Methods for active neuronal tracing in postmortem mammalian brains

Abstract

In one aspect, the invention provides a method for active neuronal tracing in postmortem mammalian brain, wherein the method comprises perfusing a mammalian brain with a perfusate using a perfusion device; introducing a tracer into the mammalian brain while the mammalian brain is being perfused with the perfusate; and monitoring movement of the tracer in the mammalian brain.

Description

[0001] Atorney Docket No. 047162-7357W01(02701)

[0002] METHODS FOR ACTIVE NEURONAL TRACING IN POSTMORTEM

[0003] MAMMALIAN BRAINS

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] The present application is entitled to priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 694,575, filed September 13, 2024, which is hereby incorporated by reference herein in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under MH 117064 awarded by the National Institute of Health. The government has certain rights in the invention.

[0008] SEQUENCE LISTING

[0009] This application is being filed electronically via Patent Center and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled ’‘047162-7357WOl .xml”, created on August 27, 2025 and having a size of 7,656 bytes. The sequence listing contained in this .xml file is part of the specification and is herein incorporated by reference in its entirety.

[0010] BACKGROUND OF THE INVENTION

[0011] The mammalian central nervous system (CNS) is arguably the most complex biological structure where diverse neuronal populations are highly interconnected to deliver information important for sensory, motor, emotional and cognitive functions. This sharing of information is mediated by structural connections, collectively known as the connectome. The brain connectome has been investigated at different levels of granularity: macroconnections between gray matter regions, mesoconnections between neurons, and microconnections at synapses.

[0012] Despite the achievement of the mesoscopic brain mapping technologies in experimental animals, the knowledge of human brain connectivity remains incomplete. In humans and other large-brained mammals, the diversity of neural cell types and neuronal wiring is immensely complex. These large brains have independently evolved numerous times across different taxonomic groups, implying that their cellular compositions and wiring patterns may exhibit more variation than those within the same group. For instance, the Atorney Docket No. 047162-7357W01(02701) human CNS alone consists of approximately 86 billion neurons and an equal number of nonneuronal cells. This vast network integrates an estimated 600 trillion synapses involved in 850 thousand kilometers of axons and dendrites, and an estimated several hundred thousands of distinct long-range neural pathways. However, the existing mesoscopic tracing methodologies, originally developed and predominantly utilized in rodents, present certain limitations when applying in large mammals due to their reliance on invasive surgical procedures including craniectomy and survival incubation. These procedures are necessary because the tracing approaches rely on the active metabolism of living cells, enabling endocytosis for the uptake of active vectors and subsequent transport / viral expression. However, these invasive procedures are not directly applicable to humans, creating a barrier to comparable information gathering in the human brain.

[0013] SUMMARY

[0014] In one aspect, provided herein is a method comprising: a. perfusing a mammalian brain with a perfusate using a perfusion device; b. introducing a tracer into the mammalian brain while the mammalian brain is being perfused with the perfusate; and c. monitoring movement of the tracer in the mammalian brain.

[0015] In certain embodiments , the method comprises, based on the monitoring, mapping the mammalian brain.

[0016] In certain embodiments, the mapping of the mammalian brain is a mesoscale connectivity mapping of the mammalian brain.

[0017] In certain embodiments, based on the monitoring, the method comprises examining long-range neuronal connections of the mammalian brain.

[0018] In certain embodiments, the tracer comprises at least one anterograde tracer and at least one retrograde tracer.

[0019] In certain embodiments, the at least one anterograde tracer comprises BDA10K, and the at least one retrograde tracer comprises BDA3K.

[0020] In certain embodiments, the tracer compnses at least one chemical tracer.

[0021] In certain embodiments, the at least one chemical tracer comprises one or more chemical tracers selected from the group consisting of: Fluorescein-conjugated dextran 10k, FluoroGold, CTb, Tetramethylrhodamine-conjugated dextran 10k, Fluorescein-conjugated dextran 3k. and combinations thereof.

[0022] In certain embodiments, the tracer comprises at least one viral tracer. In certain Atorney Docket No. 047162-7357W01(02701) embodiments, the at least one viral tracer comprises one or more viruses selected from the group consisting of: Sindbis virus. Lentivirus. Adeno-associated virus 2 (AAV2), Adeno- associated virus 9 (AAV9), and combinations thereof. In certain embodiments, wherein the at least one viral tracer comprises recombinant Sindbis virus.

[0023] In certain embodiments, the at least one viral tracer carries green fluorescent protein (GFP). In certain embodiments, the method comprises visualizing GFP RNA signals using RNAscope.

[0024] In certain embodiments, the tracer is injected into at least one of a primary motor cortex of the mammalian brain or a dorsal medial prefrontal cortex of the mammalian brain.

[0025] In certain embodiments, the mammalian brain is perfused for at least 6 hours. In certain embodiments, the mammalian brain is perfused for 6-24 hours. In certain embodiments, the mammalian brain is perfused for 12-24 hours. In certain embodiments, the mammalian brain is perfused for 18-24 hours. In certain embodiments, the method comprises removing the mammalian brain from a body of a mammal and connecting the mammalian brain to the perfusion device.

