XperCT-guided Intra-cisterna Magna Injection of Streptozotocin for Establishing an Alzheimer’s Disease Model Using the Cynomolgus Monkey (Macaca fascicularis)

Till date, researchers have been developing animal models of Alzheimer’s disease (AD) in various species to understand the pathological characterization and molecular mechanistic pathways associated with this condition in humans to identify potential therapeutic treatments. A widely recognized AD model that mimics the pathology of human AD involves the intracerebroventricular (ICV) injection with streptozotocin (STZ).However, ICV injection as an invasive approach has several limitations related to complicated surgical procedures. Therefore, in the present study, we created a customized stereotaxic frame using the XperCT-guided system for injecting STZ in cynomolgus monkeys, aiming to establish an AD model. The anatomical structures surrounding the cisterna magna (CM) were confirmed using CT/MRI fusion images of monkey brain with XperCT, the c-arm cone beam computed tomography. XperCT was used to determine the appropriate direction in which the needle tip should be inserted within the CM region. Cerebrospinal fluid (CSF) was collected to confirm the accurate target site when STZ was injected into the CM.Cynomolgus monkeys were administered STZ dissolved in artificial CSF once every week for 4 weeks via intracisterna magna (ICM) injection using XperCT-guided stereotactic system. The molecular mechanisms underlying the progression of STZ-induced AD pathology were analyzed two weeks after the final injection. The monkeys subjected to XperCT-based STZ injection via the ICM route showed features of AD pathology, including markedly enhanced neuronal loss, synaptic impairment, and tau phosphorylation in the hippocampus. These findings suggest a new approach for the construction of neurodegenerative disease models and development of therapeutic strategies.

Synaptic loss and amyloid beta alterations in the rodent hippocampus induced by streptozotocin injection into the cisterna magna / 한국실험동물학회지

To date, researchers have developed various animal models of Alzheimer’s disease (AD) to investigate its mechanisms and to identify potential therapeutic treatments. A widely recognized model that mimics the pathology of human sporadic AD involves intracerebroventricular (ICV) injection with streptozotocin (STZ). However, ICV injections are an invasive approach, which creates limitations in generalizing the results. In this study, we produced a rodent model of AD using STZ (3 mg/kg) injection via the cisterna magna (CM) once every week for 4 weeks, and analyzed at 4 weeks and 16 weeks after final injection. In the CM-STZ rodent model of AD, we observed increase in extracellular amyloid-beta (Aβ) deposition and decrease and abnormal morphology of post-synaptic protein, PSD95 in 16 weeks STZ-injected group. The model developed using our less-invasive method induced features of AD-like pathology, including significantly increased extracellular amyloid-beta deposition, and decreased synaptic protein in the hippocampus. These findings supporting the success of this alternative approach, and thus, we suggest this is a promising, less invasive model for use in future AD research.

Assessment of Hand Motor Function in a Non-human Primate Model of Ischemic Stroke

Ischemic stroke results from arterial occlusion and can cause irreversible brain injury. A non-human primate (NHP) model of ischemic stroke was previously developed to investigate its pathophysiology and for efficacy testing of therapeutic candidates; however, fine motor impairment remains to be well-characterized. We evaluated hand motor function in a cynomolgus monkey model of ischemic stroke. Endovascular transient middle cerebral artery occlusion (MCAO) with an angiographic microcatheter induced cerebral infarction. In vivo magnetic resonance imaging mapped and measured the ischemia-induced infarct lesion. In vivo diffusion tensor imaging (DTI) of the stroke lesion to assess the neuroplastic changes and fiber tractography demonstrated three-dimensional patterns in the corticospinal tract 12 weeks after MCAO. The hand dexterity task (HDT) was used to evaluate fine motor movement of upper extremity digits. The HDT was modified for a home cage-based training system, instead of conventional chair restraint training. The lesion was localized in the middle cerebral artery territory, including the sensorimotor cortex. Maximum infarct volume was exhibited over the first week after MCAO, which progressively inhibited ischemic core expansion, manifested by enhanced functional recovery of the affected hand over 12 weeks after MCAO. The total performance time decreased with increasing success rate for both hands on the HDT. Compensatory strategies and retrieval failure improved in the chronic phase after stroke. Our findings demonstrate the recovery of fine motor skill after stroke, and outline the behavioral characteristics and features of functional disorder of NHP stroke model, providing a basis for assessing hand motor function after stroke.

