Aged Rats Research Articles

Rapamycin facilitates healing of the tendon-bone interface in an aging rat model of chronic rotator cuff injury

BackgroundTendon–bone interface (TBI) healing in chronic rotator cuff injury (CRCI) in older individuals is a common clinical challenge due to cellular senescence, as well as decreased tissue repair and regeneration. Many studies have demonstrated the anti-aging, improved tissue repair, and bone regeneration properties of rapamycin (RPM) in multiple age-related diseases. This study aimed to explore the effects of RPM on TBI healing after CRCI in an aging rat model. MethodsA CRCI model was established in 60 Sprague–Dawley rats (24 months old). Rats were then randomly allocated into the control, 0.1 μg RPM, and 1 μg RPM groups. At 4 and 8 weeks post-reconstructive surgery, the supraspinatus tendon–humerus complexes were harvested for biomechanical, microimaging, histological, and immunohistochemical evaluations. ResultsBiomechanical testing results demonstrated that the failure load, ultimate strength, and stiffness of the two RPM groups were significantly higher than those of the control group at 4 and 8 weeks postoperatively. Microradiographically, both RPM groups had significantly higher values of bone mineral density and the ratio of trabecular bone volume to total volume than controls at each time point. Moreover, the RPM groups had higher histological scores and showed better regenerated TBI, characterized by better organizational tissue, more fibrocartilage cells, and more bone formation. Immunohistochemical evaluations showed that RUNX2-, SOX9-, and SCX-positive cells were significantly more in the two RPM groups than in the controls at each time point. ConclusionsRPM may effectively enhance CRCI healing after reconstruction by facilitating osteogenesis, tenogenesis, and fibrocartilage reformation at the TBI, as well as improving biomechanical properties.

Age-dependent effects of metformin on human oligodendrocyte lineage cell ensheathment capacity.

Metformin restores the myelination potential of aged rat A2B5+ oligodendrocyte progenitor cells and may enhance recovery in children with post-radiation brain injury. Human late progenitor cells (O4+A2B5+) have a superior capacity to ensheath nanofibres compared to mature oligodendrocytes, with cells from paediatric sources exceeding adults. In this study, we assessed the effects of metformin on ensheathment capacity of human adult and paediatric progenitors and mature oligodendrocytes and related differences to transcriptional changes. A2B5+ progenitors and mature cells, derived from surgical tissues by immune-magnetic separation, were assessed for ensheathment capacity in nanofibre plates over 2 weeks. Metformin (10 µM every other day) was added to selected cultures. RNA was extracted from treated and control cultures after 2 days. For all ages, ensheathment by progenitors exceeded mature oligodendrocytes. Metformin enhanced ensheathment by adult donor cells but reduced ensheathment by paediatric cells. Metformin marginally increased cell death in paediatric progenitors. Metformin-induced changes in gene expression are distinct for each cell type. Adult progenitors showed up-regulation of pathways involved in the process of outgrowth and promoting lipid biosynthesis. Paediatric progenitors showed a relatively greater proportion of down- versus up-regulated pathways, these involved cell morphology, development and synaptic transmission. Metformin-induced AMP-activated protein kinase activation in all cell types; AMP-activated protein kinase inhibitor BML-275 reduced functional metformin effects only with adult cells. Our results indicate age and differentiation stage-related differences in human oligodendroglia lineage cells in response to metformin. Clinical trials for demyelinating conditions will indicate how these differences translate in vivo.

Quantification of Aging-Related Decreases in Sensory Innervation of the Bladder Trigone in Rats.