[0026] In certain embodiments, the method comprises connecting the mammalian brain to the perfusing device comprises connecting an artery of the mammalian brain to a fluid line of the perfusion device. In certain embodiments, connecting the mammalian brain to the perfusing device comprises connecting ascending pharyngeal arteries of the mammalian brain to a fluid line of the perfusion device.

[0027] In certain embodiments, the method comprises placing the mammalian brain in a brain chamber of the perfusion device. In certain embodiments, the method further comprises warming the mammalian brain to about 36 degrees Celsius. In certain embodiments, the perfusate is an acellular and cytoprotective perfusate. In certain embodiments, the perfusate is formulated to inhibit neuronal excitability in the mammalian brain.

[0028] In certain embodiments, the method comprises monitoring arteriovenous gradients of glucose consumption and maintaining a stable glucose level. In certain embodiments, the arteriovenous gradients of glucose consumption are monitored hourly. In certain embodiments, the stable glucose level is maintained by introducing glucose to the mammalian brain via the perfusate. In certain embodiments, the method further comprises injecting vessel labeling dye through arteries of the mammalian brain at an end of the perfusion.

[0029] In certain embodiments, the method further comprises injecting Evans bl ue-al bumin Atorney Docket No. 047162-7357W01(02701) complex into the mammalian brain. In certain embodiments, the Evans blue-albumin complex is injected intra-arterially into the mammalian brain. In certain embodiments, the perfusion of the mammalian brain with the perfusate preserves a blood brain barrier provided by the mammalian brain.

[0030] In certain embodiments, the method further comprises monitoring electrical activity' via electrodes and electrocorticography (ECoG) from a dorsal cerebral cortex of the mammalian brain.

[0031] In certain embodiments, the method further comprises monitoring hemodynamics, dissolved gasses, and electrolyte levels.

[0032] In certain embodiments, the method further comprises administering one or more metabolic supplements into the perfusate solution. In certain embodiments, the one or more metabolic supplements comprise one or more members selected from the group consisting of glucose, adenosine, amino acids, vitamins, and combinations thereof.

[0033] In certain embodiments, the method further comprises injecting a dye into the mammalian brain via a fluid line of the perfusion device. In certain embodiments, the dye is Evans blue dye. In certain embodiments, the method comprises perfusing the mammalian brain with albumin after injecting the dye into the mammalian brain to remove the dye from vessels in the mammalian brain. In certain embodiments, the method further comprises sectioning the mammalian brain. In certain embodiments, the method further comprises imaging sections of the mammalian brain. In certain embodiments, the method further comprises processing samples taken from the mammalian brain.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] For the purpose of illustrating the invention, there are depicted in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

[0036] FIGS. 1 A-1F illustrate a long-term perfusion. FIG. 1A illustrates a perfusion experimental workflow. FIG. IB shows a representative perfusion flow and FIG. 1C shows brain resistance in the duration of long- term perfusion. FIG. ID shows MEA recording of the postmortem hippocampal slices treated with perfusate and in control recording solution. FIG. IE shows EPSP amplitude change between perfusate and in control recording solution. FIG. IF shows ECoG recording traces of perfused brain; representative isoelectric signals from the designated surface electrodes are shown to the right. Atorney Docket No. 047162-7357W01(02701)

[0037] FIGS. 2A-2F show cellular uptake and intracellular transport of chemical tracers. FIG. 2A is a schematic illustration of the injection site in the pig primary motor cortex. FIG. 2B is a representative neuronal uptake of conventional chemical neuroanatomic tracers (BDAlOk, CTb, and Fluogold). FIG. 2C shows cell labeling with BDA 1 Ok. FIG. 2D is a bar graph showing travel velocity' of anterograde and retrograde tracer transport (mm / h). n = 3 brains. Mean ± s.e.m. FIG. 2E is a representation of anterogradely labeled fibers and FIG. 2F is a representation of retrogradely labeled neurons.

[0038] FIGS. 3A-3C are images showing retrograde tracing in the brain stem with perfusion. Representative images of cranial nerves labeling with BDA 3K (either delivered alone or codelivered using different colors (fluorophores)) for hypoglossal nerves / nuclei are shown FIG. 3 A. facial nerves are shown in FIG. 3B, and abducens nerves / nuclei are shown in FIG. 3C. The images on the right depict enlargement of the boxed areas.

[0039] FIGS. 4A-4F depict mapping projections using MAPseq. FIG. 4A shows Sindbis viral labeling of neurons in postmortem porcine brain with perfusion. FIG. 4B is an overview of MAPseq workflow. Six target areas were chosen for analysis: ACC, dlPFC, vmPFC, c- dmPFC, c-ACC and c- dlPFC. FIG. 4C shows projection matrix in the six target areas, as well as the olfactory bulb (OB) as a negative control of 338 neurons mapped using MAPseq. FIG. 4D shows number of cells analyzed from each brain. FIG. 4E shows the percentage of neurons projected to each target. FIG. 4F shows a number of projection targets of dmPFC neurons when considering one target area only.