Age-related Effects of Heroin on Gene Expression in the Hippocampus and Striatum of Cynomolgus Monkeys

Objective@#The aim of this study was to investigate differentially expressed genes and their functions in the hippocampus and striatum after heroin administration in cynomolgus macaques of different ages. @*Methods@#Cynomolgus monkeys were divided by age as follows: 1 year (A1, n = 2); 3 to 4 years (A2, n = 2); 6 to 8 years (A3, n = 2); and older than 11 years (A4, n = 2). After heroin was injected intramuscularly into the monkeys (0.6 mg/kg), we performed large-scale transcriptome profiling in the hippocampus (H) and striatum (S) using RNA sequencing technology. Some genes were validated with real-time quantitative PCR. @*Results@#In the hippocampus, the gene expression of A1H was similar to that of A4H, while the gene expression of A2H was similar to that of A3H. Genes associated with the mitogen-activated protein kinase signaling pathway (STMN1, FGF14, and MAPT) and -aminobutyric acid-ergic synapses (GABBR2 and GAD1) were differentially expressed among control and heroin-treated animals. Differential gene expression between A1S and A4S was the least significant, while differential gene expression between A3S and A2S was the most significant. Genes associated with the neurotrophin signaling pathway (NTRK1 and NGFR), autophagy (ATG5), and dopaminergic synapses (AKT1) in the striatum were differentially expressed among control and heroin-treated animals. @*Conclusion@#These results suggest that even a single heroin exposure can cause differential gene expression in the hippocampus and striatum of nonhuman primates at different ages.

Increased CD68/TGFβ Co-expressing Microglia/Macrophages after Transient Middle Cerebral Artery Occlusion in Rhesus Monkeys

The function of microglia/macrophages after ischemic stroke is poorly understood. This study examines the role of microglia/macrophages in the focal infarct area after transient middle cerebral artery occlusion (MCAO) in rhesus monkeys. We measured infarct volume and neurological function by magnetic resonance imaging (MRI) and non-human primate stroke scale (NHPSS), respectively, to assess temporal changes following MCAO. Activated phagocytic microglia/macrophages were examined by immunohistochemistry in post-mortem brains (n=6 MCAO, n=2 controls) at 3 and 24 hours (acute stage), 2 and 4 weeks (subacute stage), and 4, and 20 months (chronic stage) following MCAO. We found that the infarct volume progressively decreased between 1 and 4 weeks following MCAO, in parallel with the neurological recovery. Greater presence of cluster of differentiation 68 (CD68)-expressing microglia/macrophages was detected in the infarct lesion in the subacute and chronic stage, compared to the acute stage. Surprisingly, 98~99% of transforming growth factor beta (TGFβ) was found colocalized with CD68-expressing cells. CD68-expressing microglia/macrophages, rather than CD206⁺ cells, may exert anti-inflammatory effects by secreting TGFβ after the subacute stage of ischemic stroke. CD68⁺ microglia/macrophages can therefore be used as a potential therapeutic target.

Abnormal Mitochondria in a Non-human Primate Model of MPTP-induced Parkinson's Disease: Drp1 and CDK5/p25 Signaling

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.

