Large- and Medium-sized Arteries Remaining in Transmural Scar Distal to Permanent Coronary Ligation Undergo Neointimal Hyperplasia and Inward Remodeling.

This study aimed to investigate the structural integrity and dynamic changes in chronically occluded residual arteries found in post-myocardial infarction (MI) scar. A transmural MI was induced in middle-aged, male Sprague-Dawley rats by left coronary artery ligation. The rats were euthanized 3 days and 1, 2, 4, 8, and 12 weeks after MI, and their hearts were processed into paraffin for histology, immunohistochemistry, and quantitative morphometry. It has been found that large- and medium-sized arteries were able to survive inside the transmural scars for 12 post-MI weeks. Furthermore, most residual arteries preserved their structural integrity for up to 2 weeks post-MI, but gradually all disused vessels had undergone neointimal hyperplasia and inward remodeling at later time periods. In addition, the replacement of vascular smooth muscle cells in the wall of residual arteries by extracellular matrix components led to a disruption of the vessel integrity and progressive obliteration of their lumen between 4 and 12 post-MI weeks. Taken together, this study demonstrate that residual arteries in post-infarcted region were capable of maintaining their structural integrity, including the patent lumen, during two post-MI weeks, suggesting that during this period they can be used as potential conduits for conceivable reflow of arterial blood within the scarred region of the heart.

Long-term treatment with spermidine increases health span of middle-aged Sprague-Dawley male rats.

Let alone calorie restriction, life span extension in higher organisms has proven to be difficult to achieve using simple drugs. Previous studies have shown that the polyamine spermidine increased the maximum life span in C. elegans and the median life span in mice. However, younger subjects (< 40 years of age) are infrequently prescribed nor self-medicating with antiaging drugs. Therefore, in the present study, we aimed at assessing the effect of long-term treatment with spermidine given in the drinking water on behavioral performance and longevity of male, middle-aged Sprague-Dawley rats. We report that spermidine given in the drinking water did not extend neither the median nor the maximum life span of the middle-aged male Sprague-Dawley rats. However, spermidine treatment had a beneficial effect on the body weight and the kidney tubules, liver, and heart morphology. Behaviorally, spermidine led to a reduction in anxiety and an increase in curiosity, as assessed by exploratory behavior. Moreover, long-term treatment with spermidine enhanced autophagy in the brain and led to a diminished expression of the inflammatory markers, Tgfb, CD11b, Fcgr1, Stat1, CR3, and GFAP mRNAs in several cortical region and hippocampus of the treated rats suggesting that one beneficial effect of the long-term treatment with spermidine is an attenuated proinflammatory state in the aged brain. Our results suggest that long-term treatment with spermidine increases health span of middle-aged rats by attenuating neuroinflammation and improving anxiety and exploratory behavior.

Cannabidiol improves metabolic dysfunction in middle-aged diabetic rats submitted to a chronic cerebral hypoperfusion.

Cannabidiol (CBD), a compound obtained from Cannabis sativa, has wide range of therapeutic properties, including mitigation of diabetes and neurodegeneration. Cerebral ischemia and consequent learning disabilities are aggravated in elderly diabetic subjects. However, there are no studies showing the effect of CBD treatment in elderly diabetes patients suffering cerebral ischemia. The present work tested the hypothesis that CBD treatment improves metabolic dysfunctions in middle-aged diabetic rats submitted to chronic cerebral hypoperfusion. In this work, 350-day-old male Wistar streptozotocin-induced diabetic rats were used. To induce cerebral ischemia was used a chronic cerebral hypoperfusion (CCH), surgically, via the four-vessel occlusion/internal carotid artery (4-VO/ICA). Four diabetic groups were established: Non-CCH Treated Diabetic (DNT), CCH Treated Diabetic (DCT), Non-CCH Vehicle Diabetic (DNV), and CCH Vehicle Diabetic (DCV). Vehicle groups were not treated with CBD. The animals were treated during 30 days with 10 mg CBD/Kg bw/day. After treatment, the animals were euthanized, and blood levels of glucose, insulin, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides, fructosamine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were evaluated. DCT group presented reduction of hyperglycemia and an increase of insulinemia. Also was observed lower fructosamine, LDL, HDL, triglycerides and total cholesterol levels. AST and ALT concentration were reduced in CBD treated groups. CBD may be used as therapeutic tool to protect metabolism against injuries from diabetes aggravated by cerebral ischemia.

The isoflavones genistein and daidzein increase hepatic concentration of thyroid hormones and affect cholesterol metabolism in middle-aged male rats.

