**1. Introduction**

We successfully established stroke-prone SHR (SHRSP, 1974) [1] by successive breeding from a spontaneously hypertensive rat (SHR, 1963) [2], which died of hemorrhagic and/or ischemic stroke. Since they developed stroke spontaneously [3], they were regarded as an appropriate model for research not only on the pathophysiological mechanisms of stroke in general [4], lacunar stroke [5] , cerebral small vessel diseases [6], and subcortical ischemic stroke [7,8], but also the post-stroke treatments [9,10]. Further, since they develop stroke genetically similar to humans, they are a useful model for the potential prediction of stroke through the analysis of genes related to stroke [11–17]. When stroke can be predicted based on the genes in SHRSP and hopefully in humans, stroke will be prevented by nutrition in humans, as first proven experimentally in SHRSP [3].

Stroke became the leading cause of death in Japan in 1960 after tuberculosis became pharmacologically treated. The nutritional situation in Japan over 60 years ago was supposed to cause stroke among cardiovascular diseases (CVD) because of traditional low protein intake characterized by limited meat supply and owing to habitually low calcium (Ca) and/or magnesium (Mg) intake due to low dairy food consumption. Therefore, we focused on the nutritional prevention of stroke and CVD in the newly established SHRSP by feeding them high-protein diets with Ca and Mg. Since the nutritional intakes were objectively estimated epidemiologically by 24 h urine (24U) analyses of the biomarkers in humans [18], we attempted to show whether experimentally beneficial nutrients for preventing stroke in SHRSP would be epidemiologically associated with cardiovascular risk reduction in humans [19].

**Citation:** Yamori, Y.; Sagara, M.; Mori, H.; Mori, M., on behalf of the CARDIAC Study Group. Stroke-Prone SHR as Experimental Models for Cardiovascular Disease Risk Reduction in Humans. *Biomedicines* **2022**, *10*, 2974. https://doi.org/ 10.3390/biomedicines10112974

Academic Editors: Josef Zicha and Ivana Vanˇeˇcková

Received: 31 August 2022 Accepted: 7 November 2022 Published: 18 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Therefore, we proposed to the World Health Organization (WHO) an international cooperative study, "Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study" [20,21], from our WHO Collaborating Center for Research on Primary Prevention of Cardiovascular Diseases, which was designated by WHO in 1983. In response to our proposal, representative researchers from 61 study sites joined the CARDIAC Study [22].

Since our cooperative studies for over 20 years revealed nutritional biomarkers in worldwide collected 24U samples were significantly associated with CVD risk reduction, we further analyzed these biomarkers in some populations known for their longevity, including Japan (J), which keeps the nearly longest average life expectancy in the world, and Mediterranean (M) countries such as Greece, Sicily of Italy, Spain, and Portugal.

In comparison with Euro-Western countries (EW) (Scotland, Ireland, Sweden, plus Canada, New Zealand, and Australia) where immigrants and descendants from their countries have been living. Among these countries, the popular diets of M countries, rich in polyphenols [23] and related nutrients from fruits, vegetables, olive oil, nuts [24], and fish, have been focused on their basic health effects [25], including cognitive function [26]. However, since no data on M diets compared with EW and J diets has been reported on nutritional biomarkers in 24U samples, we compared them with the CVD risks of M, J, and EW populations in the epidemiological study of this article.

#### **2. Materials and Methods**

(1) SHRSP had been used for various nutrition experiments [3,19,27,28], and in the present study, SHRSP from 6 groups (Table 1 and Figure 1) were given control and soy diets (CD, SD) with Mg or Ca at the age of 7 weeks, thereafter until their natural death, and autopsied for macroscopical and microscopical pathological observation [19].


**Table 1.** Contents of dietary factors in each group of SHRSP.

(2) A health examination was carried out for males and females according to the protocol of the WHO-coordinated Cardiovascular Diseases and Alimentary Comparison (CARDIAC) Study [20,21], and fasting blood and 24U samples were analyzed after anthropological and blood pressure (BP) measurements [20–22,29,30]. About 100 males and 100 females in the age range of 48–56 were randomly invited to the CARDIAC Study health examination after informed consent was obtained from the participants. The study design described in detail [20] was approved at the international committee meeting before starting the CARDIAC Study in 1985.

Informed consent was obtained at the reception of the CARDIAC Study Health Examination from volunteer participants, who were asked to sign the first page of the CARDIAC study questionnaire, and the study was conducted according to the guidelines of the Declaration of Helsinki. Urinary biomarkers such as sodium (Na) for salt intake, potassium (K) for vegetable intake, magnesium (Mg) for grains, nuts, soy, and dietary fiber intake, isoflavones for soy intakes, taurine (Tau) for seafood intakes, urea nitrogen for protein intakes, and creatinine (Cre) for checking the completeness of the collection of 24U samples were analyzed in 50 populations in the world, in total 4211 participants (49.7% females F) in 22 countries worldwide, and also 6 J (864, 53.7% F), 6 M (574, 50.2% F), and 6 EW populations (549, 45.9% F) [18,20–22].

**Figure 1.** Effect of soy protein, Ca, Mg, and combined diets on the survival rate of salt-loaded SHRSP.

Obese subjects were defined as those with body mass index (BMI) ≥ 30 kg/m2. Participants with hypertension were defined as those with systolic BP (SBP) ≥ 140 mmHg or diastolic BP (DBP) ≥ 90 mm Hg or those who were receiving anti-hypertensive drug therapy. Hypercholesterolemic subjects were defined as those with serum total cholesterol (TC) ≥ 220 mg/dL. General linear models were used to estimate adjusted mean values of BMI, SBP, DBP, and TC across quintiles of the 24 h urinary Tau/Cre (Mg/Cre) ratio after adjustment for age, sex, and use of anti-hypertensive drugs. To evaluate the association of Tau/Cre (Mg/Cre) ratio with cardiovascular disease risk factors, we estimated adjusted odds ratios for obesity, hypercholesterolemia and hypertension in relation to quintiles of Tau/Cre (Mg/Cre) using logistic regression models, adjusting for age and sex as to hypertension and additionally for anti-hypertensive drugs as to obesity and hypercholesterolemia.

ANOVA was used for the comparisons of 24U biomarkers, BMI, SBP, DBP, and TC among the J, M, and EW diets.

#### **3. Results**