Evaluation of fecal microbiomes associated with obesity in captive cynomolgus monkeys (Macaca fascicularis)

Microorganisms play important roles in obesity; however, the role of the gut microbiomes in obesity is controversial because of the inconsistent findings. This study investigated the gut microbiome communities in obese and lean groups of captive healthy cynomolgus monkeys reared under strict identical environmental conditions, including their diet. No significant differences in the relative abundance of Firmicutes, Bacteroidetes and Prevotella were observed between the obese and lean groups, but a significant difference in Spirochetes (p < 0.05) was noted. Microbial diversity and richness were similar, but highly variable results in microbial composition, diversity, and richness were observed in individuals, irrespective of their state of obesity. Distinct clustering between the groups was not observed by principal coordinate analysis using an unweighted pair group method. Higher sharedness values (95.81% ± 2.28% at the genus level, and 79.54% ± 5.88% at the species level) were identified among individual monkeys. This paper reports the association between the gut microbiome and obesity in captive non-human primate models reared under controlled environments. The relative proportion of Firmicutes and Bacteroidetes as well as the microbial diversity known to affect obesity were similar in the obese and lean groups of monkeys reared under identical conditions. Therefore, obesity-associated microbial changes reported previously appear to be associated directly with environmental factors, particularly diet, rather than obesity.

Reference values of hematological and biochemical parameters in young-adult cynomolgus monkey (Macaca fascicularis) and rhesus monkey (Macaca mulatta) anesthetized with ketamine hydrochloride / 한국실험동물학회지

Nonhuman primate models are valuable in biomedical research. However, reference data for clinical pathology parameters in cynomolgus and rhesus monkeys are limited. In the present study, we established hematologic and biochemical reference intervals for healthy cynomolgus and rhesus monkeys anesthetized with ketamine hydrochloride. A total of 142 cynomolgus monkeys (28 males and 114 females) and 42 rhesus monkeys (22 males and 20 females) were selected and analyzed in order to examine reference intervals of 20 hematological and 16 biochemical parameters. The effects of sex were also investigated. Reference intervals for hematological and biochemical parameters were separately established by species (cynomolgus and rhesus) and sex (male and female). No sex-related differences were determined in erythrocyte-related parameters for cynomolgus and rhesus monkey housed in indoor laboratory conditions. Alkaline phosphatase and gamma glutamyltransferase were significantly lower in females than males in both cynomolgus and rhesus monkeys aged 48–96 months. The reference values for hematological and biochemical parameters established herein might provide valuable information for researchers using cynomolgus and rhesus monkeys in experimental conditions for biomedical studies.

Differential expression of caveolins and myosin heavy chains in response to forced exercise in rats / 한국실험동물학회지

Exercise training can improve strength and lead to adaptations in the skeletal muscle and nervous systems. Skeletal muscles can develop into two types: fast and slow, depending on the expression pattern of myosin heavy chain (MHC) isoforms. Previous studies reported that exercise altered the distribution of muscle fiber types. It is not currently known what changes in the expression of caveolins and types of muscle fiber occur in response to the intensity of exercise. This study determined the changes in expression of caveolins and MHC type after forced exercise in muscular and non-muscular tissues in rats. A control (Con) group to which forced exercise was not applied and an exercise (Ex) group to which forced exercise was applied. Forced exercise, using a treadmill, was introduced at a speed of 25 m/min for 30 min, 3 times/day (07:00, 15:00, 23:00). Homogenized tissues were applied to extract of total RNA for further gene analysis. The expression of caveolin-3 and MHC2a in the gastrocnemius muscle of female rats significantly increased in the Ex group compared with the Con group (P<0.05). Furthermore, in the gastrocnemius muscle of male rats, the expression of MHC2x was significantly different between the two groups (P<0.05). There was an increased expression in caveolin-3 and a slightly decreased expression in TGFbeta-1 in muscular tissues implicating caveolin-3 influences the expression of MHC isoforms and TGFbeta-1 expression. Eventually, it implicates that caveolin-3 has positive regulatory function in muscle atrophy induced by neural dysfunction with spinal cord injury or stroke.

