1. Introduction
The rapid aging of the world’s population presents one of the most significant challenges that we face today. Aging is a universal phenomenon, characterized by a multifaceted biological process that induces various changes across all organs and tissues of living beings. The brain, in particular, is among the organs most affected by aging. In both humans and animal models, age-related cognitive decline is associated with changes in specific brain structures. The aging brain exhibits alterations at multiple levels, from molecular and cellular levels to the level of networks, all of them contributing to adverse modifications in neuroplasticity, and thus negatively influencing learning and memory. Aging promotes cognitive decline and is a major risk factor for the development of common neurological diseases [
1,
2]. As the life expectancy continues to increase worldwide, age-related cognitive disturbances are becoming increasingly impactful on society and are likely to pose even greater challenges in the future. Numerous studies have reported significant cognitive variability among aged individuals, with chronic stress exposure throughout life being one of the factors influencing cognitive preservation. Chronic exposure to stress hormones impacts brain structures involved in cognition and other aspects of mental health, which may be particularly important in the aging brain due to its increased vulnerability to extreme factors [
3,
4].
In the 1990s, several studies established spontaneously hypertensive rats (SHRs) as a model for aging-related comorbid pathologies based on age-dependent hypertension [
5,
6]. Although normotensive at birth, SHRs develop sustained hypertension by 6 months of age. SHRs exhibit compensated cardiac hypertrophy, with eventual transition to heart failure in the final quartile of their lifespan; this makes them a valuable model for studying the progression from stable compensated hypertrophy to decompensated heart failure in the context of aging [
7]. Aged SHRs naturally develop a reduced coronary flow reserve, an increased coronary vascular resistance, cardiac fibrosis, and impaired cardiac function, along with glomerular hypertension and ischemia, proteinuria, glomerular sclerosis, and interstitial fibrosis, similar to what is observed in hypertension in humans [
8]. Hypertension is a critical risk factor for cerebrovascular diseases, including stroke, and is involved in the development of vascular cognitive impairment (VCI) and vascular dementia (VD). SHRs are widely regarded as the gold standard animal model for studying the association between hypertension and VCI/VD. Cerebrovascular changes, brain atrophy, the loss of nerve cells in cerebrocortical areas, and glial reactions have been documented in SHRs [
9], with several changes resembling those found in in vivo imaging studies of patients with essential hypertension. Microanatomical, neurochemical, and behavioral data on SHRs suggest that this strain is a suitable model for age-related vascular brain diseases [
10,
11].
Age-associated cognitive disturbances, including dementia and mild cognitive impairment, necessitate effective treatments, making the development of drugs that improve and preserve cognition in the elderly a critical priority [
12]. Various molecules with neuroprotective properties have been proposed, including neuropeptides. A number of peptide drugs with potential cognitive benefits are currently available, most of which are administered parenterally due to poor absorption from the gastrointestinal tract. Since peptide drugs are typically prescribed for chronic conditions, the need for continuous, repetitive daily injections presents a significant disadvantage compared to the oral route, which is associated with higher patient compliance [
13].
N-Pep-Zn is a formulation based on N-PEP-12, a compound derived from purified neuronal proteins. N-PEP-12 contains peptides that have been shown to support brain function, particularly in the context of aging. These peptides promote neuronal survival, stimulate neurite outgrowth, and protect against metabolic stress, as evidenced in various experimental models [
14]. In N-PEP-Zn, zinc is added to N-PEP-12 to enhance its potential cognitive benefits. Zinc, an essential trace element, plays a crucial role in synaptic plasticity and neurotransmission, further contributing to the preservation of cognitive function in aging populations [
15]. This formulation combines the neuroprotective effects of N-PEP-12 with the well-known cognitive support provided by zinc [
16].
The aim of this study was to explore the effects of chronic isolation stress in aging SHRs on their cognitive functions and stress response, as well as the impact of the chronic oral administration of N-Pep-Zn, the zinc derivative of N-PEP-12 [
16]. This study is the first experimental investigation with N-PEP-Zn in vivo.
4. Discussion
SHRs are regarded as an animal model of genetic hypertension which, with aging, develops heart failure similarly to humans. The influence of the CNS on the cardiovascular system and blood pressure regulation was described in detail, including the role of the CNS in hypertension [
25]. In studies of the myocardial tissue dysfunction mechanisms, a central role for neurohormonal activation, including the HPA axis and the renin–angiotensin–aldosterone system, was confirmed [
26].
