Effects of isoflurane and xylazine on inducing cerebral ischemia by the model of middle cerebral artery occlusion in mice / 한국실험동물학회지

Preclinical ischemic stroke studies extensively utilize the intraluminal suture method of middle cerebral artery occlusion (MCAo). General anesthesia administration is an essential step for MCAo, but anesthetic agents can lead to adverse effects causing death and making a considerable impact on inducing cerebral ischemia. The purpose of this study was to comparatively assess the effect of isoflurane and xylazine on transient cerebral ischemia in a mouse model of MCAo. Twenty animals were randomly divided into four groups: sham group (no MCAo), control group (MCAo under isoflurane, no agent till reperfusion), isoflurane group (MCAo under isoflurane continued till reperfusion), xylazine group (MCAo under isoflurane, and administration of xylazine till reperfusion). The survival rate, brain infarct volume, and neurologic deficits were studied to assess the effect of isoflurane and xylazine on the stroke model. Our results showed that the body weight showed statistically significant change before and 24 h after surgery in the control and Isoflurane groups, but no difference in the Xylazine group. Also, the survival rate, brain infarct volume, and neurologic deficits were slightly reduced in the isoflurane group at 24 h after reperfusion injury. However, the xylazine and control groups showed similar BIV and neurologic deficits. Interestingly, a high survival rate was observed in the xylazine group. Our results indicate that the modified method of inhalation anesthetics com‑ bined with xylazine can reduce the risk of mortality and develop a reproducible MCAo model with predictable brain ischemia. In addition, extended isoflurane anesthesia after MCAo is associated with the risk of mortality.

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.

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.

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.