ±16.33 177.71 ±7.80 275.16 ±19.83 153.10 ±4.18
Biology 2023,12, 196 21 of 28
Based on the subsequent quantitative evaluation, NMDAR1-immunoreactivity, con-
sidered in terms of both cell density and OD, significantly enhanced in P mice compared to
C animals in both considered areas with the stronger effect measured in the CA1 region. In
detail, considering the DG, an extremely significant enhancement of immunolabeled cell
density was reported in P mice compared to controls (Figure 9and Table 5). Further, in the
same region, a significant increase in NMDAR1-immunopositive cell OD was determined
comparing P and C animals (Figure 9and Table 5). Displaying a similar trend, even with
more pronounced effects, the NMDAR1-immunoreactivity, evaluated in terms of both cell
density and OD, significantly enhanced in the CA1 area of P mice compared to controls
(Figure 9and Table 5).
With regard to mGluR2, its cellular localization and distribution bared an extensive
spreading both in the DG and the CA1 region of the Ammon’s horn of P mice compared
to controls (Figures 8and 9and Table 5). Control mice showed a weaker and much less
widespread immunoreactivity, thus revealing an immunostaining pattern analogous to
that described above for the ionotropic receptor NMDAR1. In particular, in P mice the
DG was characterized by some heavily mGluR2-immunopositive cells localized both in
the width of the GL as well as close to the SGZ. Additionally, in the same area some
mGluR2-immunoreactive large neurons were noticeable in the PL (Figure 8). Considering
the CA1 region in P animals, several mGluR2-immunopositive neurons were discernible in
the SP, frequently characterized by immunoreactive prolongations, deepening beneath in
the underlying SR (Figure 8).
Notably, comparing P mice with controls, the quantitative analysis documented a
significant increase in mGluR2-immunoreactive cell density in both considered areas, i.e.,
DG and CA1, with the latter region showing the most striking effect (Figure 9and Table 5).
Diversely, focusing on mGluR2-immunopositive cell OD, any significant difference was
measured neither in the DG nor in the CA1 area comparing P and C animals (Figure 9and
Table 5).
4. Discussion
Hippocampus is one of the most vulnerable organs of the CNS to neurodegenera-
tion during aging and inflammation, and the contiguous and interconnected regions of
hippocampus show different, region-specific, reactivity to aging [54].
He2 primordium extract contains a pure amount of ERGO, a powerful antioxidant,
about three times higher compared to that measured in previously described extracts [
7
,
41
].
In a previous paper we tested the preventive effect of this primordium extract on locomotor
frailty during normal aging and its antioxidants profile on cerebellar cortex [6].
Currently, with the aim of assessing the preventive effect of ERGO on cognitive frailty
development during aging, we performed a longitudinal study monitoring the cognitive
decline both in the knowledge and the remember components of the recognition memory.
We focused on the consequences for
in vivo
recognition memory during physiological
aging, and in parallel demonstrating changes in CA1 and DG regions on different path-
ways, crucially linked to this cognitive function, i.e., inflammation, oxidative stress and
glutamatergic neurotransmission.
ERGO-rich He2 primordium was administered to mice starting from 15 months of age,
during the adulthood life phase, until 23 months of age, corresponding to the senescence
period. In search of a translational approach “from bench to bedside”, we intentionally
chose the dose of 1 mg/day to mirror the usual human oral intake (1 g/day) and we chose
spontaneous behavioral tests resembling those used in clinical practice. All reported results
were obtained in age-matched supplemented and non-supplemented control mice.
Wide consensus among scientists agrees that recognition memory is composed of two
components: (i) the knowledge/familiarity component; and (ii) the remember/recollection
component, which can be accessed via the use of different spontaneous object exploration
paradigms. During aging, the two components of recognition memory declined in a differ-
ent manner. Notably, the preventive effect of ERGO on the physiological age-related decline
Biology 2023,12, 196 22 of 28
of the two components was different: the knowledge frailty index increased less than in
non-supplemented mice, whereas the remember component reached a “gain of function”
even compared to young animals (Figure 10). A debate is still open regarding the role of
the medial temporal area of the brain in the two components of the recognition memory.
The so-called “unitary strength model” theory asserts that recognition memory could be
a unitary declarative system recognizing different strength in memory traces. Diversely,
the so-called “dual-process model” theory postulates that recollection and familiarity are
placed in anatomically and functionally distinct regions in medial temporal brain areas.
