Classification of Microglial Morphological Phenotypes Using Machine Learning

Microglia are the brain’s immunocompetent macrophages with a unique feature that allows surveillance of the surrounding microenvironment and subsequent reactions to tissue damage, infection, or homeostatic perturbations. Thereby, microglia’s striking morphological plasticity is one of their prominent characteristics and the categorization of microglial cell function based on morphology is well established. Frequently, automated classification of microglial morphological phenotypes is performed by using quantitative parameters. As this process is typically limited to a few and especially manually chosen criteria, a relevant selection bias may compromise the resulting classifications. In our study, we describe a novel microglial classification method by morphological evaluation using a convolutional neuronal network on the basis of manually selected cells in addition to classical morphological parameters. We focused on four microglial morphologies, ramified, rod-like, activated and amoeboid microglia within the murine hippocampus and cortex. The developed method for the classification was confirmed in a mouse model of ischemic stroke which is already known to result in microglial activation within affected brain regions. In conclusion, our classification of microglial morphological phenotypes using machine learning can serve as a time-saving and objective method for post-mortem characterization of microglial changes in healthy and disease mouse models, and might also represent a useful tool for human brain autopsy samples

Protective microglial activation in Alzheimer’s disease pathogenesis

Here it was of interest to determine the spatiotemporal relationships between Aβ, tau, and microglial pathological changes in post-mortem human AD brains by comparing differentially affected brain regions. Immunohistochemistry and fluorescence immunohistochemistry targeting Aβ, tau, and the pan-microglia marker ionised calcium binding adaptor molecule 1 (Iba1) was performed in four regions of decreasing pathological severity: inferior temporal cortex, superior frontal cortex, primary visual cortex, and primary motor cortex of ten controls, five controls with Alzheimer changes (CAc), and eight AD cases. Following a validated modified disector sampling approach, using manual and corroborative automated methods, the results showed that activated microglia predominated in the inferior temporal cortex of CAc. AD brains were characterised by increased clustering of activated microglia in the primary visual cortex and a substantial loss of clustering and ramified healthy microglia in the inferior temporal cortex. Activated microglia were found to internalise Aβ pathology but not tau pathology. Further, microglia were found to phagocytose greater quantities of pre-synapses in AD compared to both CAc and controls in a study using super-resolution microscopy. Gene amplification studies of a number of candidate genes were performed in coronal neonatal mouse brain slice cultures treated with synthetic preparations of Aβ. Findings demonstrated the upregulation of select phagocytic and anti-inflammatory markers in response to low-dose Aβ monomers. Additionally, a validation amplification study confirmed findings from an RNA-Seq study which demonstrated the upregulation of gene transcripts related to immune pathways and phagocytosis in mildly affected regions of the AD brain. Taken together, these findings are indicative of neuroprotective activation of microglia early in the pathogenesis of AD

Effect of hypertension on the structural and functional integrity of the young and aged brain in an inducible transgenic model