The prevalence of lower urinary tract symptoms (LUTS), characterized by problems regarding storage and/or voiding of urine, is known to significantly increase with age. Effective communication between the lower urinary tract and the central nervous system (CNS) is essential for the optimal function of this system, and heavily relies on the efficient interaction between the bladder urothelium and the afferent nerve fibers situated in close proximity to the urothelium within the lamina propria. We aimed to quantify aging-related differences in the expression of calcitonin gene-related peptide (CGRP, an established marker for sensory nerve fibers) in the trigonal mucosal layers of young (3-4 months) and aged (25-30 months) rats. We evaluated trigonal tissue from 3 animals per age group. Tissue was serially sectioned at 10 μm and stained for CGRP. Images were taken along the full length of the tissue. For each image we computed the total CGRP-positive area (μm2) and the median value for each animal was used for further analysis. Upon statistical analysis the aged rats show a significantly lower CGRP-positive area compared to young rats (P=0.0049). These results indicate that aging has a negative effect on the area of CGRP-positive signal in the trigone. The structural and functional integrity of the sensory web in the trigonum of rats is negatively affected by the aging process, potentially leading to impaired communication between the bladder urothelium the CNS. Consequently, these perturbations in the sensory system may contribute to the pathogenesis or exacerbation LUTS.

Effects of Aging and Nerve Growth Factor on Neuropeptide Expression and Cholinergic Innervation of the Rat Basolateral Amygdala.

The basolateral amygdala (BLA) contains interneurons that express neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP), both of which are involved in the regulation of functions and behaviors that undergo deterioration with aging. There is considerable evidence that, in some brain areas, the expression of NPY and VIP might be modulated by acetylcholine. Importantly, the BLA is one of the brain regions that has one of the densest cholinergic innervations, which arise mainly from the basal forebrain cholinergic neurons. These cholinergic neurons depend on nerve growth factor (NGF) for their survival, connectivity, and function. Thus, in this study, we sought to determine if aging alters the densities of NPY- and VIP-positive neurons and cholinergic varicosities in the BLA and, in the affirmative, if those changes might rely on insufficient trophic support provided by NGF. The number of NPY-positive neurons was significantly reduced in aged rats, whereas the number of VIP-immunoreactive neurons was unaltered. The decreased NPY expression was fully reversed by the infusion of NGF in the lateral ventricle. The density of cholinergic varicosities was similar in adult and old rats. On the other hand, the density of cholinergic varicosities is significantly higher in old rats treated with NGF than in adult and old rats. Our results indicate a dissimilar resistance of different populations of BLA interneurons to aging. Furthermore, the present data also show that the BLA cholinergic innervation is particularly resistant to aging effects. Finally, our results also show that the reduced NPY expression in the BLA of aged rats can be related to changes in the NGF neurotrophic support.

Stevia rebaudiana Bertoni, an American plant used as sweetener: Study of its effects on body mass control and glycemia reduction in Wistar male and female rats.

Stevia rebaudiana Bertoni water extracts have been used as a natural sweetener and customary medicine by the indigenous inhabitants of South America for several hundred years. This plant was sent to Europe in the 16th century and was described by Peter Jacob Esteve in Spain. Recently the food industry has started to employ S. rebaudiana as sweetener using its glycosides after purification. Advertisement claims that Stevia glycosides is good for controling body mass and reducing glycemia. This study's objective was to evaluate the effect of S. rebaudiana leaf extract on Wistar rats as animal model to prove its effectiveness on body mass control, glycemia reduction, and other biochemical parameters. Three groups were randomly formed with 24 males and 24 females: A blank group without any sweetener, a control group drinking water with 10% glucose, and the test group ingesting a 0.94% water extract of S. rebaudiana. Body mass measurements as well as food and drink consumption were daily performed. The experiment lasted 120 days after the specimens were weaned and got used to eating solid food. Euthanasia was done and blood serum was collected to evaluate the following biochemical parameters: Glucose, triglycerides, cholesterol, insulin, glucagon, leptin, ghrelin, and glucose-dependent insulinotropic peptide, GIP. Results indicated that only female rats had statistical differences in body mass gain. No relevant effects either positive or negative were found in the biochemical parameters measured. The crude extracts of S. rebaudiana did not show any relevant changes in biochemical and hormonal profiles, changes nor body mass with respect to the blank and control groups of young and healthy rats in the age range of infancy to youth. According to the results obtained, the therapeutic properties that have been associated to S. rebaudiana consumption especially for body mass control and glycemia reduction, did not occur in young and healthy male and female rats in equivalent age to infants, young children, and youths.

The role of melatonin deficiency induced by pinealectomy on motor activity and anxiety responses in young adult, middle-aged and old rats.