[0040] FIGS. 5A-5D are images showing restoration of cerebral microcirculation during the perfusion. Representative images of labeled vasculature by lectin during the perfusion in the prefrontal cortex are shown in FIG. 5A, motor cortex are shown in FIG. 5B, hippocampus are shown in FIG. 5C and cerebellum are shown in FIG. 5D.

[0041] FIGS. 6A-6D are images showing preservation of the cytoarchitectural integrity and are representative images of neurons (NeuN, Nissl), astrocytes (GFAP), microglia (IBA1), and oligodendrocytes (MBP) in the prefrontal cortex (FIG. 6A), motor cortex (FIG. 6B), hippocampus (FIG. 6C) and cerebellum (FIG. 6D).

[0042] FIGS. 7A-7G show preservation of the blood-brain-barrier. FIGS. 7A-7C show representative images of the infiltration of EB- Al bumin complex in different brain regions (from anterior to posterior) in the wide field (top) and under fluorescence (bottom Quantitation of the infiltrated EB-Albumin complex into the brain tissue in cortex is show n in FIG. 7D, into subcortical regions is shown in FIG. 7E, in to brain stem is shown in FIG. 7F, and in to cerebellum si shown in FIG. 7G. Atorney Docket No. 047162-7357W01(02701)

[0043] FIGS. 8A-8D show neuronal labeling of different viral vectors. FIG. 8 A shows representative images of the Sinbis vims labeled neurons with preserved endogenous RNA expression in brain cells using layer specific marker Cux2 and Fezf2. Representative images of neurons labeled by Lentivirus, AAV2 and AAV9 are shown in FIGS. 8B, 8C, and 8D, respectively.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] Mapping of the brain connectivity is fundamental for unraveling the complexities of the nervous system and its function. Various approaches have been developed and advanced to achieve brain-wide, cellular-level connectivity mapping for rodents. However, their application to larger mammals and humans is hindered by invasive procedures, limiting the understanding of connectivity in the larger brain. Reported herein is active neuronal tracing in isolated pig brains hours after death, providing a novel approach for mapping brain connectome at mesoscale in postmortem large mammals. Leveraging previously developed BrainEx technology, a pulsatile perfusion system was developed to sustain prolonged cell viability and blood-brain barrier (BBB) integrity in the postmortem pig brain under ex vivo conditions. Extending normothermic perfusion from 6 to 24 hours, a robust cellular uptake of chemical neuroanatomic tracers and their active intracellular transport in major brain regions is demonstrated. Significantly, provided herein is the evidence that viruses could infect neurons in ex vivo postmortem brain and express viral genetic payloads, suggesting the possibility of cell-type-specific targeting and high throughput brain-wide connectivity sequencing. These findings highlight the untapped potential of postmortem large mammalian brains for high-resolution mapping when cell viability is preserved. This proof- of-concept study also provides a translatable approach that could be adapted for human’s or any other mammal’s brain connectivity mapping.

[0046] Definitions

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described. As used herein, each of the following terms has the meaning associated with it in this section. Atorney Docket No. 047162-7357W01(02701)

[0048] As used herein, the singular form “a.” “an.” and “the” include plural references unless the context clearly dictates otherwise.

[0049] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%. 1%, 0.5%, 0.1%. 0.05%. or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0050] As used in the specification and claims, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U.S. patent law and can mean “includes,” “including,” and the like.

[0051] Unless specifically stated or obvious from context, the term “or,” as used herein, is understood to be inclusive.

[0052] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2. 3, 4, 5, 6, 7, 8, 9. 10. 11, 12, 13, 14, 15, 16. 17. 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29. 30. 31. 32. 33. 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).

[0053] Abbreviations:

[0054] MEA: multi-electrode arrays

[0055] EPSP: excitatory post synaptic potentials

[0056] ACC: anterior cingulate cortex dlPFC-: dorsolateral PFC vmPFC: ventromedial prefrontal cortex c-dmPFC: dorsomedial prefrontal cortex c-ACC: contralateral anterior cingulate cortex c- dlPFC: dorsal lateral prefrontal cortex, and ECoG: Electrocorticography

[0057] Methods

[0058] In one aspect, provided herein is a method comprising perfusing a brain with a perfusate using a perfusion device; introducing a tracer into the brain while the brain is being perfused with the perfusate; and monitoring movement of the tracer in the brain. In certain embodiments, the brain is a mammalian brain. Atorney Docket No. 047162-7357W01(02701)

[0059] In certain embodiments, based on the monitoring, the method further comprises mapping the brain. In certain embodiments, the mapping is a mesoscale connectivity mapping of the brain.

[0060] In certain embodiments, based on the mapping, the method further comprises examining long-range neuronal connections of the brain. In certain embodiments, the step of examining is performed using optical and / or genomic methods.

[0061] In certain embodiments, the tracer comprises at least one anterograde tracer and / or at least one retrograde tracer. In certain embodiments, the at least one anterograde tracer comprises, for example, BDA10K, and the at least one retrograde tracer comprises, for example, BDA3K. In certain other embodiments, the tracer is at least one selected from the group consisting of biotinylated dextran amine (BDA), phaseolus vulgaris leucoagglutinin (PHAL), cholera toxin subunit B (CTB), Dil, fluorogold, and horseradish peroxidase (HRP).