Hypertension has been associated with causing deleterious effects to the cerebrovasculature, which are thought to underlie the formation of white matter lesions (WML) and predispose individuals to age related cognitive decline. In humans hypertension frequently occurs concomitantly with other vascular risk factors making it difficult to ascertain the primary mechanisms of hypertension in isolation. Animal models of hypertension have been used in an aid to establish the mechanisms of hypertension in isolation. To date the knowledge gleaned from animal models has undoubtedly provided an insight as to the role of hypertension and cerebrovasculature remodelling but, these models have limitations such as lack of genetically matched controls and the inability to control the severity of hypertension, restricting the understanding of the underlying mechanisms. All studies within this thesis used the Cyp1a1 Ren2 inducible hypertensive rat model, induced by dietary addition of Indole-3-carbinol (I3C), allowing the severity and duration of hypertension to be tightly controlled and compared to genetically matched controls. This thesis set out to address the hypothesis that sustained hypertension will lead to alterations to the structural integrity of the cerebrovasculature and white matter, which will be exacerbated with age and that hypertension will be associated with alterations to gene expression and cognitive function. Initially this thesis sought to investigate the effect of hypertension on the structural integrity of the vasculature in the Cyp1a1 Ren2 rat model. Firstly, blood pressure in the Cyp1a1 Ren2 rat model was characterised and it was found that the dietary addition of I3C, caused a sustained level of increased blood pressure in all three cohorts. Cerebrovascular alterations were found to consist of increased eNOS expression in the young brain, which progressed with increased duration of hypertension to vascular morphological alterations of decreased vessel width and a redistribution of tight junction protein claudin-5. With age, hypertensive vascular alterations consisted of increased eNOS expression and vascular density. Additionally, there was evidence that hypertension caused a vascular inflammatory response in the young and aged brain. Secondly, this thesis investigated the effect of hypertension on gene expression. Overall it was found that hypertension altered genes related to collagen growth factors, ion channels, eNOS related Map-Kinase pathway and inflammatory genes. Thirdly, this thesis sought to investigate the impact of hypertension on the overall structural integrity of the brain and white matter examining neurons, myelin, oligodendrocytes, axons and microglia, in several regions of the young and aged brain. In general, this study found that hypertension did not cause overt structural or myelin alterations in the majority of regions analysed, with only evidence of myelin alterations occurring within the subcortex of hypertensive animals from each of the young cohorts analysed. However, an adverse subcortical inflammatory response was found in hypertensive animals of the young 6-month cohort and also in hypertensive animals from the aged 4-month cohort, where the inflammatory response was not exclusive to the subcortex of hypertensive animals but also occurred in multiple white matter tracts. Lastly this thesis chose to examine the effect of hypertension on cognitive function, specifically spatial reference and working memory using the Morris water maze and found no evidence of alterations in the cognitive functions examined. Conclusions The results presented within this thesis demonstrated that hypertension in isolation leads to modest alterations to the integrity of the cerebrovasculature and white matter, with no evidence of alterations to specific cognitive functions examined, demonstrating the importance of studying hypertension in isolation. Additionally, this study highlights the initial hypertensive induced alterations to the cerebrovasculature, such as endothelial signalling, vascular structure and inflammation, providing a window for therapeutic intervention at a time point when there are minimal alterations to the overall structural integrity of the brain. Future studies in this model should concentrate on examining different severities of hypertension and also hypertension concomitantly with other vascular risk factors to try and recapitulate pathological alterations found in humans

Rapid prototyping of soft bioelectronic implants for use as neuromuscular interfaces

Neuromuscular interfaces are required to translate bioelectronic technologies for application in clinical medicine. Here, by leveraging the robotically controlled ink-jet deposition of low-viscosity conductive inks, extrusion of insulating silicone pastes and in situ activation of electrode surfaces via cold-air plasma, we show that soft biocompatible materials can be rapidly printed for the on-demand prototyping of customized electrode arrays well adjusted to specific anatomical environments, functions and experimental models. We also show, with the monitoring and activation of neuronal pathways in the brain, spinal cord and neuromuscular system of cats, rats and zebrafish, that the printed bioelectronic interfaces allow for long-term integration and functional stability. This technology might enable personalized bioelectronics for neuroprosthetic applications

Regulation of microglia polarization after cerebral ischemia

Stroke ranks second as a leading cause of death and permanent disability globally. Microglia, innate immune cells in the brain, respond rapidly to ischemic injury, triggering a robust and persistent neuroinflammatory reaction throughout the disease’s progression. Neuroinflammation plays a critical role in the mechanism of secondary injury in ischemic stroke and is a significant controllable factor. Microglia activation takes on two general phenotypes: the pro-inflammatory M1 type and the anti-inflammatory M2 type, although the reality is more complex. The regulation of microglia phenotype is crucial to controlling the neuroinflammatory response. This review summarized the key molecules and mechanisms of microglia polarization, function, and phenotypic transformation following cerebral ischemia, with a focus on the influence of autophagy on microglia polarization. The goal is to provide a reference for the development of new targets for the treatment for ischemic stroke treatment based on the regulation of microglia polarization

A Themed Issue in Honor of Professor Raphael Mechoulam: The Father of Cannabinoid and Endocannabinoid Research