Aging affects anxiety levels in rats while the pineal gland, via its hormone melatonin, could modulate their inherited life "clock." The present study aimed to explore the impact of plasma melatonin deficiency on anxiety responses and the possible involvement of the hypothalamic-pituitary-adrenocortical (HPA) axis and heat shock proteins (Hsp) 70 and 90 in the frontal cortex (FC) and the hippocampus in young adult, middle-aged and elderly rats with pinealectomy. Melatonin deficiency induced at different life stages did not affect the lifespan of rats. Pinealectomy abolished the circadian rhythm of motor activity, measured for 48h in the actimeter, in young adult but not in middle-aged rats. Pinealectomy reduced the motor activity of the young adult rats during the dark phase and impaired the diurnal activity variations of old rats. The same generations (3- and 18month-old rats with pinealectomy) had lower anxiety levels than the matched sham groups, measured in three tests: elevated-plus maze, light-dark test, and novelty-suppressed feeding test. While the activity of the HPA axis remained intact in young adult and middle-aged rats with melatonin deficiency, a high baseline corticosterone level and blunted stress-induced mechanism of its release were detected in the oldest rats. Age-associated reduced Hsp 70 and 90 levels in the FC but not in the hippocampus were detected. Pinealectomy diminished the expression of Hsp 70 in the FC of middle-aged rats compared to the matched sham rats. Our results suggest that while melatonin hormonal dysfunction impaired the motor activity in the actimeter and emotional behavior in young adult and elderly rats, the underlying pathogenic mechanism in these generations might be different and needs further verification.

Cognitive performance in aged rats is associated with differences in distinctive neuronal populations in the ventral tegmental area and altered synaptic plasticity in the hippocampus.

Deterioration of cognitive functions is commonly associated with aging, although there is wide variation in the onset and manifestation. Albeit heterogeneity in age-related cognitive decline has been studied at the cellular and molecular level, there is poor evidence for electrophysiological correlates. The aim of the current study was to address the electrophysiological basis of heterogeneity of cognitive functions in cognitively Inferior and Superior old (19-20 months) rats in the ventral tegmental area (VTA) and the hippocampus, having Young (12 weeks) rats as a control. The midbrain VTA operates as a hub amidst affective and cognitive facets, processing sensory inputs related to motivated behaviours and hippocampal memory. Increasing evidence shows direct dopaminergic and non-dopaminergic input from the VTA to the hippocampus. Aged Superior and Inferior male rats were selected from a cohort of 88 animals based on their performance in a spatial learning and memory task. Using in vivo single-cell recording in the VTA, we examined the electrical activity of different neuronal populations (putative dopaminergic, glutamatergic and GABAergic neurons). In the same animals, basal synaptic transmission and synaptic plasticity were examined in hippocampal slices. Electrophysiological recordings from the VTA and hippocampus showed alterations associated with aging per se, together with differences specifically linked to the cognitive status of aged animals. In particular, the bursting activity of dopamine neurons was lower, while the firing frequency of glutamatergic neurons was higher in VTA of Inferior old rats. The response to high-frequency stimulation in hippocampal slices also discriminated between Superior and Inferior aged animals. This study provides new insight into electrophysiological information underlying compromised cerebral ageing. Further understanding of brain senescence, possibly related to neurocognitive decline, will help develop new strategies towards the preservation of a high quality of life.

Age-related changes in hematological and biochemical profiles of Wistar rats

BackgroundWistar rats are extensively used as the model for assessing toxicity and efficacy in preclinical research. Hematological and biochemical laboratory data are essential for evaluating specific variations in the physiological and functional profile of a laboratory animal. Establishing hematological and biochemical reference values for Wistar (han) rats at various age intervals was the goal of this work. Male and female Wistar rats (n = 660) of ages 6–8 weeks, 10–14 weeks and > 6 months were used in the experiment. Blood and serum were collected from these rats under fasting conditions.ResultsWe observed that the majority of hematological and biochemical parameters were significantly influenced by sex and age. Hematological changes were significantly correlated to aging were increased red blood cells, hemoglobin, hematocrit, neutrophils, monocytes and eosinophils in both sexes, as well as decreased platelet, mean corpuscular volume, mean corpuscular hemoglobin and lymphocytes in both sexes. White blood cells of male rats were considerably higher than those of female rats in all age ranges. For biochemistry, increase in glucose, total protein and creatinine were seen in both sexes, along with increases in urea in females and alanine aminotransferase in males. Age was significantly associated with decreased alkaline phosphatase in both sexes.ConclusionsWhen using Wistar rats as a model, these reference values may be useful in evaluating the results.

