Long-Term Alterations in Motor Skills, Neurogenesis and Astrocyte Numbers following Transient Cerebral Ischemia in Mice.

Background and Objectives. Neurogenesis is an integral process in post-stroke recovery, involving the recruitment of proliferating neuroblasts from neurogenic niches of the mammal brain. However, the role of neurogenesis in the long-term restoration following ischemic stroke is fragmented. Post-stroke motor dysfunction includes challenges in the proper, coordinated use of hands and is present in roughly two-thirds of human patients. In this study, we investigated chronic behavioral and biochemical alterations after transient cerebral ischemia in adult male mice. Materials and Methods: Twelve-week-old C57BL/6N male mice were used, and fMCAo lasting 60 min was induced. At multiple timepoints after fMCAo induction, a single pellet reaching task was performed. Six months after the procedure, we immunohistochemically determined the number of proliferating neuroblasts (BrdU and DCX-positive) and the number of differentiated astrocytes (GFAP-positive) in both brain hemispheres. Results: The reaching ability of fMCAo mice was impaired from one month to six months after the induction of ischemia. Neuroblast proliferation was increased in the ipsilateral SVZ, whereas GFAP+ cell count was elevated in the hippocampal DG of both hemispheres of the fMCAo group mice. Conclusions: Our current report demonstrates the long-term effects of transient cerebral ischemia on mice functional parameters and neurogenesis progression. Our data demonstrate that transient cerebral ischemia promotes a long-lasting regenerative response in the ipsilateral brain hemisphere, specifically in the neurogenic SVZ and DG regions.

Focal Cerebral Ischemia Induces Global Subacute Changes in the Number of Neuroblasts and Neurons and the Angiogenic Factor Density in Mice.

Background and Objectives: Dissecting the complex pathological cascade of an ischemic stroke in preclinical models is highly warranted to understand the course of this disease in humans. Neurogenesis and angiogenesis are integral for post-stroke recovery, yet it is not clear how these processes are altered months after an ischemic stroke. In this study, we investigated the changes that take place subacutely after focal cerebral ischemia in experimental adult male mice. Materials and Methods: Male 12-week-old C57BL/6 mice underwent a 60 min long fMCAo or sham surgery. Two months after the procedure, we examined the immunohistochemistry to assess the changes in neuroblast (DCX) and differentiated neuron (NeuN) numbers, as well as the density of the pro-angiogenic factor VEGF. Results: We found decreased neuroblast numbers in both brain hemispheres of the fMCAo mice: by more than 85% in the dentate gyrus and by more than 70% in the subventricular zone. No neuroblasts were found in the contralateral hemisphere of the fMCAO mice or the sham controls, but a small population was detected in the ipsilateral ischemic core of the fMCAo mice. Intriguingly, the number of differentiated neurons in the ipsilateral ischemic core was lower by 20% compared to the contralateral hemisphere. VEGF expression was diminished in both brain hemispheres of the fMCAo mice. Conclusions: Our current report shows that focal cerebral ischemia induces changes in neuroblast numbers and the pro-angiogenic factor VEGF in both cerebral hemispheres 2 months after an fMCAo in mice. Our data show that focal cerebral ischemia induces a long-term regenerative response in both brain hemispheres.

Long-Term Region-Specific Mitochondrial Functionality Changes in Both Cerebral Hemispheres after fMCAo Model of Ischemic Stroke.

Cerebral ischemia/reperfusion (I/R) refers to a secondary brain injury that results in mitochondrial dysfunction of variable extent, leading to neuronal cell damage. The impact of this process has mainly been studied in the short term, from the early hours up to one week after blood flow reperfusion, and in the ischemic hemisphere only. The focus of this study was to assess the long-term impacts of I/R on mitochondrial functionality using high-resolution fluorespirometry to evaluate state-dependent activities in both ischemic (ipsilateral) and non-ischemic (contralateral) hemispheres of male mice 60, 90, 120, and 180 days after I/R caused by 60-min-long filament-induced middle cerebral artery occlusion (fMCAo). Our results indicate that in cortical tissues, succinate-supported oxygen flux (Complex I&II OXPHOS state) and H2O2 production (Complex II LEAK state) were significantly decreased in the fMCAo (stroke) group ipsilateral hemisphere compared to measurements in the contralateral hemisphere 60 and 90 days after stroke. In hippocampal tissues, during the Complex I&II ET state, mitochondrial respiration was generally lower in the ipsilateral compared to the contralateral hemisphere 90 days following stroke. An aging-dependent impact on mitochondria oxygen consumption following I/R injury was observed 180 days after surgery, wherein Complex I&II activities were lowest in both hemispheres. The obtained results highlight the importance of long-term studies in the field of ischemic stroke, particularly when evaluating mitochondrial bioenergetics in specific brain regions within and between separately affected cerebral hemispheres.