Our previous
in vivo
investigations demonstrated a differential effect on the two described
components of the recognition memory. In agreement with our previous data of He1
extracts, the differential effect of this ERGO-rich extract supports the “dual process model”
theory [42,43].
Biology 2023, 12, x 22 of 29
Based on the subsequent quantitative evaluation, NMDAR1-immunoreactivity, con-
sidered in terms of both cell density and OD, significantly enhanced in P mice compared
to C animals in both considered areas with the stronger effect measured in the CA1 region.
In detail, considering the DG, an extremely significant enhancement of immunolabeled
cell density was reported in P mice compared to controls (Figure 9 and Table 5). Further,
in the same region, a significant increase in NMDAR1-immunopositive cell OD was de-
termined comparing P and C animals (Figure 9 and Table 5). Displaying a similar trend,
even with more pronounced effects, the NMDAR1-immunoreactivity, evaluated in terms
of both cell density and OD, significantly enhanced in the CA1 area of P mice compared
to controls (Figure 9 and Table 5).
With regard to mGluR2, its cellular localization and distribution bared an extensive
spreading both in the DG and the CA1 region of the Ammon’s horn of P mice compared
to controls (Figures 8 and 9 and Table 5). Control mice showed a weaker and much less
widespread immunoreactivity, thus revealing an immunostaining pattern analogous to
that described above for the ionotropic receptor NMDAR1. In particular, in P mice the DG
was characterized by some heavily mGluR2-immunopositive cells localized both in the
width of the GL as well as close to the SGZ. Additionally, in the same area some mGluR2-
immunoreactive large neurons were noticeable in the PL (Figure 8). Considering the CA1
region in P animals, several mGluR2-immunopositive neurons were discernible in the SP,
frequently characterized by immunoreactive prolongations, deepening beneath in the un-
derlying SR (Figure 8).
Notably, comparing P mice with controls, the quantitative analysis documented a
significant increase in mGluR2-immunoreactive cell density in both considered areas, i.e.,
DG and CA1, with the latter region showing the most striking effect (Figure 9 and Table
5). Diversely, focusing on mGluR2-immunopositive cell OD, any significant difference
was measured neither in the DG nor in the CA1 area comparing P and C animals (Figure
9 and Table 5).
Figure 10. Pictographic drawing summarizing main findings and take-home message.
4. Discussion
Hippocampus is one of the most vulnerable organs of the CNS to neurodegeneration
during aging and inflammation, and the contiguous and interconnected regions of hippo-
campus show different, region-specific, reactivity to aging [54].
He2 primordium extract contains a pure amount of ERGO, a powerful antioxidant,
about three times higher compared to that measured in previously described extracts
[7,41]. In a previous paper we tested the preventive effect of this primordium extract on
locomotor frailty during normal aging and its antioxidants profile on cerebellar cortex [6].
Currently, with the aim of assessing the preventive effect of ERGO on cognitive
frailty development during aging, we performed a longitudinal study monitoring the
Figure 10. Pictographic drawing summarizing main findings and take-home message.
Furthermore, we cannot exclude that other brain areas involved in the recognition
memory through a multisynaptic loop [
55
], and less vulnerable to aging process, could
be differently exploited by young and aged mice. As recently published, young rats
predominately used hippocampal solutions while old rats employed striatal solutions
on different recognition memory tasks. In particular, it was described as an enhanced
performance on a striatal object replacement task over time with increasing aging [56].
Cytokine dysregulation deeply affects the remodeling of the immune system in the
elderly, triggering to uncontrolled systemic inflammation, the so-called inflammaging, a
typical hallmark of unsuccessful aging. For instance, high levels of IL6 are associated in the
aged subject with increased risk of morbidity and mortality [57,58].
As regards inflammation, we currently revealed a significant lessening of both IL6
and TGF
β
1 in hippocampus of supplemented mice. In detail, IL6 significantly diminished
in both the DG and CA1 (mainly in SP layer) of P animals, while TGF
β
1 levels decreased
in the DG region only (predominantly in GL and POL layers). In parallel, we unveiled
a marked reduction in GFAP in hippocampus of P mice, both in DG and CA1 regions,
thus supporting the reduction in the age-related reactive gliosis typically observed in
physiological aging in non-supplemented animals (Figure 10). These data are in line with
the notion that reactive gliosis during aging could be caused by an increased number
and/or activity of astrocytes [59].