The hippocampus, a selectively vulnerable brain region, is sensitive to the effects of hypertension, and this has been confirmed in SHRs [
11]. Overactivation of the HPA axis and the overproduction of glucocorticoid and mineralocorticoid neuroactive steroids in SHRs may be detrimental to the hippocampus, which is enriched in glucocorticoid and mineralocorticoid receptors. Dysfunction of the HPA axis in aging and age-associated diseases (diabetes, hypertension) induces hippocampal damage associated with impairments in learning, memory, and emotional sphere [
27,
28]. In aging and age-associated diseases, adrenocortical steroid overdrive sensitizes the hippocampus to the pathological milieu imposed by a pre-existing degeneration or illness [
29]. Some abnormalities commonly found in the hippocampus of aging, diabetic, and hypertensive animals (in particular, SHRs) include decreased neurogenesis, astrogliosis and neuronal loss in the dentate gyrus, a low expression of brain-derived neurotrophic factor (BDNF), and a decreased number of neurons in the hilus [
30]. In the hypothalamus, SHRs demonstrate increased expression of the hypertensinogenic peptide arginine vasopressin (AVP) and its V1b receptor. Hypertension and ageing upregulate the NO pathway in structures involved in the regulation of blood pressure in SHRs [
31]. Alterations in neurotransmitter systems in SHRs have been reported, including abnormally low levels of dopamine in the neostriatum and nucleus accumbens [
32]. Neurohumoral, neurochemical, and neurometabolic abnormalities in the brains of SHRs affect neuroplasticity, inducing learning and memory deficits and making this rat strain a clinically relevant model of dementia [
11].
Vascular cognitive impairment dementia (VCID), an increasingly important cause of dementia in the elderly, embraces a number of diagnoses, including large vessel disease with multiple strokes, small vessel disease (SVD) with lacunar infarcts, and white matter disease. Because of its progressive course, SVD is thought to be the optimal form of VCID for treatment. Its pathophysiology involves hypoxic hypoperfusion resulting in injury to the white matter and neuronal death. Since hypertension leads to SVD, resulting in progressive damage to the white matter, neocortex, and hippocampus, SHRs may be regarded as a good model of SVD [
33]. Importantly, similar ultrastructural breakdowns of cerebrocortical capillaries were revealed in Alzheimer’s disease, Parkinson’s disease, and SHRs, indicating that cerebral capillary damage is not exclusive to AD but occurs in other neurodegenerative disorders and hypertension as well. These ultrastructural abnormalities of cerebral capillaries may be causally related to decreased cerebral blood flow and create conditions which favor neurodegenerative mechanisms and eventually cause the development of dementia [
34]. Thus, SHR is the rat strain most extensively investigated and used for assessing hypertensive brain damage and therapy. A high arterial blood pressure, brain atrophy, the loss of nerve cells, and glial reaction are phenomena shared with hypertensive brain damage in humans. Alterations the in neurotransmitter systems of SHRs are believed to have functional and behavioral relevance. In particular, the impaired cholinergic neurotransmission characteristic of SHRs is similar to that reported in patients affected by vascular diseases.
Communication is considered as one of the vital human requirements. Loneliness and social isolation are more than just a psychosocial problem. Chronic social isolation affects the clinical course of different diseases, including diabetes and cardiovascular and cerebrovascular diseases, as well as the average life expectancy and the risk of death caused by any causes, and is comparable with the effects of major risk factors like smoking, alcohol consumption, physical inactivity, hypertension, obesity, hypercholesterolemia, and various medical interventions [
35]. The recent COVID-19-related social isolation and a more sedentary life in all age groups was most harmful for the elderly population who are the most vulnerable to infections and chronic neurodegenerative diseases. It is expected that the elderly patients with chronic neurodegenerative diseases who survived SARSCoV-2 infection will show aggravation of their neurodegenerative conditions [
36,
37]. Since social isolation and loneliness are believed to provoke consequent cognitive decline, specifically in the selectively vulnerable elderly population, we have chosen to use chronic isolation as a major challenge, a chronic stress inducing cognitive decline in SHRs, and explore the potential therapeutic effects of a new dietary peptide, N-Pep-Zn, on the behavioral and some biochemical indices of SHRs.