The complex relationship between late-onset caloric restriction and synaptic plasticity in aged Wistar rats.

Age-related reduction in spine density, synaptic marker expression, and synaptic efficiency are frequently reported. These changes provide the cellular and molecular basis for the cognitive decline characteristic for old age. Nevertheless, there are several approaches that have the potential to ameliorate these processes and improve cognition, caloric restriction being one of the most promising and widely studied. While lifelong caloric restriction is known for its numerous beneficial effects, including improved cognitive abilities and increased expression of proteins essential for synaptic structure and function, the effects of late-onset and/or short-term CR on synaptic plasticity have yet to be investigated. We have previously documented that the effects of CR are strongly dependent on whether CR is initiated in young or old subjects. With this in mind, we conducted a long-term study in aging Wistar rats to examine changes in the expression of several key synaptic markers under the regimen of CR started at different time points in life. We found a significant increase in the expression of both presynaptic and postsynaptic markers. However, taking into account previously reported changes in the behavior detected in these animals, we consider that this increase cannot represent beneficial effect of CR.

Restorative antiaging influence and chemical profile of Prunus domestica L. (European plum) seed extract in a D-galactose-induced rat model

Background Prunus domestica L., also known as European plums or prunes, is a member of the Rosaceae family. Recently, fruit byproducts like seeds have been used as a novel and affordable source of bioactive compounds that may be an affordable source of substances. These substances hold potential benefit to the food and pharmaceutical industries. Objective The study aims to evaluate the chemical composition of P. domestica L. seeds and their safety and efficiency as an antiaging agent. Materials and methods The phenolic content in ethanolic seed extract was determined using the Folin–Ciocalteu reagent. Chlorophyll a, chlorophyll b, and β-carotene concentrations were measured by high performance liquid chromatography analysis. Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) negative ion mode was carried out to investigate the phytoconstituents, in addition to the isolation of the main active compounds on preparative TLC as well as evaluation of cognitive capabilities, measurement of antioxidants and neurotransmitters and histopathological brain examination. Results and conclusion The total phenolic and flavonoid contents of ethanolic extract were 136.55 mg GAE/g and 89 mg CE/g, respectively. Also, high performance liquid chromatography analysis led to the identification of β-carotene as the main natural pigment (59.376 mg/100 g), followed by chlorophyll b (41.745 mg/100 g) and finally chlorophyll a (20.824 mg/100 g). In addition, UPLC/ESI-MS analysis using the negative ion mode led to the identification of 24 compounds, including two flavonols, one flavanone, two flavones, 10 flavonoid-O-glycosides, two methylated flavonoids, one isoflavonoidC-glycosides, two phenolic acids, two aldehydes, one stilbene, and one coumarin. Preparative TLC chromatographic technique for ethanolic P. domestica seed extract resulted in the isolation of γ-tocopherol, kaempferol-3-O-rutinoside, luteolin, and luteolin-7-O-glucoside. The ethanolic extract proved to have a remarkable scavenging effect against 1,1-Diphenyl-2-picryl-hydrazyl in a dose-dependent manner compared with vitamin C. Improvement in behavioral assessment was noticed posttreatment of rats with the extract suggesting amelioration in cognitive ability and spatial memory of rats compared with standard drug. In addition, noticeable improvements were observed in neurotransmitter levels, acetylcholinesterase, noradrenaline, dopamine, and serotonin, in the brain tissue of aged rats. Besides, remarkable improvements in oxidative stress biomarkers (nitric oxide, reduced glutathione and lipid peroxide, total antioxidant capacity) and in histopathological pictures of different brain regions posttreatment of aged rats with the extract compared with donepezil standard drug were recorded.