Concerning oxidative stress pathway, Nrf2 plays different key roles depending on
ongoing oxidative conditions. This crucial pleiotropic transcription factor, pivotally impli-
cated in aging and lifespan regulation, is able to both up- and down-regulate the expression
of several gene and enzymes, also boosting the maintenance of cellular resistance against
oxidants [
17
,
60
,
61
]. Our findings demonstrated a decrease in Nrf2 expression levels both in
the CA1 area, as well as in the DG (mainly closed to SGZ and in POL) of P mice compared
to the same regions of age-matched not supplemented animals (Figure 10).
Biology 2023,12, 196 23 of 28
Several investigations also explored age-related changes in SOD activity, but often
the reported results are inconsistent, or even contradictory. In fact,
in vivo
studies showed
an age-dependent decrease in SOD1 activity in the brains of old rats. Differently, other
investigations even reported that SOD1 activity increased with aging in some tissues
such as mouse skeletal muscles and brain [
62
,
63
]. Therefore, it remains elusive which
mechanism may cause the controversy over age-related changes in SOD1 activity. Presently,
we revealed a significant lessening in the SOD1 expression levels in supplemented mice in
the DG only, with the CA1 remaining unaffected (Figure 10).
The oxidative/nitrosative imbalance, characterized by continuous ROS generation
as a typical hallmark of an age-related persistent condition of oxidative stress, is able to
provoke chronic inflammation through lipid peroxidation and pro-inflammatory cytokines
enhancement [
64
,
65
]. In particular, the excessive NO production was described in nu-
merous pathological conditions such as neurodegenerative diseases, inflammaging, and
ischemia [
66
]. Our current findings proved a significant selective diminishing of both NOS2
and COX2 expression levels in the CA1 area (Figure 10). This expression pattern, immuno-
histochemically disclosed in the hippocampus, is similar to that previously observed in
the cerebellum of aged mice fed with the same ERGO-rich supplementation. Further, our
current findings are in line with previous literature revealing that natural extract-enriched
diet diminished age-associated increased expression of COX2 and NOS2 [
67
]. It should also
be mentioned that enzymatic sources of oxidative mediators in the brain are controlled by
the glutamatergic NMDARs activation, suggesting that excitatory amino acids determined
stress-induced NOS2 and COX2 expression/activity in the brain [67,68].
The glutamatergic pathway is crucially involved in learning, memory formation/storage
and synaptic plasticity [
23
]. Several studies in pre-clinical models revealed during aging a
decrease in the number of glutamatergic neurons [
69
], a decline in glutamate level in the
hippocampus [
5
] paralleled by hippocampal detrimental changes in synaptic composition
and function [
70
]. It has also been demonstrated that a disrupted glutamate balance guides
to the perturbation of glutamate neurotransmission which may bring severe consequences,
possibly leading to the onset of dementia and neurodegenerative diseases [71–74].
Glutamatergic receptors, both ionotropic and metabotropic, keep the ability to induce
synaptic plasticity in the hippocampus [
75
]. In murine models the characteristic high-
density amount of NMDAR1 in the hippocampus plays a key role in the initiation steps of
learning and memory [
5
,
76
]. In fact, NMDA receptors are known to be pivotally involved
in the performance of many different memory tasks including those using spatial, reference,
working and passive avoidance memory, being also essential to LTP in many different brain
regions [
77
,
78
]. A wide scientific consensus exists that NMDARs declined both in binding
densities as well as in functions with increasing age, also differing the NMDARs subunit
compositions in the aged individuals [
5
]. Furthermore, behavioral and electrophysiological
evidence supported the role of NMDARs [
51
] and G protein coupled metabotropic receptors
in neural plasticity changes with aging [
26
,
30
,
31
]. In the current study we revealed that
ERGO-rich He2 primordium was strikingly effective to improve NMDAR1 expression levels
(in terms of both cell density and OD) in the hippocampus of aged mice, both in the DG
and CA1. In a similar manner, after the eight-month He2 oral supplementation a marked
enhancement of mGluR2-immunoreactive cell density in the same hippocampal regions
was revealed in aged mice (Figure 10). Based on these findings, we postulated that ERGO-
rich He2 primordium extract was able to better glutamatergic pathway/neurotransmission
in aged mice.