[0062] In certain embodiments, the tracer comprises at least one chemical tracer. Nonlimiting examples of the chemical tracers include, for example, Fluorescein-conjugated dextran 10k, FluoroGold, CTb, Tetramethylrhodamine-conjugated dextran 10k, Fluorescein- conjugated dextran 3k. and combinations thereof. In certain embodiments, non-limiting examples of the at least one viral tracer include, for example, viruses selected from the group consisting of: Sindbis vims, Lentivirus, Adeno-associated virus 2 (AAV2), Adeno-associated virus 9 (AAV9), and combinations thereof. In certain embodiments, the at least one viral tracer comprises recombinant Sindbis virus. In certain embodiments, the at least one viral tracer carries green fluorescent protein (GFP).

[0063] In certain embodiments, the method comprises visualizing GFP RNA signals using RNAscope.

[0064] In certain embodiments, the tracer is injected into at least one of a primary motor cortex of the brain or a dorsal medial prefrontal cortex of the brain.

[0065] In certain embodiments, the brain is perfused for at least 6 hours. In certain embodiments, the brain is perfused for 6-24 hours. In certain embodiments, the brain is perfused for 12-24 hours. In certain embodiments, the brain is perfused for 18-24 hours. In certain embodiments, the brain is perfused for 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours.

[0066] In certain embodiments, the method comprises removing the brain from a body of a mammal and connecting the brain to the perfusion device. In certain embodiments, connecting the brain to the perfusing device comprises connecting an artery of the brain to a fluid line of the perfusion device. In certain embodiments, connecting the brain to the Atorney Docket No. 047162-7357W01(02701) perfusing device comprises connecting ascending pharyngeal arteries of the brain to a fluid line of the perfusion device.

[0067] In certain embodiments, the method comprises placing the brain in a brain chamber of the perfusion device. In certain embodiments, the method comprises warming the brain to about 32-38 degrees Celsius. In certain embodiments, the method comprises warming the brain to about 32, 33, 34, 35, 36. 37. or 38 degrees Celsius. In certain embodiments, the method comprises warming the brain to about 36 degrees Celsius.

[0068] In certain embodiments, the perfusate is an acellular and cytoprotective perfusate. In certain embodiments, the perfusate is formulated to inhibit neuronal excitability in the brain. In certain embodiments, the method comprises monitoring arteriovenous gradients of glucose consumption and maintaining a stable glucose level. In certain embodiments, the arteriovenous gradients of glucose consumption are monitored hourly.

[0069] In certain embodiments, the stable glucose level is maintained by introducing glucose to the brain via the perfusate.

[0070] In certain embodiments, the method further comprises injecting vessel labeling dye through arteries of the brain at an end of the perfusion. In certain embodiments, the method comprises, injecting Evans blue-albumin complex into the brain. In certain embodiments, the Evans blue-albumin complex is injected intra-arterially into the brain.

[0071] In certain embodiments, the perfusion of the brain with the perfusate preserves a blood brain barrier provided by the brain.

[0072] In certain embodiments, the method further comprises monitoring electrical activity via electrodes and electrocorticography (ECoG) from a dorsal cerebral cortex of the brain.

[0073] In certain embodiments, the method further comprises monitoring hemodynamics, dissolved gasses, and electrolyte levels.

[0074] In certain embodiments, the method further comprises administering one or more metabolic supplements into the perfusate solution. In certain embodiments, the one or more metabolic supplements comprise one or more members selected from the group consisting of glucose, adenosine, amino acids, vitamins, and combinations thereof.

[0075] In certain embodiments, the method further comprises injecting a dye into the brain via a fluid line of the perfusion device. In certain embodiments, the dye is Evans blue dye.

[0076] In certain embodiments, the method further comprises perfusing the brain with albumin after injecting the dye into the brain to remove the dye from vessels in the brain. Atorney Docket No. 047162-7357W01(02701)

[0077] In certain embodiments, the method further comprises sectioning the brain. In certain embodiments, the method further comprises imaging sections of the brain. In certain embodiments, the imaging comprises optical and / or fluorescent imaging.

[0078] In certain embodiments, the method further comprises processing samples taken from the brain. In certain embodiment, the processing comprises, for example, histological processing.

[0079] In certain embodiments, the mammalian brain is a pig brain. In certain embodiments, the mammalian brain is a human brain.

[0080] All references throughout this application (for example, patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material) are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference, to the extent each reference is at least partially not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).

[0081] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size / volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing / oxidizing agents, with art- recognized alternatives and using no more than routine experimentation, are within the scope of the present application. Further examples include application of this system to other intact mammalian organs, e.g. liver, lung, heart, pancreas, kidney, etc. In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, j oumal references and contexts known to those skilled in the art. Any- preceding definitions are provided to clarify their specific use in the context of the invention.