Vitamin D Protects Against Cardiac Hypertrophy Through the Regulation of Mitochondrial Function in Aging Rats.

Cardiac aging is defined as mitochondrial dysfunction of the heart. Vitamin D (VitD) is an effective agent in ameliorating cardiovascular disorders. In this study, we indicated the protective effects of VitD against cardiac aging. Male Wistar rats were randomly divided into four groups: control (CONT), D-galactose (D-GAL): aged rats induced by D-GAL, D-GAL + Ethanol: aged rats treated with ethanol, and D-GAL + VitD aged rats treated with VitD. Aging was induced by D-GAL at 150 mg/kg via intraperitoneal injection for 8 weeks. Aged rats were treated with VitD (D-GAL + VitD) by gavage for 8 weeks. The serum samples were used to evaluate biochemical factors, and heart tissues were assessed to determine oxidative stress and gene expression. The D-GAL rats exhibited cardiac hypertrophy, which was associated with decreased antioxidant enzyme activity, enhanced oxidative marker, and changes in the expression of mitochondrial genes in comparison with the control rats. Co-treatment with VitD ameliorated all these changes. In conclusion, VitD could protect the heart against D-GAL-induced aging via enhancing antioxidant effects, and the expression of mitochondrial genes.

Evaluation of the wound healing effect of Nishamalaki, an Ayurvedic formulation comprising Curcuma longa and Phyllanthus emblica in aging rats

BackgroundThere are very few drugs available for healing wounds in the aged population, which is more prone to chronic cutaneous wounds that are particularly hard to heal and require a long healing process. This study which deals with age-related wound healing, investigates the healing effect of Nishamalaki, a classic antidiabetic Ayurvedic formulation comprising turmeric (Curcuma longa) and Indian gooseberry (Phyllanthus emblica), on cutaneous wounds in aging rats.MethodsRats with excision wounds of 7 mm created on their dorsal side received Nishamalaki (500 mg/kg p.o) daily, or a combination of Nishamalaki (500 mg/kg p.o) with 1% Nishamalaki gel applied on the wound or the reference standard metformin (2 μmol) applied on the wound daily till the scabs fell off.ResultsAll treatments enhanced the rate of formation of granulation tissue and wound contraction. All treated rats showed lower blood glucose levels compared with their 1st-day values and significantly lower blood glucose levels when compared with the Aged Control rats. A significant restoration of the aging-depleted L-hydroxyproline, hexosamine, ascorbic acid, PDGF, AMPK, and mTOR levels, and attenuation of the aging-elevated IL-6 and TNF-α levels was elicited by all treatments. The treatments significantly restored the aging-depleted endogenous antioxidants. The Nishamalaki combination treatment of the oral extract and topical gel displayed a better wound-healing effect than the oral treatment alone. The histopathological studies on skin ulceration, hair follicles, granulation tissue, and collagen fiber formation of the wound tissues corroborated the biochemical findings.ConclusionCurcumin and other antioxidant polyphenolic components of Nishamalaki may be responsible for its wound-healing effect. For the first time, the present study has investigated the action of PDGF, AMPK, and mTOR on cutaneous wounds. They seem to be acting together to promote wound healing and repair.Graphical

Fear extinction is impaired in aged rats.

Normal aging is accompanied by broad loss of cognitive function in humans and rodents, including declines in cognitive flexibility. In extinction, a conditional stimulus (CS) that was previously paired with a footshock is presented alone. This procedure reliably reduces conditional freezing behavior in young adult rats. Here, we aimed to investigate how normal aging affects extinction learning. Using young (3months) and aged (20months) male and female Long Evans rats, we compared extinction (using 20 CS-alone presentations) to a no extinction control (equal exposure to the conditioning chamber without CS presentations) following delay fear conditioning. We found that young animals in the extinction group showed a decrease in freezing following extinction; aged animals did not. We next examined changes in neural activity using expression of the immediate early gene zif268. In young animals, extinction corresponded with decreased expression of zif268 in the basolateral amygdala and anterior retrosplenial cortex; this was not observed in aged animals. Further, aged animals showed increased zif268 expression in each region examined, suggesting that dysfunction in neural activity precedes cognitive deficits. These results demonstrate that aging impacts both extinction learning and neural activity.

