Biology subjects

Garrood, M.

Publications and source records attributed to Garrood, M..

5 recordsLinked to original sources

bioRxiv2026

Comparative effects of perfusate composition on rat brain histology following transcardial perfusion fixation

Background: Transcardial perfusion fixation is widely used to preserve rodent brains for histological and ultrastructural analysis, but protocols can vary in the use of pre-fixation washout, fixative formulation, and osmotic additives. Relatively few studies have directly compared how the chemical composition of the perfusate affects tissue preservation. Methods: Male Sprague-Dawley rats underwent transcardial perfusion using a series of aldehyde-based fixative formulations that varied in fixative composition, use of a phosphate buffered saline washout, and the addition of mannitol or polyethylene glycol 35 kDa (PEG35). Perfusion outcomes were assessed using gross brain morphology, semi-quantitative grading of vascular blood clearance, quantitative detection of residual erythrocytes in whole-slide histological images, and light microscopic measures of cellular visualization and morphology. Perfusate osmolality was measured, and selected specimens were examined by electron microscopy. Results: Substantial vascular blood clearance was frequently achieved with fixative-only perfusion, indicating that a pre-fixation washout was not required to achieve high levels of blood clearance under the conditions tested. Routine light microscopy-based measures of cellular visibility and morphology were broadly similar across treatment groups. Adding mannitol and PEG35 to the fixative solution both led to concentration-dependent gross tissue shrinkage, particularly with PEG35, without correspondingly large or consistent changes on light microscopy. A substantial degree of case-to-case variation in perfusion quality was also observed among animals undergoing nominally similar procedures. Conclusions: Substantial variation in perfusate composition produced relatively modest differences in the light microscopy outcomes examined, despite pronounced effects of osmotic additives on gross brain morphology. There were some cases where perfusion with fixative alone led to near-complete blood vessel clearance, and the use of a preceding phosphate buffered saline washout was not associated with higher vascular clearance scores. The degree of case-to-case variability observed within treatment conditions suggests that procedural factors can play an important role on perfusion quality in addition to perfusate composition.

neuroscience↗

bioRxiv2026

Perfusion quality does not necessarily predict ultrastructural preservation after hyperosmotic brain perfusion

Perfusion fixation is widely used in neuroscience to prepare mammalian brain tissue for histological and ultrastructural analysis. Perfusion protocols are commonly assessed using macroscopic indicators such as gross appearance and neuroimaging, which assess the extent to which perfusate has been distributed throughout the brain. There is a critical need to determine to what extent these metrics can accurately predict high-quality ultrastructural preservation, particularly as new perfusion protocols are developed for connectomics. In this technical report, we describe evidence that these two measures can be decoupled by the addition of dehydrating agents to the perfusate solution. In three human brain donors and one canine brain donor perfused with a fixative solution containing 10% mannitol and 10% polyethylene glycol 35 kDa, macroscopic and radiological indicators of perfusion quality appeared adequate or favorable. However, electron microscopy revealed expanded extracellular space, shrunken cellular processes, and distorted cell membranes, consistent with an osmotic shock artifact resulting from severe hyperosmotic dehydration. Similar ultrastructural artifacts were observed in a canine brain donor perfused with 20% mannitol in 20% neutral buffered formalin without PEG. We compare these ultrastructural findings with findings from previously reported cases perfused with standard neutral buffered formalin without osmotic additives. These findings illustrate a risk of optimizing brain perfusion protocols designed to preserve neural circuitry based on macroscopic or radiological perfusion quality metrics alone, since these metrics can be satisfied while the ultrastructure is severely compromised.

