MORPHIOUS: an unsupervised machine learning workflow to detect the activation of microglia and astrocytes.

BACKGROUND: In conditions of brain injury and degeneration, defining microglial and astrocytic activation using cellular markers alone remains a challenging task. We developed the MORPHIOUS software package, an unsupervised machine learning workflow which can learn the morphologies of non-activated astrocytes and microglia, and from this information, infer clusters of microglial and astrocytic activation in brain tissue. METHODS: MORPHIOUS combines a one-class support vector machine with the density-based spatial clustering of applications with noise (DBSCAN) algorithm to identify clusters of microglial and astrocytic activation. Here, activation was triggered by permeabilizing the blood-brain barrier (BBB) in the mouse hippocampus using focused ultrasound (FUS). At 7 day post-treatment, MORPHIOUS was applied to evaluate microglial and astrocytic activation in histological tissue. MORPHIOUS was further evaluated on hippocampal sections of TgCRND8 mice, a model of amyloidosis that is prone to microglial and astrocytic activation. RESULTS: MORPHIOUS defined two classes of microglia, termed focal and proximal, that are spatially adjacent to the activating stimulus. Focal and proximal microglia demonstrated activity-associated features, including increased levels of ionized calcium-binding adapter molecule 1 expression, enlarged soma size, and deramification. MORPHIOUS further identified clusters of astrocytes characterized by activity-related changes in glial fibrillary acidic protein expression and branching. To validate these classifications following FUS, co-localization with activation markers were assessed. Focal and proximal microglia co-localized with the transforming growth factor beta 1, while proximal astrocytes co-localized with Nestin. In TgCRND8 mice, microglial and astrocytic activation clusters were found to correlate with amyloid-ß plaque load. Thus, by only referencing control microglial and astrocytic morphologies, MORPHIOUS identified regions of interest corresponding to microglial and astrocytic activation. CONCLUSIONS: Overall, our algorithm is a reliable and sensitive method for characterizing microglial and astrocytic activation following FUS-induced BBB permeability and in animal models of neurodegeneration.

The human brain endothelial barrier: transcytosis of AAV9, transduction by AAV2: An Editorial Highlight for 'Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells'.

Read the highlighted article 'Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells' on page 216.

A Blinded Comparison of Three Generative Artificial Intelligence Chatbots for Orthopaedic Surgery Therapeutic Questions.

Objective To compare the quality of responses from three chatbots (ChatGPT, Bing Chat, and AskOE) across various orthopaedic surgery therapeutic treatment questions. Design We identified a series of treatment-related questions across a range of subspecialties in orthopaedic surgery. Questions were "identically" entered into one of three chatbots (ChatGPT, Bing Chat, and AskOE) and reviewed using a standardized rubric. Participants Orthopaedic surgery experts associated with McMaster University and the University of Toronto blindly reviewed all responses. Outcomes The primary outcomes were scores on a five-item assessment tool assessing clinical correctness, clinical completeness, safety, usefulness, and references. The secondary outcome was the reviewers' preferred response for each question. We performed a mixed effects logistic regression to identify factors associated with selecting a preferred chatbot. Results Across all questions and answers, AskOE was preferred by reviewers to a significantly greater extent than both ChatGPT (P<0.001) and Bing (P<0.001). AskOE also received significantly higher total evaluation scores than both ChatGPT (P<0.001) and Bing (P<0.001). Further regression analysis showed that clinical correctness, clinical completeness, usefulness, and references were significantly associated with a preference for AskOE. Across all responses, there were four considered as having major errors in response, with three occurring with ChatGPT and one occurring with AskOE. Conclusions Reviewers significantly preferred AskOE over ChatGPT and Bing Chat across a variety of variables in orthopaedic therapy questions. This technology has important implications in a healthcare setting as it provides access to trustworthy answers in orthopaedic surgery.

Transgene distribution and immune response after ultrasound delivery of rAAV9 and PHP.B to the brain in a mouse model of amyloidosis.

Efficient disease-modifying treatments for Alzheimer disease, the most common form of dementia, have yet to be established. Gene therapy has the potential to provide the long-term production of therapeutic in the brain following a single administration. However, the blood-brain barrier poses a challenge for gene delivery to the adult brain. We investigated the transduction efficiency and immunological response following non-invasive gene-delivery strategies to the brain of a mouse model of amyloidosis. Two emerging technologies enabling gene delivery across the blood-brain barrier were used to establish the minimal vector dosage required to reach the brain: (1) focused ultrasound combined with intravenous microbubbles, which increases the permeability of the blood-brain barrier at targeted sites and (2) the recombinant adeno-associated virus (rAAV)-based capsid named rAAV-PHP.B. We found that equal intravenous dosages of rAAV9 combined with focused ultrasound, or rAAV-PHP.B, were required for brain gene delivery. In contrast to rAAV9, focused ultrasound did not decrease the rAAV-PHP.B dosage required to transduce brain cells in a mouse model of amyloidosis. The non-invasive rAAV delivery to the brain using rAAV-PHP.B or rAAV9 with focused ultrasound triggered an immune reaction including major histocompatibility complex class II expression, complement system and microglial activation, and T cell infiltration.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease.

Gene therapy can be designed to efficiently counter pathological features characteristic of neurodegenerative disorders. Here, we took advantage of the glial fibrillary acidic protein (GFAP) promoter to preferentially enhance transgene expression near plaques composed of amyloid-beta peptides (Aß), a hallmark of Alzheimer's disease (AD), in the TgCRND8 mouse model of amyloidosis. Methods: The delivery of intravenously injected recombinant adeno-associated virus mosaic serotype 1/2 (rAAV1/2) to the cortex and hippocampus of TgCRND8 mice was facilitated using transcranial MRI-guided focused ultrasound in combination with microbubbles (MRIgFUS), which transiently and locally increases the permeability of the blood-brain barrier (BBB). rAAV1/2 expression of the reporter green fluorescent protein (GFP) under a GFAP promoter was compared to GFP expression driven by the constitutive human beta actin (HBA) promoter. Results: MRIgFUS targeting the cortex and hippocampus facilitated the entry of rAAV1/2 and GFP expression under the GFAP promoter was localized to GFAP-positive astrocytes. Adjacent to Aß plaques where GFAP is upregulated, the volume, surface area, and fluorescence intensity of the transgene GFP were greater in rAAV1/2-GFAP-GFP compared to rAAV1/2-HBA-GFP treated animals. In peripheral organs, GFP expression was particularly strong in the liver, irrespective of the promoter. Conclusion: The GFAP promoter enhanced transgene expression in proximity of Aß plaques in the brain of TgCRND8 mice, and it also resulted in significant expression in the liver. Future gene therapies for neurological disorders could benefit from using a GFAP promoter to regulate transgene expression in response to disease-induced astrocytic reactivity.