Effects of the protective and therapeutic effect of yogurt enriched with Citrus sinensis essential oil against ibuprofen-induced gastric ulcer in elderly rats.

The aim of this study was to evaluate the effect of Citrus sinensis essential oil (EO) on the proximate composition of yogurt over a 28-day shelf life and to investigate the therapeutic and prophylactic effects of functional yogurt on ibuprofen-induced gastric ulcers in a rat model. It was observed that the yogurt group containing C. sinensis EO had higher acidity, total solids, and ash values. Histologic evaluation of the stomachs of rats with gastric ulcers revealed that rats fed with functional yogurt had fewer lesions compared to the control group. The treatment group had fewer lesions than the positive control (p>0.05). Lesions in the glandular mucosa of the prophylactic group were significantly lower than those in the positive control group (p<0.05). Yogurt with C. sinensis EO may be beneficial in reducing the severity of ulcers and improving overall health.

The prevention effect of pulsed electromagnetic fields treatment on senile osteoporosis in vivo via improving the inflammatory bone microenvironment

ABSTRACT This study aimed to assess PEMF in a rat model of senile osteoporosis and its relationship with NLRP3-mediated low-grade inflammation in the bone marrow microenvironment. A total of 24 Sprague Dawley (SD) rats were included in this study. Sixteen of them were 24-month natural-aged male SD rats, which were randomly distributed into the Aged group and the PEMF group (n = 8 per group). The remaining 8 3-month -old rats were used as the Young positive control group (n = 8). Rats in the PEMF group received 12 weeks of PEMF with 40 min/day, five days per week, while the other rats received placebo PEMF intervention. Bone mineral density/microarchitecture, serum levels of CTX-1 and P1CP, and NLRP3-related signaling genes and proteins in rat bone marrow were then analyzed. The 12-week of PEMF showed significant mitigation of aging-induced bone loss and bone microarchitecture deterioration, i.e. PEMF increased the bone mineral density of the proximal femur and L5 vertebral body and improved parameters of the proximal tibia and L4 vertebral body. Further analysis showed that PEMF reversed aging-induced bone turnover, specifically, decreased serum CTX-1 and elevated serum P1CP. Furthermore, PEMF also dramatically inhibited NLRP3-mediated low-grade inflammation in the bone marrow, i.e. PEMF inhibited the levels of NLRP3, proCaspase1, cleaved Caspase1, IL-1β, and GSDMD-N. The study demonstrated that PEMF could mitigate the aging-induced bone loss and reverses the deterioration of bone microarchitecture probably through inhibiting NLRP3-mediated low-grade chronic inflammation to improve the inflammatory bone microenvironment in aged rats.

Restoration of norepinephrine release, cognitive performance, and dendritic spines by amphetamine in aged rat brain.

Age-related dysfunctions in specific neurotransmitter systems likely play an important role in cognitive decline even in its most subtle forms. Therefore, preservation or improvement of cognition via augmentation of neurotransmission is a potential therapeutic strategy to prevent further cognitive deficits. Here we identified a particular neuronal vulnerability in the aged Fischer 344 rat brain, an animal model of neurocognitive aging. Specifically, we demonstrated a marked impairment in glutamate-stimulated release of norepinephrine (NE) in the hippocampus and cerebral cortex of aged rats, and established that this release was mediated by N-methyl-D-aspartate (NMDA) receptors. Further, we also demonstrated that this decrease in NE release is fully rescued by the psychostimulant drug amphetamine (AMPH). Moreover, we showed that AMPH increases dendritic spine maturation, and importantly shows preclinical efficacy in restoring memory deficits in the aged rat through its actions to potentiate NE neurotransmission at β-adrenergic receptors. Taken together, our results suggest that deficits in glutamate-stimulated release of NE may contribute to and possibly be a determinant of neuronal vulnerability underlying cognitive decline during aging, and that these deficits can be corrected with currently available drugs. Overall these studies suggest that repurposing of psychostimulants for age-associated cognitive deficits is a potential avenue to delay or prevent cognitive decline and/or frank dementia later in life.