We examined whether isoflavones interfere with thyroid homeostasis, increase hepatic thyroid hormone concentrations and affect cholesterol metabolism in middle-aged (MA) male rats. Thirteen-month-old Wistar rats were injected subcutaneously with 35 mg/kg b.w./day of genistein, daidzein or vehicle (controls) for four weeks. Hepatic Dio1 gene expression was up-regulated by 70% (p < 0.001 for both) and Dio1 enzyme activity increased by 64% after genistein (p < 0.001) and 73% after daidzein treatment (p < 0.0001). Hepatic T3 was 75% higher (p < 0.05 for both), while T4 increased only after genistein treatment. Serum T4 concentrations were 31% lower in genistein- and 49% lower in dadzein-treated rats (p < 0.001 for both) compared with controls. Hepatic Cyp7a1 gene expression was up-regulated by 40% after genistein and 32% after daidzein treatment (p < 0.05 for both), in agreement with a 7α-hydroxycholesterol increase of 50% (p < 0.01) and 88% (p < 0.001), respectively. Serum 24- and 27-hydroxycholesterol were 30% lower (p < 0.05 for both), while only 24-hydroxycholesterol was decreased in the liver by 45% after genistein (p < 0.05) and 39% (p < 0.01) after dadzein treatment. Serum concentration of the cholesterol precursor desmosterol was 32% (p < 0.05) lower only after dadzein treatment alone, while both isoflavones elevated this parameter in the liver by 45% (p < 0.01). In conclusion, isoflavones increased T3 availability in the liver of MA males, despite decreasing serum T4. Hepatic increase of T3 possibly contributes to activation of the neutral pathway of cholesterol degradation into bile acids in the liver. While isoflavones obviously have the potential to trigger multiple mechanisms involved in cholesterol metabolism and oxysterol production, they failed to induce any hypocholesterolemic effect.

Restraint stress exacerbates cell degeneration induced by acute binge ethanol in the adolescent, but not in the adult or middle-aged, brain.

Restraint stress (RS) induces neurotoxicity in the hippocampus, yet most of the studies have employed protracted RS (i.e., ≈ 21 days). Binge ethanol can induce brain toxicity, an effect affected by age. It could be postulated that RS may facilitate ethanol-induced neurotoxicity, perhaps to a greater extent in adolescent vs. older subjects. We analyzed whether adolescent, adult or middle-aged male rats exposed to five episodes of RS followed, 72h later, by binge ethanol (i.e., two administrations of 2.5 g/kg ethanol) exhibited hippocampal neurotoxicity. Adolescents, but not adult or middle-aged rats, exhibited sensitivity to the neurotoxic effects of ethanol at dorsal CA2, ventral CA3 and ventral DG, and a neurotoxic effect of stress at dorsal CA1. Moreover, the combination of ethanol and stress exerted a synergistic effect upon cell degeneration at ventral CA1 and CA2, which was restricted to adolescents. Ethanol also increased cell degeneration, irrespective of age or stress, in dorsal CA3 and in dorsal DG; and ethanol and stress had, across all ages, a synergistic effect upon cell degeneration at the dorsal CA1. The greater neurotoxic response of adolescents to ethanol, stress, or ethanol+stress can put them at risk for the development of alcohol problems.

Effects of Cannabidiol on Diabetes Outcomes and Chronic Cerebral Hypoperfusion Comorbidities in Middle-Aged Rats.

Diabetes and aging are risk factors for cognitive impairments after chronic cerebral hypoperfusion (CCH). Cannabidiol (CBD) is a phytocannabinoid present in the Cannabis sativa plant. It has beneficial effects on both cerebral ischemic diseases and diabetes. We have recently reported that diabetes interacted synergistically with aging to increase neuroinflammation and memory deficits in rats subjected to CCH. The present study investigated whether CBD would alleviate cognitive decline and affect markers of inflammation and neuroplasticity in the hippocampus in middle-aged diabetic rats submitted to CCH. Diabetes was induced in middle-aged rats (14 months old) by intravenous streptozotocin (SZT) administration. Thirty days later, the diabetic animals were subjected to sham or CCH surgeries and treated with CBD (10 mg/kg, once a day) during 30 days. Diabetes exacerbated cognitive deficits induced by CCH in middle-aged rats. Repeated CBD treatment decreased body weight in both sham- and CCH-operated animals. Cannabidiol improved memory performance and reduced hippocampal levels of inflammation markers (inducible nitric oxide synthase, ionized calcium-binding adapter molecule 1, glial fibrillary acidic protein, and arginase 1). Cannabidiol attenuated the decrease in hippocampal levels of brain-derived neurotrophic factor induced by CCH in diabetic animals, but it did not affect the levels of neuroplasticity markers (growth-associated protein-43 and synaptophysin) in middle-aged diabetic rats. These results suggest that the neuroprotective effects of CBD in middle-aged diabetic rats subjected to CCH are related to a reduction in neuroinflammation. However, they seemed to occur independently of hippocampal neuroplasticity changes.