During the last 60 years the relevance of cannabis (Cannabis sativa or Cannabis indica) ingredients, like the psychoactive Δ9-tetrahydrocannabinol (THC), cannabidiol, 120+ additional cannabinoids and 440+ non-cannabinoid compounds, for human health and disease has become apparent. Approximately 30 years after the elucidation of THC structure the molecular reasons for the biological activity of these plant extracts were made clearer by the discovery of endocannabinoids, that are endogenous lipids able to bind to the same receptors activated by THC. Besides endocannabinoids, that include several N-acylethanolamines and acylesters, a complex array of receptors, metabolic enzymes, transporters (transmembrane, intracellular and extracellular carriers) were also discovered, and altogether they form a so-called “endocannabinoid system” that has been shown to finely tune the manifold biological activities of these lipid signals. Both plant-derived cannabinoids and endocannabinoids were first discovered by the group led by Prof. Dr. Raphael Mechoulam, who has just celebrated his 90th birthday and clearly stood out as a giant of modern science. The many implications of his seminal work for chemistry, biochemistry, biology, pharmacology and medicine are described in this special issue by the scientists who reached during the last 20 years the highest recognition in the field of (endo)cannabinoid research, receiving the Mechoulam Award for their major contributions. I thank them for having accepted my invitation to be part of this honorary issue of Molecules, and Raphi for continuing to illuminate our field with his always inspiring investigations and new ideas

Evaluation of a novel approach to promoting post-ischemic recovery of upper extremity function in the rat

Following stroke, impairments in arm function are common motor deficits in survivors, affecting thousands of people each year. A useful technique used in clinical rehabilitation of patients with arm impairments is to force use of the impaired arm through constraint of the unaffected (or less affected) one. The success of this ‗constraint induced movement therapy‘ (CIMT) is believed to be due to neuroplastic changes that take place on a cellular level in surviving brain tissue, however, little is understood about the mechanisms involved. Appropriate animal models are necessary to study how rehabilitation affects neuroplasticity. Previous literature has described several models of forced use following stroke in rats which have resulted in varying success. Animal stress and lack of behavioural pressure may have contributed to the inconsistency of prior forced use models. The purpose of the research presented in this thesis was to optimize a surgical model of post-ischemic upper extremity impairment, determine whether it would be possible to force use of the impaired forelimb using a novel appetitively motivated protocol, and then to investigate the effects of this novel model on markers of neuroplasticity. First, the endothelin-1 (ET-1) model of focal unilateral ischemia was optimized by attempting a previously unpublished protocol of injections along the motor cortex and to the striatum. Male Sprague Dawley rats were subjected to ET-1 or sham surgery. Ensuing forelimb functional deficits were measured using a battery of behavioural tests, which were compared to intact sham surgical control performance. The stroke model resulted in reliable and reproducible lesions to forelimb motor regions of the brain, and deficits that lasted up to the end of the 21 day study period in some tests. Next, this ET-1 stroke surgery was used to evaluate a novel form of forced use rehabilitation in which rats engaged the impaired limb to move voluntarily in commercial pet activity balls. Animals were subjected to ET-1 or sham surgery, and then received either rehabilitation or a control treatment. Behavioural tests revealed that animals receiving rehabilitation recovered to sham levels of performance sooner than animals receiving the control treatment. Stroke, but not rehabilitation, affected the proportion of cells expressing brain derived neurotrophic factor (BDNF) and the presence of doublecortin-positive neuroblasts, but had no effect on the expression of the growth inhibiting protein NOGOA. Finally, the novel forced use model was developed further to more closely resemble clinical CIMT with the addition of a task-specific reaching component. Animals were subjected to either ET-1 followed by rehabilitation, ET-1 followed by a control treatment, or sham surgery. Again, behavioural tests revealed that animals that had undergone ET-1 surgery had significant deficits that recovered sooner in the group that received rehabilitation. Rehabilitation did not affect the proportion of BDNF-expressing cells, but did appear to cause a shift in the cellular origin of the BDNF that was present. Further, rehabilitation resulted in more doublecortin-positive cells in the damaged hemisphere. This novel approach to rehabilitation represents a useful model of forced use therapy which results in accelerated functional recovery following ischemic injury. The mechanisms underlying this effect may be related to changes in BDNF expression and increased generation or survival of new born cells