Reduced SV2A and GABAA receptor levels in the brains of type 2 diabetic rats revealed by [18F]SDM-8 and [18F]flumazenil PET

PurposeType 2 diabetes mellitus (T2DM) is associated with a greater risk of Alzheimer's disease. Synaptic impairment and protein aggregates have been reported in the brains of T2DM models. Here, we assessed whether neurodegenerative changes in synaptic vesicle 2 A (SV2A), γ-aminobutyric acid type A (GABAA) receptor, amyloid-β, tau and receptor for advanced glycosylation end product (RAGE) can be detected in vivo in T2DM rats. MethodsPositron emission tomography (PET) using [18F]SDM-8 (SV2A), [18F]flumazenil (GABAA receptor), [18F]florbetapir (amyloid-β), [18F]PM-PBB3 (tau), and [18F]FPS-ZM1 (RAGE) was carried out in 12-month-old diabetic Zucker diabetic fatty (ZDF) and SpragueDawley (SD) rats. Immunofluorescence staining, Thioflavin S staining, proteomic profiling and pathway analysis were performed on the brain tissues of ZDF and SD rats. ResultsReduced cortical [18F]SDM-8 uptake and cortical and hippocampal [18F]flumazenil uptake were observed in 12-month-old ZDF rats compared to SD rats. The regional uptake of [18F]florbetapir and [18F]PM-PBB3 was comparable in the brains of 12-month-old ZDF and SD rats. Immunofluorescence staining revealed Thioflavin S-negative, phospho-tau-positive inclusions in the cortex and hypothalamus in the brains of ZDF rats and the absence of amyloid-beta deposits. The level of GABAA receptors was lower in the cortex of ZDF rats than SD rats. Proteomic analysis further demonstrated that, compared with SD rats, synaptic-related proteins and pathways were downregulated in the hippocampus of ZDF rats. ConclusionThese findings provide in vivo evidence for regional reductions in SV2A and GABAA receptor levels in the brains of aged T2DM ZDF rats.

The bone morphogenetic protein 2 analogue L51P enhances spinal fusion in combination with BMP2 in an in vivo rat tail model

Bone morphogenic protein 2 (BMP2) is known to induce osteogenesis and is applied clinically to enhance spinal fusion despite adverse effects. BMP2 needs to be used in high doses to be effective due to the presence of BMP2 inhibitors. L51P is a BMP2 analogue that acts by inhibition of BMP2 inhibitors. Here, we hypothesized that mixtures of BMP2 and L51P could achieve better spinal fusion outcomes regarding ossification. To test whether mixtures of both cytokines are sufficient to improve ossification, 45 elderly Wistar rats (of which 21 were males) were assigned to seven experimental groups, all which received spinal fusion surgery, including discectomy at the caudal 4–5 level using an external fixator and a porous β-tricalcium phosphate (βTCP) carrier. These βTCP carriers were coated with varying concentrations of BMP2 and L51P. X-rays were taken immediately after surgery and again six and twelve weeks post-operatively. Histological sections and µCT were analyzed after twelve weeks. Spinal fusion was assessed using X-ray, µCT and histology according to the Bridwell scale by voxel-based quantification and a semi-quantitative histological score, respectively. The results were congruent across modalities and revealed high ossification for high-dose BMP2 (10 µg), while PBS induced no ossification. Low-dose BMP2 (1 µg) or 10 µg L51P alone did not induce relevant bone formation. However, all combinations of low-dose BMP2 with L51P (1 µg + 1/5/10 µg) were able to induce similar ossificationas high-dose BMP2. These results are of high clinical relevance, as they indicate L51P is sufficient to increase the efficacy of BMP2 and thus lower the required dose for spinal fusion. Statement of SignificanceSpinal fusion surgery is frequently applied to treat spinal pathologies. Bone Morphogenic Protein-2 (BMP2) has been approved by the U .S. Food and Drug Administration (FDA-) and by the “Conformité Européenne” (CE)-label. However, its application is expensive and high concentrations cause side-effects. This research targets the improvement of the efficacy of BMP2 in spinal fusion surgery.