Genistein and daidzein treatments differently affect uterine homeostasis in the ovary-intact middle-aged rats.

This study aimed to investigate the effects of soy isoflavones, genistein (GEN) and daidzein, (DAI) on the uterine function in ovary-intact middle-aged rats. GEN and DAI (35mg/kg) were subcutaneously administrated to acyclic (12-month-old) Wistar females, daily, for 4weeks. Control group received either vehicle (olive oil and ethanol, 9:1) or remained intact. We found that GEN and DAI differently affect uterine morphophysiology. GEN significantly increased the uterine wet weight which was associated with hyperplastic changes, revealed by stereological and histomorphometrical analyses. Also, PCNA immunoexpression was increased, whereas expression of apoptotic marker (caspase-3) was decreased. Protein and gene expressions of ERα were down-regulated, while PR and ERß were up-regulated after GEN application. Also, GEN caused an increase of LAC and VEGF mRNA expression, together with an up-regulation of Akt activity. In contrast, DAI did not change the uterine wet weight and stereological features of the main uterine compartments as well as LAC and VEGF gene expression. Absence of hyperplastic changes were illustrated by an increase in caspase-3 immunoexpression, associated with reduced PCNA expression. DAI up-regulated only the expression of ERß, while the expression levels of ERα and PR remain unaffected. Also, DAI inhibited the activation of Akt due to down-regulation of phosphorylated and total form of Akt protein expression. Compared to GEN, DAI did not promote events associated with the endometrial cell proliferation in the conducted study, figuring as the compound with a potential safety profile, which justifies further investigation.

Preservation of Functional Microvascular Bed Is Vital for Long-Term Survival of Cardiac Myocytes Within Large Transmural Post-Myocardial Infarction Scar.

This study was aimed to understand the mechanism of persistent cardiac myocyte (CM) survival in myocardial infarction (MI) scars. A transmural MI was induced in 12-month-old Sprague-Dawley rats by permanent coronary artery ligation. The hearts were collected 3 days, 1, 2, 4, 8, and 12 weeks after MI and evaluated with histology, immunohistochemistry, and quantitative morphometry. Vasculature patency was assessed in 4-, 8-, and 12-week-old scars by infusion of 15-micron microspheres into the left ventricle before euthanasia. The infarcted/scarred area has a small continually retained population of surviving CMs in subendocardial and subepicardial regions. Surprisingly, whereas the transverse area of subepicardial CMs remained relatively preserved or even enlarged over 12 post-MI weeks, subendocardial CMs underwent progressive atrophy. Nevertheless, the fractional volume of viable CMs remained comparable in mature scars 4, 8, and 12 weeks after MI (3.6 ± 0.4%, 3.4 ± 0.5%, and 2.5 ± 0.3%, respectively). Despite the opposite dynamics of changes in size, CMs of both regions displayed sarcomeres and gap junctions. Most importantly, surviving CMs were always accompanied by patent microvessels linked to a venous network composed of Thebesian veins, intramural sinusoids, and subepicardial veins. Our findings reveal that long-term survival of CMs in transmural post-MI scars is sustained by a local microcirculatory bed.

Sex Differences in Macrophage Functions in Middle-Aged Rats: Relevance of Estradiol Level and Macrophage Estrogen Receptor Expression.

The aim of this study was to examine the influence of sex on age-related changes in phenotype and functional capacity of rat macrophages. The potential role of estradiol as a contributing factor to a sex difference in macrophage function with age was also examined. Thioglycollate-elicited peritoneal macrophages derived from the young (2 months old) and the naturally senescent intact middle-aged (16 months old) male and female rats were tested for cytokine secretion and antimicrobial activity (NO and H2O2 production and myeloperoxidase activity). Serum concentration of estradiol and the expression of estrogen receptor (ER)α and ERß on freshly isolated peritoneal macrophages were also examined. Decreased secretion of IL-1ß and IL-6 by macrophages from middle-aged compared to the young females was accompanied with the lesser density of macrophage ERα expression and the lower systemic level of estradiol, whereas the opposite was true for middle-aged male rats. Macrophages in the middle-aged females, even with the diminished circulating estradiol levels, produce increased amount of IL-6, and comparable amounts of IL-1ß, TNF-α, and NO to that measured in macrophages from the middle-aged males. Age-related changes in macrophage phenotype and the antimicrobial activity were independent of macrophage ERα/ERß expression and estradiol level in both male and female rats. Although our study suggests that the sex difference in the level of circulating estradiol may to some extent contribute to sex difference in macrophage function of middle-aged rats, it also points to more complex hormonal regulation of peritoneal macrophage activity in females.