Autophagy contributes to retardation of cardiac growth in diabetic rats / 한국실험동물학회지

Diabetes mellitus is a major predictor of heart failure, although the mechanisms by which the disease causes cardiomyopathy are not well understood. The purpose of this study was to determine whether prolonged exposure of cardiomyocytes to high glucose concentrations induces autophagy and contributes to cardiomyopathy. Interestingly, there were no differences in the autophagic activation produced by different glucose concentrations. However, cell viability was decreased by high glucose. In the diabetic rats, we found a higher level of microtubule-associated protein light chain 3 (LC3) expression and a reduction in the size of the left ventricle (LV) (P<0.05) caused by growth retardation, suggesting activated autophagy. Our in vitro findings indicate that hyperglycemic oxidative stress induces autophagy, and our in vivo studies reveal that autophagy is involved in the progression of pathophysiological remodeling of the heart. Taken together, the studies suggest that autophagy may play a role in the pathogenesis of juvenile diabetic cardiomyopathy.

Prophylactic effects of swimming exercise on autophagy-induced muscle atrophy in diabetic rats / 한국실험동물학회지

Diabetes decreases skeletal muscle mass and induces atrophy. However, the mechanisms by which hyperglycemia and insulin deficiency modify muscle mass are not well defined. In this study, we evaluated the effects of swimming exercise on muscle mass and intracellular protein degradation in diabetic rats, and proposed that autophagy inhibition induced by swimming exercise serves as a hypercatabolic mechanism in the skeletal muscles of diabetic rats, supporting a notion that swimming exercise could efficiently reverse the reduced skeletal muscle mass caused by diabetes. Adult male Sprague-Dawley rats were injected intraperitoneally with streptozotocin (60 mg/kg body weight) to induce diabetes and then submitted to 1 hr per day of forced swimming exercise, 5 days per week for 4 weeks. We conducted an intraperitoneal glucose tolerance test on the animals and measured body weight, skeletal muscle mass, and protein degradation and examined the level of autophagy in the isolated extensor digitorum longus, plantaris, and soleus muscles. Body weight and muscle tissue mass were higher in the exercising diabetic rats than in control diabetic rats that remained sedentary. Compared to control rats, exercising diabetic rats had lower blood glucose levels, increased intracellular contractile protein expression, and decreased autophagic protein expression. We conclude that swimming exercise improves muscle mass in diabetes-induced skeletal muscle atrophy, suggesting the activation of autophagy in diabetes contributes to muscle atrophy through hypercatabolic metabolism and that aerobic exercise, by suppressing autophagy, may modify or reverse skeletal muscle wasting in diabetic patients.

Acute gastrointestinal dilation in laboratory rhesus monkeys in the Korea National Primate Research Center / 한국실험동물학회지

Acute gastrointestinal dilation is a medical condition in which the stomach and intestine become overstretched by excessive gas content. In laboratory monkeys, cases of bloating involving gastrointestinal dilation are rarely seen, and the cause thereof is not clearly defined. Two rhesus monkeys in the Korea National Primate Research Center were found to suffer from acute gastrointestinal dilation. One of the monkeys showed severe gastric bloating after recovering from general anesthesia with isoflurane, where after it died suddenly. During necropsy, severe congestion of the lung was observed. The other monkey showed gastrointestinal dilation and died after treatment. During necropsy, severe dilation of the large intestine was observed. Severe congestion was detected in small and large intestines. Histopathologically, erythrocytes were found to fill the alveoli and alveolar capillaries of the lung. In stomach, epithelial cells were found to be sloughed from the mucosal layer, and erythrocytes were found to fill the blood vessels of the submucosal and mucosal layers. In small and large intestines, epithelial cells were also found to be sloughed from the mucosal layer, and inflammatory cells were found to have infiltrated in the submucosa (only large intestine) and mucosa. Microbiologically, Enterococcus faecalis and the pathogenic Staphylococcus haemolyticus, which do not form gas in the gastrointestinal tract, were detected in the gastrointestinal contents of both monkeys. These results suggest that the cause of the acute gastrointestinal dilation in these monkeys was not infection by gas-forming bacteria, but rather multiple factors such as diet, anesthesia, and excessive water consumption.