5. Conclusions
In conclusion, our findings demonstrated the functional neuroprotective role of an
eight-month lasting oral supplementation with He2 primordium in preventing cognitive
deterioration (Figure 10). A bulk of literature data demonstrated
in vivo
the beneficial
effects of ERGO on learning, cognition, and memory using different models of age-related
and neurodegenerative diseases [
79
–
83
]. Based on all these data we reasonably supposed
Biology 2023,12, 196 24 of 28
that the nootropic described effects could be ascribable to ERGO, since the well-known
erinacines and hericenones were not present [
40
], although we cannot exclude the neuro-
protective effects due to other metabolites. The recovery and boosting of both components
of the recognition memory were paralleled by a neuroprotective action on hippocampus
that was characterized by a lessening of inflammation and oxidative stress accompanied by
an increase in ionotropic and metabotropic glutamate receptors expression. In different
hippocampal areas, ERGO-rich He2 primordium elicited selective changes in the explored
biomarkers.
As a concern to inflammation, the supplementation protects both DG and CA1 regions
leading to a diminished IL6 production, with a measured 7-8-time folder lessening of
IL6-immunopositive cell density. Diversely, only the DG displayed a significant decrease
in TGFbeta1 and GFAP in P mice, while the CA1 region appeared unaffected (Figure 10).
Therefore, we highlighted a selective preventive and neuroprotective efficacy of ERGO-rich
He2 primordium against inflammaging process in the DG.
Similarly, ERGO-rich He2 primordium displayed a differential selective preventive
and neuroprotective effect against oxidative stress on the two hippocampal areas, i.e., DG
and Ca1. Specifically, the DG of P mice was characterized by reduced Nrf2 and SOD1 levels
(about three and four times lower, respectively, compared to those measured in C animals),
with NOS2 and COX2 remaining unaffected. Differently, in the CA1 area we proved an
ERGO-rich He2 primordium-induced significant diminishing of NOS2 and COX2 (about
three and 10 times lower, respectively, compared to those assessed in C mice), paralleled by
a lessening of Nrf2 levels without any changes in SOD1 expression levels (Figure 10).
Furthermore, our data indicated that ERGO-rich He2 primordium was effective to
lead to an increase in NMDAR1 and mGluR2 glutamatergic receptors, both in CA1 and DG
regions (Figure 10).
Taken as a whole, these data led us to assume that as these molecules are pivotally
involved in age-associated damages the assessed expression changes induced by ERGO-
rich He2 primordium could feasibly boost lifetime, or even better life quality. Strikingly, it
must be emphasized that the reported modifications in the expression patterns of these key
molecules were accompanied by the amelioration of cognitive performances.
Author Contributions:
Conceptualization, P.R., E.S. and E.R.; methodology, D.R. and E.C.P.; software,
E.C.P.; formal analysis, E.R., F.D.L., D.R.; investigation, D.R.; data curation, F.D.L., D.R. and E.C.P.;
writing—original draft preparation, E.R., F.D.L., D.R.; writing-review and editing, E.R. and P.R.;
supervision, P.R., E.R., M.G.B. and P.R.; project administration, P.R. All authors have read and agreed
to the published version of the manuscript.
Funding:
This research was funded by the University of Pavia: Fondi Ricerca Giovani (FRG 2018).
This research was also supported by Italian Ministry of Education, University and Research (MIUR):
Dipartimenti di Eccellenza Program (2018–2022) Dept. of Biology and Biotechnology “L. Spallanzani”,
University of Pavia.
Institutional Review Board Statement:
The study was conducted in accordance with the guidelines
laid out by the institution’s animal welfare committee, the Ethics Committee of Pavia University
(Ministry of Health, License number 774/2016-PR, 4th august 2018), also in compliance with the
European Council Directive 2010/63/EU on the care and use of laboratory animals. All animals used
in this research have been treated humanely, with due consideration for the alleviation of distress
and discomfort.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments:
The authors wish to thank: (i) Valentina Cesaroni and the staff of Centro Grandi
Strumenti, University of Pavia for their valuable technical assistance; (ii) Daniela Rossi and Liliana
Brambilla (Laboratory for Research on Neurodegenerative Disorders, ICS Maugeri IRCCS Pavia), who
kindly provided anti-NMDAR1 and anti-mGluR2 primary antibodies; (iii) the animal facility “Centro
di servizio per la gestione unificata delle attivitàdi stabulazione e di radiobiologia”, University of
Biology 2023,12, 196 25 of 28
Pavia, to host the animals; (iv) the OPBA of the University of Pavia for support in animal protocol
drawing up; (v) TETRAHEDRON, Paris, France to provide them the ergothioneine standard.
Conflicts of Interest: The authors declare no conflict of interest.
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