neuroscience↗

bioRxiv2026

A canine brain bank for comparative neuroscience and brain aging research

Companion animal brain banking has been recognized as a valuable approach for translational aging and dementia research. However, realizing the full value of canine brain banks depends on optimizing the methods that are used to collect and preserve the tissue. Whole brain perfusion fixation is one promising approach, but it is not yet well described in dogs. Here we describe the development of methods for a canine brain bank (currently n = 55), including whole brain perfusion fixation via aortic cannulation and brain extraction. We assessed perfusion quality using gross examination, post-perfusion CT, and histological clearance of blood vessels. We found that body weight and average flow rate per body weight were each significantly correlated with perfusion quality in our cohort. To illustrate the kind of analysis the bank could facilitate, we next performed a preliminary study of brain aging, one of our primary planned research applications. Using a pixel classifier applied to whole slide images, we quantified lipofuscin burden, and in this preliminary cohort found that it increased strongly with age in both the thalamus and hippocampus. In the hippocampus, lipofuscin burden was also elevated in dogs with owner-reported cognitive dysfunction, although the current cohort is too small to determine to what extent this association is independent of age. Preliminary electron microscopy studies also confirmed that perfusion fixed tissue from the bank is amenable to ultrastructural analysis. This work describes one approach for canine brain perfusion fixation and introduces a brain tissue resource that may help support future neuroscience research.

neuroscience↗

bioRxiv2026

Aldehyde-based cryopreservation of whole brains

Long-term storage of aldehyde-fixed brain tissue is commonly performed in the fluid state. This has the potential to maintain morphology for many decades, but has been found to cause progressive loss of antigenicity over time for some biomolecules. While cryoprotection and subzero storage has been successfully used for brain tissue sections or blocks, methods for preserving whole brains using this approach have not been widely characterized. Here we present a protocol for the preservation of fixed whole brains using graded immersion cryoprotection and subzero temperature storage, which is one type of a more general approach that we refer to as aldehyde-based cryopreservation (ABC). Our method uses a gradual ramp-up of the osmotic concentration of cryoprotectants, leading to a final solution containing 50% (v/v) ethylene glycol and 30% (w/v) sucrose. We used CT imaging to track cryoprotectant penetration, finding that with the use of our protocol, approximately 10 months is required to reach equilibration throughout whole human brains. In our initial histological validation, we found that insufficient equilibration time prior to freezing led to apparent ice crystal artifacts seen on ultrastructural imaging of the white matter. After refining the protocol to allow adequate diffusion time, histologic data at both the light and electron microscopic levels showed preserved cellular architecture and ultrastructure after the process of cryoprotectant loading, freezer storage, and unloading. This protocol can be implemented using laboratory freezers or freezer rooms and provides a degree of resilience against freezer failures because the morphology of the fixed tissue is expected to remain preserved long-term in the fluid state even if rewarmed. Our approach may be valuable for laboratories seeking to enhance the long-term preservation of antigenicity in large brain tissue specimens for future research applications.

neuroscience↗

bioRxiv2025

Evaluating ultrastructural preservation quality in banked brain tissue

The ultrastructural analysis of postmortem brain tissue can provide important insights into cellular architecture and disease-related changes. For example, connectomics studies offer a powerful emerging approach for understanding neural circuit organization. However, electron microscopy (EM) data is difficult to interpret when the preservation quality is imperfect, which is common in brain banking and may render it unsuitable for certain research applications. One common issue is that EM images of postmortem brain tissue can have an expansion of regions that appear to be made up of extracellular space and/or degraded cellular material, which we call ambiguous interstitial zones. In this study, we report a method to assess whether EM images have ambiguous interstitial zone artifacts in a cohort of 10 postmortem brains with samples from each of the cortex and thalamus. Next, in matched samples from the contralateral hemisphere of the same brains, we evaluate the structural preservation quality of light microscopy images, including immunostaining for cytoskeletal proteins. Through this analysis, we show that on light microscopy, cell membrane morphology can be largely maintained, and neurite trajectory visualized over micrometer distances, even in specimens for which there are ambiguous interstitial zone artifacts on EM. Taken together, our analysis may assist in maximizing the usefulness of donated brain tissue by informing tissue selection and preparation protocols for various research goals.

neuroscience↗