[0082] It is to be understood that wherever values and ranges are provided herein, all values and ranges encompassed by these values and ranges, are meant to be encompassed within the scope of the present invention. Moreover, all values that fall within these ranges, as w ell as the upper or lower limits of a range of values, are also contemplated by the present application. Atorney Docket No. 047162-7357W01(02701)

[0083] The following examples further illustrate aspects of the present invention. However, they are in no way a limitation of the teachings or disclosure of the present invention as set forth herein.

[0084] EXAMPLES

[0085] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0086] Without further description, it is believed that one of ordinary' skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out certain embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure. The materials and methods employed in the experiments disclosed herein are now described.

[0087] Materials and Methods

[0088] Tissue procurement and surgical preparation

[0089] The study utilized domestic pigs (Sus scrofa domesticus; aged 6-8 months, approximately 30-50 kg) that had been raised for food production. The animals were humanely stunned using electrical shock and euthanized by exsanguination, following the regulations set by the USDA. After the animals were deceased, the decapitation was performed at the C3 vertebrae. The removal of skin, connective tissue, and musculature from the skull was performed. Tissue preparation and surgical procedures were previously described in detail. Upon arrival at the laboratory’s surgical suite, the procedure commenced by carefully removing excess bone and soft tissue from the pig skull. Careful bone cuts were performed at the front, back, and sides of the skull, ensuring no harm was inflicted on the brain. Excess soft tissue was subsequently detached using bone periosteal elevators, scalpel blades, and electrocautery (Bovie. Symmetry Surgical). After removing excessive soft and bone tissue, the arterial vasculature was carefully sutured and cauterized. Following this, the ascending pharyngeal arteries (analogus to internal carotid arteries) were isolated and prepared for cannulation using microsurgical instruments. These arteries were then connected to the perfusion device to allow whole brain perfusion, as previously reported. To isolate the brain, a craniectomy was performed while ensuring the head remained as chilled as possible. Atorney Docket No. 047162-7357W01(02701)

[0090] Perfusion protocol

[0091] The perfusion protocol was described in detail previously. It was further optimized with proprietary' steps to allow for improved stability and control overtime. Briefly, the perfusion device was assembled according to our previous reports (Vrselja. Z. et al. Nature 568, 336-343 (2019). Detailed descriptions of the perfusion device, system, and platform can be found in US Patent Application Publication No. 2021 / 0360913 and International Publication No. WO 2023 / 044014, which are incorporated by reference herein. The device is primed with proprietary perfusion solution, and the device is prepared to receive the isolated brain. Following surgical preparation, the brain is placed into the brain chamber and connected to the device, at which point the perfusion protocol is initiated. The brain was slowly rewarmed in a stepwise manner from 20°C to 36°C to prevent thermal shock and allow for metabolic recovery. Over time, key physiological and perfusion parameters are controlled via multiple circuits as described, to achieve physiological equilibrium. The brain was perfused for a minimum of 24 hours and up to 48 hours. The perfusion was terminated before significant changes in global tissue integrity occurred, ensuring that the organ could be used for post hoc tracing analysis. Throughout the perfusion, the platform continuously monitored hemodynamics, dissolved gasses, and electrolyte levels. Key parameters such as arteriovenous consumption of oxygen and glucose, pH, and potassium levels were closely monitored, and appropriate metabolic supplements were administered to maintain normal physiological conditions. Arterial and venous samples were collected from the respective ports, and immediate analysis was performed using the i-STAT clinical blood analyzer system (Abbott) and GEM4000 clinical blood analyzer system (Instrumentation Laboratories).

[0092] Tracer injection

[0093] Following the connection of the brain to the platform, local injections of tracers using a Hamilton syringe and a stereotaxic instrument (Kopf) were performed. The injections were targeted at specific regions, including the primary motor cortex and dorsal medial prefrontal cortex. In the case of cranial nerves, the injections were administered prior to connecting the brain through the foramen magnum, targeting regions that included the facial, abducens, and hypoglossal nerves.

[0094] To assess tracer uptake, three different tracers were co-injected into the primary motor cortex: Fluorescein-conjugated dextran 10k (Invitrogen, 5%), FluoroGold (1%, Atorney Docket No. 047162-7357W01(02701)

[0095] Fluorochrome, LLC), and CTb (647 conjugate, 0.25%). These tracers’ selections were made to facilitate distinct labeling capabilities and enable comprehensive analysis. To evaluate active tracer transport in both the cortex and brainstem, Tetramethylrhodamine-conjugated dextran 10k (Invitrogen, 5%), Fluorescein-conjugated dextran 3k (Invitrogen, 5%), and CTb were utilized.

[0096] Tissue processing and histology

[0097] After completing the perfusion process, the brains were carefully removed from the perfusion platform while remaining cannulated to the custom-designed support housing. This allowed for intravascular perfusion to facilitate subsequent histological evaluation. A 4% paraformaldehyde solution (PF A) was injected into the brains for fixation. Following fixation, the brains were postfixed in 4% PFA at 4 °C for 48 hours and then transferred to a 30% sucrose solution supplemented with 200 mM glycine. The specific regions of interest were embedded in OCT compound and coronally sectioned at a thickness of 100pm using a cryostat.