Cumulative Deleterious Effects of Tetrahydrocannabinoid (THC) and Ethanol on Mitochondrial Respiration and Reactive Oxygen Species Production Are Enhanced in Old Isolated Cardiac Mitochondria.

Delta 9 tetrahydrocannabinol (THC), the main component of cannabis, has adverse effects on the cardiovascular system, but whether concomitant ethanol (EtOH) and aging modulate its toxicity is unknown. We investigated dose responses of THC and its vehicle, EtOH, on mitochondrial respiration and reactive oxygen production in both young and old rat cardiac mitochondria (12 and 90 weeks). THC dose-dependently impaired mitochondrial respiration in both groups, and such impairment was enhanced in aged rats (-97.5 ± 1.4% vs. -75.6 ± 4.0% at 2 × 10-5 M, and IC50: 0.7 ± 0.05 vs. 1.3 ± 0.1 × 10-5 M, p < 0.01, for old and young rats, respectively). The EtOH-induced decrease in mitochondrial respiration was greater in old rats (-50.1 ± 2.4% vs. -19.8 ± 4.4% at 0.9 × 10-5 M, p < 0.0001). Further, mitochondrial hydrogen peroxide (H2O2) production was enhanced in old rats after THC injection (+46.6 ± 5.3 vs. + 17.9 ± 7.8%, p < 0.01, at 2 × 10-5 M). In conclusion, the deleterious cardiac effects of THC were enhanced with concomitant EtOH, particularly in old cardiac mitochondria, showing greater mitochondrial respiration impairment and ROS production. These data improve our knowledge of the mechanisms potentially involved in cannabis toxicity, and likely support additional caution when THC is used by elderly people who consume alcohol.

Abstract WMP111: Intra-Arterial Administration of Cerebral Endothelial Cell Derived Small Extracellular Vesicles Reduces Neurovascular Damage and Improves Neurological Outcome After Acute Ischemia in the Aged Rat

Stroke is a leading cause of death and disability worldwide, mainly affecting the elderly. Endovascular thrombectomy (EVT) is a standard of care therapy for stroke patients with large-vessel occlusion, however, the majority of these patients do not achieve full functional improvement. The present study tested the hypothesis that intra-arterial (IA) administration of cerebral endothelial cell derived small extracellular vesicles (CEC-sEVs) following transient middle cerebral artery occlusion (tMCAO) improves stroke outcome in the aged rat. CEC-sEVs (1x10 11 particles/injection) isolated from healthy adult rats were employed. Male aged rats (18 months, n=10/group) subjected to 3h tMCAO were randomly divided into the experimental groups. To examine whether IA injection of CEC-sEVs improves stroke outcome, CEC-sEVs were administered via the internal carotid artery (ICA) immediately following reperfusion by withdrawal of the occlusion filament. To examine whether intravenous administration (IV) of CEC-sEVs during tMCAO and then followed by IA CEC-sEVs upon reperfusion further enhances stroke outcome, CEC-sEVs were given at 30 min of tMCAO via a tail vein and upon reperfusion via the ICA. The ischemic rats treated with IA saline were used as a control. Neurological outcome and infarct volume were assessed in a blinded manner. IA CEC-sEVs upon reperfusion significantly reduced infarct volume (24±5% vs 31±6% in saline, p&lt;0.05) which was associated with robust improvement of neurological function from weeks 2 to 4 after tMCAO compared to saline treated rats. Moreover, rats treated with CEC-sEVs IV at 30min followed by IA treatment upon reperfusion further reduced infarct volume (16±6%) compared to rats treated with IA saline or CEC-sEVs. These rats also exhibited robust and early improvement of neurological function starting at 1d after tMCAO. In conclusion, IA administration of CEC-sEVs upon reperfusion significantly reduces ischemic damage, while an additional early (30min post stroke) dose of IV CEC-sEVs further enhances and accelerates the therapeutic effect of IA CEC-sEVs in the aged rats after tMCAO. Our data suggest that CEC-sEVs could potentially may be a therapy to amplify the efficacy of EVT even for elderly patients with acute ischemic stroke.