An Improved Surgical Approach for Complete Interhemispheric Corpus Callosotomy Combined with Extended Frontoparietal Craniotomy in Mice.

Callosotomy is an invasive method that is used to study the role of interhemispheric functional connectivity in the brain. This surgical approach is technically demanding to perform in small laboratory animals, such as rodents, due to several methodological challenges. To date, there exist two main approaches for transecting the corpus callosum (CC) in rodents: trephine hole(s) or unilateral craniotomy, which cause damage to the cerebral cortex or the injury of large vessels, and may lead to intracranial hemorrhage and animal death. This study presents an improved surgical approach for complete corpus callosotomy in mice using an interhemispheric approach combined with bilateral and extended craniotomy across the midline. This study demonstrated that bilateral and extended craniotomy provided the visual space required for hemisphere and sinus retraction, thus keeping large blood vessels and surrounding brain structures intact under the surgical microscope using standardized surgical instruments. We also emphasized the importance of good post-operative care leading to an increase in overall animal survival following experimentation. This optimized surgical approach avoids extracallosal tissue and medium- to large-sized cerebral blood vessel damage in mice, which can provide higher study reproducibility/validity among animals when revealing the role of the CC in various neurological pathologies.

Long-term behavioural alterations in mice following transient cerebral ischemia.

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Acute and chronic post-stroke changes have variable effects on the functional outcomes of the disease. Therefore, it is imperative to identify what daily activities are altered after stroke and to what extent, keeping in mind that ischemic stroke patients often have long-term post-stroke complications. Translational studies in stroke have also been challenging due to inconsistent study design of animal experiments. The objective of this study was to clarify whether and to what extent mouse behaviour was altered during a 6 months period after cerebral stroke. Experimental stroke was induced in mice by intraluminal filament insertion into the middle cerebral artery (fMCAo). Neurological deficits, recovery rate, motor performance, and circadian activity were evaluated following ischemia. We observed severe neurological deficits, motor impairments, and delay in the recovery rate of mice during the first 14 days after fMCAo. Aberrant circadian activity and distorted space map were seen in fMCAo mice starting one month after ischemia, similarly to altered new and familiar cage activity and sucrose preference using the IntelliCage, and was still evident 60- and 180- days following stroke in the voluntary running wheel using the PhenoMaster system. A preference towards ipsilateral side turns was observed in fMCAo mice both acutely and chronically after the stroke induction. Overall, our study shows the importance of determining time-dependent differences in the long-term post-stroke recovery (over 180 days after fMCAo) using multiple behavioural assessments.

Hemispheric analysis of mitochondrial Complex I and II activity in the mouse model of ischemia-reperfusion-induced injury.

Brain ischemia/reperfusion injury results in a variable mixture of cellular damage, but little is known about possible patterns of mitochondrial dysfunction from the scope of hemispheric processes. The current study used high-resolution fluorespirometry to compare ipsi- and contralateral hemispheres' linked respiration and ROS emission after 60-minutes of filament induced middle cerebral artery occlusion (fMCAo) and 2, 24, 72, and 168 h after reperfusion in mice. Our findings highlight that experimental ischemic stroke resulted in higher mitochondrial respiration in the contralateral compared to the ipsilateral hemisphere and highest ROS emission in ipsilateral hemisphere. The largest difference between the ipsilateral and contralateral hemispheres was observed 2 h after reperfusion in Complex I and II ETS state. Oxygen flux returns to near baseline 72 h after reperfusion without any changes thereafter in Complex I and II respiration. Studying the effects of brain mitochondrial functionality after ischemic stroke in each cerebral hemisphere separately provides a better understanding about the molecular and compensatory processes of the contralateral hemisphere, a region of the brain often neglected in stroke research.