[0098] In order to visualize the microvasculature, brilliant blue FCF was injected through the arteries in several brains and visualized at far red. Immunostaining for pan-neuronal markers (NeuN) was also performed to examine the cytoarchitecture of neurons following perfusion. For the evaluation of neuronal cell morphology, the primary motor cortex was sectioned and subjected to staining with rabbit NeuN (Abeam, 1 : 1000) for 24 hours at 4°C. This was followed by incubation with anti-rabbit IgG conjugated with Alexa Fluor 647 (Invitrogen) for two hours at room temperature. The sections were then counterstained with DAPI (Invitrogen, 1 : 1000), mounted, and cover-slipped using a mounting medium (Vectorlab / FOCM). For optical clearing of the thick brain tissue, the Ultrafast Optical Clearing Method (FOCM) was employed. FOCM reagent was prepared by dissolving 30% urea and 20% D-sorbitol in DMSO at room temperature overnight followed by mixing of 5% glycerol. Lastly, the tissue sections were imaged using a confocal microscope LSM800 (Zeiss) with lOx. 20x, and 40x objectives and four diode lasers (405. 488, 561. and 640 nm). The confocal images included in this work are representative tile scans with a maximum intensity z-stack projection (~20 micro stacks; ~1 micron per z-step). Alternatively, high- resolution virtual slide images (VSIs) were acquired as serial images using a VS2000 slide scanner (Olympus) with a 4x objective lens.

[0099] Evaluation ofBBB permeability Atorney Docket No. 047162-7357W01(02701)

[0100] For analysis of BBB permeability 24h into perfusion, each brain was injected with 10 ml of 2% Evans blue dye (EB, dissolved and prewarm in 5% BSA to form EB-Albumin complex; Sigma- Aldrich) through an arterial organ line of the perfusion system at the speed of 1 ml / min to infuse with the arterial brain perfusion flow. 10 mins later, the brain was continuously perfused with 5% of albumin for an extra 10 mins to remove the remaining EB-Albumin complex in the vessels. For the fresh brain tissue (TOD), the brain was harvested right after the death of the animals, and cannulated the common carotid artery (CCA) to a gravity flush solution containing 0.2% of EB with 5% of BSA. The brain was flushed for 10 min followed by an extra 10 min of wash with 5% of BSA. For the negative control (NG), the brains were treated with a hyperosmolar solution (around 600 mOsm / L) where 0.2% of EB and 5% of BSA were dissolved and flushed through the brain for 10 min followed by 10 min of 5% BSA wash. After that, the brain was removed and perfused it with 4% paraformaldehyde with 48h of post-fixation. When EB forms a complex with albumin, it undergoes a conformational change characterized by cis-trans isomerization. This structural alteration limits the dye's rotational freedom, enhancing its photophysical properties to produce fluorescence in the red to far-red spectrum. For qualitative assessment, images of the brain coronal sections were taken (excitation at 620 nm, emission at 680 nm) using Keyence BZ-X series. Images were processed with ImageJ for mean intensity7across different brain regions (cerebral cortex, subcortical regions, brainstem and cerebellum).

[0101] RNAscope In Situ Hybridization

[0102] RNAscope ISH was performed using the RNAscope® Multiplex Fluorescent Detection Reagents Kit v2 (ACD Biotechne) on PFA fixed brain tissue according to the manufacturer’s instructions with some modifications. Briefly, after treatment with boiling 1 x target retrieval (which is the reagent used to expose protein epitope for better recognition by the antibody for staining and it needs to be boiled and remain boiling during the entire retrieval process) the sections were incubated with Protease IV for 20 min at RT, followed by washing and the indicated hybridization and amplification steps. Probes used in this study were designed for EGFP (ACD Biotechne. 400281). Ss-Fezf2-C3 (ACD Biotechne. 1088091-C3) and Ss-Cux2-C2 (ACD Biotechne, 1071781-C2).

[0103] Global brain activity monitoring

[0104] Electrocorticographic activity was monitored using aNatus long-term monitoring system and Natus Xltek Sleep / EEG Breakout Box (Natus Medical Inc., Middleton, Atorney Docket No. 047162-7357W01(02701)

[0105] Wisconsin). Signals from a 5 x 4 platinum-iridium electrode grid with 10-mm contact spacing (AD- Tech; Racine, Wisconsin; FG20C-SP10X-000) placed on the dorsal convexity were sampled at 256 Hz and continuously displayed with up to 2 pV / mm resolution and 1 to 70 Hz bandpass.