N-Pep-12, a compound consisting of biopeptides and amino acids, is the parent substance for N-Pep-Zn. N-Pep-12 is a dietary supplement with neuroprotective and pro-cognitive effects confirmed in experimental models and clinical studies [
38]. In cultures, N-PEP-12 prevents the neuronal cell death of cortical neurons [
39]. In animal experiments, the chronic administration of N-PEP-12 promotes neuronal plasticity in the limbic system of aged animals [
40], partially reduces brain endothelial dysfunction [
41], enhances cognitive function, and reduces neurodegenerative events associated with aging [
42,
43]. N-PEP-12 favors the neurorecovery of middle-aged and older adults with cognitive impairments after ischemic stroke [
44,
45,
46] and may be an effective supplement to secure a healthy memory function in older adults [
47]. The Zn-derivative of N-PEP-12 used in this study, N-Pep-Zn [
16], is a novel development combining the beneficial effects of the N-PEP-12 peptides with the physiologically essential properties of zinc for proper CNS function [
15].
Although chronic isolation increased the mortality of the SHRs during the experiment threefold (31.3% in SHRiso vs. 10.5% in SHRsoc), this difference, though impressive, was not statistically significant. The same is true for the beneficial effect N-Pep-Zn; although the mortality in the SHRisoP group (11.8%) was similar to that in the SHRsoc group, this striking effect could not be confirmed by statistical analysis, possibly due to the limited sample size of this study. The differences in mortality were more expressed in the aging of the 12–13-month-old SHRs. Importantly, in the surviving SHRiso animals, the effect of chronic isolation on the behavior of the SHRs was surprisingly diminutive. Indeed, we did not find a major influence of chronic social isolation on the indices of activity, exploration, anxiety, depression-like behavior, and social behavior in the aging SHRs (
Table S1). This suggests that, in spite of the obviously pathological phenotype, the surviving SHRs were highly resilient to a chronic stress of this nature. Moreover, this may have been the expression of extensive multi-level adaptive mechanisms that allows this strain to survive in the situation of high arterial pressure and neurohumoral disturbances providing resistance to some other types of stress. We did not observe an effect of N-Pep-Zn in these behavioral tests, and the absence of effects of chronic isolation may be the main reason for this. It should be stressed that N-Pep-Zn did not exert adverse effects on physiological indices either.
The stability of major inflammatory markers in the SHRs subjected to chronic isolation (
Table S2) confirms the resilience of SHRs to this kind of chronic stress. Interestingly, the SHR is an experimental model of salivary hypofunction, and these rats show a lesser flow and salivary protein concentration as well as lower dental mineralization [
48,
49]. Loneliness and chronic social stress may lead to activation of the sympathetic nervous system and reduced activity of the parasympathetic nervous system with enhanced sympathetic reactivity and/or delayed sympathetic recovery after exposure to an acute stressor [
50]. The salivary alpha-amylase activity is a suitable measure of change in the sympathetic tone after the action of a psychosocial stressor, although it is not strongly related to the other indices of a stress response [
51]. Chronic isolation abolished the normal pattern of salivary amylase in the SHRs, which tended to increase during aging and isolation in the SHRsoc group, but was restored in the isolated SHRs receiving N-Pep-Zn.
An important result of this study was the fact that social isolation did affect the cognitive function of the SHRs, and this was confirmed in the experiments with learning and memory in the Barnes maze (
Figure 3,
Figure 4,
Figure 5,
Figure 6 and
Figure 7,
Table 1,
Table 2,
Table 3,
Table 4 and
Table 5). Clear evidence for impairments in learning and memory was found in the SHRiso group as compared with the SHRsoc group. N-Pep-Zn prevented isolation-induced cognitive decline according to indices of learning and memory in the Barnes maze. Chronically isolated SHRs did not prefer the maze quadrant with target holes, while N-Pep-Zn diminished this effect of social isolation, bringing these animals close to the SHRsoc group. The reversal learning appeared unsuccessful in the SHRs, and this may reflect worse learning abilities, as reported by many groups. However, chronic isolation specifically provoked a preference for the opposite quadrant, confirming the further impairment of cognitive function in the SHRiso group, whereas N-Pep-Zn intake stimulated the preference for the target quadrant. Thus, in aged SHRs, the N-Pep-Zn significantly improved the spatial learning and memory affected by chronic social isolation.