A New Tool for the Analysis of the Effect of Intracerebrally Injected Anti-Amyloid-ß Compounds.

BACKGROUND: A wide range of techniques has been developed over the past decades to characterize amyloid-ß (Aß) pathology in mice. Until now, no method has been established to quantify spatial changes in Aß plaque deposition due to targeted delivery of substances using ALZET® pumps. OBJECTIVE: Development of a methodology to quantify the local distribution of Aß plaques after intracerebral infusion of compounds. METHODS: We have developed a toolbox to quantify Aß plaques in relation to intracerebral injection channels using Zeiss AxioVision® and Microsoft Excel® software. For the proof of concept, intracerebral stereotactic surgery was performed in 50-day-old APP-transgenic mice injected with PBS. At the age of 100 days, brains were collected for immunhistological analysis. RESULTS: The toolbox can be used to analyze and evaluate Aß plaques (number, size, and coverage) in specific brain areas based on their location relative to the point of the injection or the injection channel. The tool provides classification of Aß plaques in pre-defined distance groups using two different approaches. CONCLUSION: This new analytic toolbox facilitates the analysis of long-term continuous intracerebral experimental compound infusions using ALZET® pumps. This method generates reliable data for Aß deposition characterization in relation to the distribution of experimental compounds.

Subchronic administration of auranofin reduced amyloid-ß plaque pathology in a transgenic APPNL-G-F/NL-G-F mouse model.

Alzheimer's disease (AD) is the most common cause of dementia. Neuropathological processes, including the accumulation of amyloid-ß (Aß) plaques and neurofibrillary tangles, and neuroinflammation, lead to cognitive impairment at middle and eventually later stages of AD progression. Over the last decade, focused efforts have explored repurposed drug approaches for AD pathophysiological mechanisms. Recently, auranofin, an anti-inflammatory drug, was shown to have therapeutic potential in a number of diseases in addition to rheumatoid arthritis. Surprisingly, no data regarding the effects of auranofin on cognitive deficits in AD mice or the influence of auranofin on Aß pathology and neuroinflammatory processes are available. In the present study, we used 14-month-old transgenic male APPNL-G-F/NL-G-F mice to assess the effects of subchronic administration of auranofin at low doses (1 and 5 mg/kg, intraperitoneal) on spatial memory, Aß pathology and the expression of cortical and hippocampal proteins (glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule-1 (Iba-1)) and proteins related to synaptic plasticity (glutamic acid decarboxylase 67 (GAD67), homer proteins homologue-1 (Homer-1)). The data demonstrated that auranofin significantly decreased Aß deposition in the hippocampus and the number of Aß plaques in the cingulate cortex, but it did not have memory-enhancing effects or induce changes in the expression of the studied proteins. Our current results highlight the importance of considering further pre-clinical research to investigate the possible beneficial effects of auranofin on the other pathological aspects of AD.

Improved method for cannula fixation for long-term intracerebral brain infusion.

BACKGROUND: Implanted osmotic minipumps are commonly used for long-term, brain-targeted delivery of a wide range of experimental agents by being connected to a catheter and a cannula. During the stereotactical surgery procedure, the cannula has to be placed correctly in the x-y directions and also with respect to the injection point in the z-direction (deepness). However, the flat fixation base of available cannula holders doesn't allow an easy, secure fixation onto the curve-shaped skull. NEW METHOD: We have developed a modified method for a better fixation of the cannula holder by using an easy-to-produce, skull-shaped silicone spacer as fixation adapter. RESULTS: We describe the application and its fast and reliable production in the lab. COMPARISON WITH EXISTING METHOD(S): Superglue or cement is currently being used as the method of choice. However, the curve-shaped skull surface does not fit well with the flat and rigid cannula adapter which leads to fixation problems over time causing wide infusion channels and often also to leakage problems from intracerebrally applied agents towards the surface meninges. As another consequence of the inappropriate fixation, the cannula may loosen from the skull before the end of the experiment or it causes damage to the brain tissue, harming the animals with leading to a failure of the whole experiment. CONCLUSIONS: The easy-to-produce spacer facilitates the crucial step of long-term, stereotactic brain infusion experiments with intracerebral catheters in a highly secure and reproducible way.