[0106] MAPseq sample processing and data analysis

[0107] Each fresh frozen brain area of interest was dissected and kept in RNAse-free tubes at - 80°C before RNA extraction. Tissues were homogenized in 400ul of Trizol (Thermo Fisher) with a Pellet Pestle Motor, and then added 600ul of Trizol to make 1 ml of Trizol / sample. RNA was extracted according to the manufacturer's protocol of Trizol Reagent. RNA of each tissue sample was dissolved into 13 ul of H2O. Reverse transcription (RT) was conducted with Superscript IV (Thermo Fisher), tw o RT reactions per sample. From each tissue sample, 4 pl of RNA w ere mixed with spike-in RNA and gene-specific RT primer for reverse transcription reaction, to produce cDNA. Spike-in RNA (GTC ATG ATC ATA ATA CGA CTC ACT ATA GGG GAC GAG CTG TAC AAG TAA ACG CGT AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC TNN NNN NNN NNN NNN NNN NNN NNN NAT CAG TCA TCG GAG CGG CCG CTA CCT AAT TGC CGT CGT GAG GTA CGA CCA CCG CTA GCT GTA CA (SEQ ID NO: 1), where ATCAGTCA (SEQ ID NO: 2) is the barcode tag of the spike-in.) was transcribed in vitro by T7 RNA polymerase (Thermo Fisher) and diluted into 10A3 molecules / pl for target sites and 10 5 molecules / pl for injection sites. Each genespecific RT primer contains a random 12-nt unique molecular identifier (UMI) to tag each individual barcode mRNA molecule and an 8-nt slice specific identifier (SSI) to tag each sample (5 -CTT GGC ACC CGA GAA TTC CAN NNN NNN NNN NNX XXX XXXXT GTA CAG CTA GCG GTG GTC G-3’(SEQ ID NO:3), where X8 is SSI and N12 is the UMI.) Double-strand cDNA was synthesized with Second Strand cDNA Synthesis Kit (Thermo Fisher). Double-strand cDNA was then purified and treated withExonucleasel (New England Biolabs) to remove the remaining RT primers. Barcodes amplicons were then amplified by nested PCR with primers 5’- CTG TAC AAG TAA ACG CGT AAT G -3’ (SEQ ID NO: 4) and 5 - CAA GCA GAA GAC GGC ATA CGA GAT CGT GAT GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA -3 ’(SEQ ID NO: 5) for the first PCR and primers 5 -AAT GAT ACG GCG ACC ACC GA-3’(SEQ ID NO: 6) and 5 - CAA GCA GAA GAC GGC ATA CGA-3’(SED ID NO: 7) for the second PCR with AccuPrime™ Pfx DNA Polymerase (Thermo Fisher). The final 233bp barcode amplicons Atorney Docket No. 047162-7357W01(02701) were purified from 2% agarose gel with MinElute Gel Extraction Kit (Qiagen) and verified on a DNA bioanalyzer chip using High Sensitivity DNA Kit (Agilent). Purified barcode libraries were sequenced by an Illumina NextSeq500 high output run at paired-end 36 using the SBS3T sequencing primer for paired-end 1 and the Illumina small RNA sequencing primer 2 for paired-end 2.

[0108] The preprocessing of the raw MAPseq data is following the previous bioinformatic pipeline. Briefly, the two fastq files from paired-end sequencing are trimmed and merged into a single file. The merged reads are demultiplexed based on the SSI-barcode and filtered to remove the ambiguous bases. Then the reads are collapsed and sorted to get the unique reads only. Then the read counts are converted to UMI counts by removing the 12-nt UMI-barcode and collapsing the remaining reads. The spike-in reads are splitted by the unique barcodes. Then the real UMI counts for each sample are merged to the UMI count matrix for analyzing the projection patterns.

[0109] To exclude low confidence projection patterns from analysis, we required each barcode to have more than 10 counts in the injection site and at least one target area with 2 more counts than the technical controls (water) or biological controls (OB). At the same time, it was required for each barcode to have less than 2 counts in all negative controls. The UMI are normalized by the maximum count in each inject site and a heatmap was made by the normalized UMI counts in each inject site.

[0110] Statistical analysis and reproducibility

[0111] All data are reported as mean ± s.e.m. with data analysis being conducted using oneway ANOVA with Dunnett’s post hoc multiple-comparisons adjustment (in reference to the fresh brain tissue in BBB integrity assessment), or unpaired t-tests for comparisons between two groups (anterograde and retrograde tracer active transport). Significance was set at P < 0.05. The statistical analysis and plotting were performed in GraphPad 9 (GraphPad). All of the figures were processed using ImageJ (Schindelin et al. 2012) or Qupath (Bankhead et al. 2017) and readapted using Adobe Illustrator (Adobe Systems).

[0112] Hippocampal slice preparation and Electrophysiology.