Chronic isolation stress is able to modify the humoral and cellular immunity in rodents, although the data on this are quite contradictory and depend on the strain, age, sex, and a number of other experimental conditions [
52,
53,
54,
55]. The experiments with the acute immobilization challenge aimed to explore whether chronic isolation in SHRs specifically affects the acute stress response and whether N-Pep-Zn intake can control this response. In 1936, the first scientific article of Hans Selye on ‘general adaption syndrome’ was published in
Nature, and the data based on experiments in rats that were exposed to severe insults/stressors suggested a ‘nonspecific bodily response’ with three major, grossly visible changes: hyperemia and enlargement of the adrenals, atrophy of the thymus and lymph nodes, and hemorrhagic gastric ulcers (the “stress triad”) [
56]. The thymus is the central organ of the immune system; it is essential for the development and maintenance of normal immune system, especially cell-mediated immunity. The neuroendocrine system regulates early T-cell differentiation by the transcription of neuroendocrine genes in the stromal network [
57]. Since thymopoiesis is essential for the development and maintenance of a robust and healthy immune system, the thymus size and function change dramatically with age as well as in response to stressors, while acute thymic atrophy is a complication of many infections, environmental stressors, and other clinical conditions [
58,
59,
60]. Complex relations exist among important molecules belonging to the central nervous system and immune system, both in norm and during the chronic stress response [
61]. Stress may transform the inflammatory signal of cytokines into a nervous signal (neurotransmitters); in turn, this process uses the endocrine system signals (e.g., cortisol) to counterbalance against the immune system. Such molecular links could explain how stress plays a role in the etiopathogenesis of several diseases through this complex interplay. The microbiota–gut–immune–brain axis was recently postulated to promote mental health or disorders. A dysregulated immune system can shift to an autoimmune response with concomitant neuropsychological consequences in the context of this axis [
62].
The isolation stress did not significantly affect the response of glucose and corticosterone to the acute restraint, confirming our above suggestions about effective mechanisms of adaptation in SHRs providing their successful viability in spite of developing gross pathological changes in many systems and organs. The adaptation of the HPA to the conditions of SHR organisms may explain why acute restrained stress did not induce adrenal hypertrophy. However, the response of the thymus turned out to be the weak link, manifesting a pathological influence of chronic isolation stress: unlike the SHRsoc group, the SHRiso group demonstrated acute thymus involution within 1 h of restraint. N-Pep-Zn intake fully prevented the expression of this well-known marker of the response to a severe acute stress (
Figure 10). Moreover, the N-Pep-Zn significantly smoothed the response of glucose and cortisol to acute stress in SHRs with chronic isolation experience (
Figure 8 and
Figure 9). This may indicate an alleviating effect of N-Pep-Zn on both the HPA axis response to acute stress (corticosterone) and sympathetic reactivity (glucose). As was mentioned above, alterations in the amylase activity, especially during stress, also reflect the sympathetic reactivity. Similarly to corticosterone and glucose, neither the SHRsoc nor the SHRiso group responded to the acute restraint with changes in their amylase activity (
Figure S1b). However, the amylase activity decreased in the stressed SHRisoP rats as compared to the control SHRisoP animals, which confirms our suggestion about the reduction in sympathetic reactivity to acute stress caused by N-Pep-Zn in animals which have experienced chronic isolation stress. Thus, N-Pep-Zn intake alleviated the response to acute stress by acting on the sympathetic system and HPA axis, both essential first-line systems which provide urgent adaptive signals in response to stress, and protects the thymus from stress-induced involution, thus exerting potential beneficial effects on the immune system.
Since only a few data are available on the effects of N-Pep-Zn (this is the first experimental in vivo study), we can only speculate about the specific mechanisms underlying beneficial stress-protective effects of this peptide. We can only hypothesize which N-Pep-Zn components are most active and how they reach their targets: in general, the intestinal absorption of peptide drugs is severely hindered by both biochemical and physical barriers. Although enzymatic hydrolysis can take place in the gastrointestinal tract, reducing the stability of polypeptides, while the plasma membranes of cells form a selective physical barrier [
13], gastrointestinal peptide uptake and the corresponding effects have been widely described [
63,
64,
65,
66]. We can currently merely guess about the nature of the specific targets of N-Pep-Zn; however, from the data that have been reported on analogous parenterally administered substances such as cerebrolysin [
67] and the parent substance N-PEP-12 [
38], we can suggest that these brain-derived peptide preparations exert their neurotrophic factor-like favorable effects on neuroplasticity. Similarly to these peptides, N-Pep-Zn may selectively protect vulnerable limbic structures, specifically the hippocampus, involved in learning, memory, and emotions. In unfavorable conditions, an uncontrolled stress response induces signal transduction, triggering pathways leading to neuroinflammation, neurodegeneration, and, eventually, to cognitive and emotional disturbances. We suggest that N-Pep-Zn is able to modulate the stress response affecting multiple essential systems, promoting adaptation (HPA axis, sympathetic and immune systems) and thus modifying the stress response, shifting it toward more resilient and rational reactions. This potential of N-Pep-Zn, together with a number of other favorable effects on different levels of neuroplasticity described for other brain-derived neuropeptides [
38,
67], may contribute to enhancing memory and cognitive performance.