Strain-dependent response to stimulation in middle-aged rat macrophages: A quest after a useful indicator of healthy aging.

Rats of Albino Oxford (AO) strain in our animal facility exhibit a longer average healthy life span than rats of Dark Agouit (DA) strain. Since chronic activation of macrophages contributes to chronic low level inflammation common in older age, elucidation of the changes in middle-aged rats could be useful in prevention of unbalanced inflammatory response in advanced age. We have analysed the phenotype of unelicited and thioglycollate-elicited peritoneal macrophages from young and middle-aged DA and AO rats and tested functions of these cells following stimulation with lipopolysaccharide (LPS) in vitro. Unelicited cells from middle-aged DA rats produced higher amounts of proinflammatory mediators interleukin-6 (IL-6) and nitric oxide (NO), but have a diminished response to LPS stimulation then cells from young rats, in spite of increased frequency of TLR4- and CD14-expressing mature macrophages. Injection of thioglycollate robustly increased overall cytokine production in young rats' macrophages, while diminishing their response to LPS stimulation. In middle-aged DA rats injection of thioglycollate diminished IL-6 production, but increased it in response to LPS stimulation. Quite the contrary to DA rats, the macrophages from middle-aged AO rats have released diminished levels of TNF-α and NO, whereas urea production was strongly increased, when compared to the macrophages from young rats. Although the thioglycollate injection has increased the proportion of CD86+MHCII+ mature macrophages in young rats, and percentages of activated TLR4+ macrophages in both age groups of AO rats, it has not affected the cytokine production in young rats' macrophages, and the TNF-α production in middle-aged rats' macrophages. Moreover, the injection of thioglycollate has robustly increased the production of urea in macrophages derived from both age groups of AO rats. Although middle-aged rats of both strains were healthy during experiment, differences between the inflammatory responses of peritoneal macrophages of middle-aged rats of these strains might be one of the contributing factors defining their health in their advanced age. Development of strategies for the prevention of undesirable inflammatory changes in the elderly would benefit from the prospective study of the middle-aged.

Influence of age on osteoarthritis progression after anterior cruciate ligament transection in rats.

The anterior cruciate ligament transection (ACLT) model of osteoarthritis (OA) in young rats is widely used to study the pathogenesis of OA and possible treatment approaches. As aging is a key factor in the progression of this condition, it is hypothesized that animals may vary in their responses to ACLT according to their age. The histopathological features of young (2month-old) and middle-aged (12month-old) rats in the presence or absence of ACLT were compared. The results indicated that moderate degradative changes can be detected in the knee joints of sham-operated middle-aged rats compared with young animals. After ACLT, cartilage degradation was significantly higher in middle-aged rats in relation to young animals. An increase in interleukin(IL)-1ß and IL-17 suggests the presence of a local inflammatory response represented by synovitis in ACLT rats which is not dependent on age. Our study indicates that age is an important factor affecting the pathogenesis of OA changes after ACLT and it should be considered in studies using this experimental model.

Structural composition of myocardial infarction scar in middle-aged male and female rats: does sex matter?

The present study was designed to determine whether the structural composition of the scar in middle-aged post-myocardial infraction (MI) rats is affected by the biological sex of the animals. A large MI was induced in 12-month-old male (M-MI) and female (F-MI) Sprague-Dawley rats by ligation of the left coronary artery. Four weeks after the MI, rats with transmural infarctions, greater than 50% of the left ventricular (LV) free wall, were evaluated. The extent of LV remodeling and fractional volumes of fibrillar collagen (FC), myofibroblasts, vascular smooth muscle (SM) cells, and surviving cardiac myocytes (CM) in the scars were compared between the two sexes. The left ventricle of post-MI male and female rats underwent a similar degree of remodeling as evidenced by the analogous scar thinning ratio (0.46 ± 0.02 vs. 0.42 ± 0.05) and infarct expansion index (1.06 ± 0.07 vs. 1.12 ± 0.08), respectively. Most important, the contents of major structural components of the scar revealed no evident difference between M-MI and F-MI rats (interstitial FC, 80.74 ± 2.08 vs. 82.57 ± 4.53; myofibroblasts, 9.59 ± 1.68 vs.9.56 ± 1.15; vascular SM cells, 2.27 ± 0.51 vs. 3.38 ± 0.47; and surviving CM, 3.26 ± 0.39 vs. 3.05 ± 0.38, respectively). Our data are the first to demonstrate that biological sex does not influence the structural composition of a mature scar in middle-aged post-MI rats.