[0113] A 1 cm long block of dorsal hippocampus was dissected orthogonal to the long hippocampal axis, glued to a metal vibratome insert and kept in ice-cold cutting solution (in mM): Gibco EBSS cat#14155, supplemented with CaC120.5 mM; MgC125 mM; Dextrose 5 mM: NaCl 117 mM; NaHCO3 26 mM; NaH2PO4 1 mM; KC1 5 mM; CaC12 0.5 mM; MgC12 5 mM; Dextrose 10.6 mM, bubbled with carbogen (5%C02 / 95%02). 400 pm slices Atorney Docket No. 047162-7357W01(02701) were cut transverse to the hippocampal long axis in ice-cold cutting solution using Leica VT1200S vibratome. transferred to a custom- made brain slice keeping chamber filled with carbogen-bubbled ACSF and left to recover for at least 1 hr at RT. Hippocampal slices were transferred to MED Probe recording chamber (catalog# MED-P545A, Alpha MED Scientific), gently positioned with a paint brush on a 64-electrodes array of 450 pm electrode spacing to cover DG-CA1 areas, under visual guidance using an inverted Nikon TMS-F microscope equipped with 4xobjective, and were held in place by a stainless-steel harp with 1.0 mm spacing nylon strings (Warner Instruments SHD-42 / 10, WI 64-1421). The slices were let to equilibrate for 5-10 minutes before recordings in ACSF (in mM): Gibco EBSS cat#14155, supplemented with CaC12 2.5 mM; MgC12 1.3 mM; Dextrose 5 mM: NaCl 117 mM; NaHCO3 26 mM; NaH2PO4 1 mM; KC1 5 mM; CaC12 2.5 mM; MgC12 1.3 mM; Dextrose 10.6 mM, bubbled with carbogen. Field excitatory post synaptic potentials (fEPSPs) were recorded from pig hippocampal slices using MED64-Plex4 system (Alpha MED Scientific). fEPSPs were evoked by 0.2 ms square current pulses delivered by MED64-Plex4 stimulus isolator through one of the 64- electrodes positioned in the middle third of the CAI stratum radiatum (CAI recordings) or in the DG region, while simultaneously recording from the rest of 64-electrodes grid. The baseline fEPSPs were evoked every' 20 seconds at 40%-50% of maximal fEPSP amplitude calculated from the input-output (I-O) curve; 1-0 curve was from 0 to 120 A in 5 A steps. The extracellular solution was ACSF as described above. Recordings were performed at RT=21-23°C.

[0114] Example 1: Long-term isolated porcine brain perfusion

[0115] To achieve long-term postmortem porcine brain perfusion, the perfusion platform was built and used with multiple enhancements, including the simplification and standardization of surgical procedures, improvement of the perfusate composition, and upgrading and automation of the brain operation system. To advance the experimental procedures, post- mortem porcine (Sus scrofa domesticus) brain specimens sourced from USDA-regulated food processing facilities, which would have otherwise been discarded, were employed. After a 4h post- mortem period, referring to the time required for brain acquisition and isolation, the carotid arteries were connected to the platform. Following priming the device with a proprietary acellular and cytoprotective perfusate, the ex vivo brain circulation was initiated (FIG. 1 A). Throughout the perfusion, the essential hemodynamic parameters were monitored in real-time and observed stable brain perfusion flow and brain resistance, indicating successful restoration of the global brain circulation Atorney Docket No. 047162-7357W01(02701) and minimal brain edema developed during the extended perfusion (FIG. IB). To ensure sufficient support to brain metabolic activity, arteriovenous gradients of glucose consumption were monitored hourly and a stable glucose level was maintained (FIG. 5A). In addition to the global circulation, a vessel labeling dye was injected through arteries at the end of the perfusion, and a robust signal labeling of the vascular tree was observed, indicating successful recirculation of microvasculature during the entire perfusion (FIGS. 5B-5C).

[0116] Following the successful restoration of both global and microcirculation within the brain, it was investigated whether the Blood-Brain Barrier (BBB) remained intact. Using an intra-arterial injection of Evans blue-albumin complex, BBB permeability' was measured based on the extent of dye leakage into the major brain regions. For comparative analysis, brains procured immediately postmortem (Time of death, TOD) and those exposed to a hyperosmolar solution (HP) were used to serve as positive and negative control respectively. Following the long-term perfusion, minimal dye penetration was detected across multiple brain regions including cerebral cortex, subcortical regions, brainstem and cerebellum. These findings were comparable to those in freshly harvested brain tissue, suggesting that perfusion can not only restore vessel circulation but also preserve neurovascular architecture, postmortem. In contrast, brains subjected to hyperosmolar treatments (HP) exhibited substantial dye diffusion into the tissue, with unequal levels in the two hemispheres, indicating BBB leakage and a potential collapse in the circulation (FIGS. 6A- 6D ). In summary, these results indicate that the perfusion platform can effectively extend brain tissue viability through the successful re-establishment of microcirculation and the preservation of blood-brain barrier integrity, both of which are crucial for maintaining metabolic function over an extended period.

[0117] Next examined was the cytoarchitectural changes of the major neural cells in the regions that are highly susceptible to ischemia, namely neocortex, hippocampus and cerebellum. Immunostaining w as performed for pan-neuronal markers (NeuN), astrocytes (GFAP), microglia (Ibal) and myelin sheath of oligodendrocyte (MBP). Similar to previously reported, robust staining of all of the markers in multiple brain regions was observed, with preserved cell architecture following the perfusion (FIGS. 5A-5D).

[0118] Use of the perfusion system described herein has revealed the postmortem brain's potential as a novel tool to improve the understanding of the brain, thereby positioning it as an emerging tool in neuroscience. It is imperative that such experiments are conducted under stringent ethical guidelines, particularly to prevent any reemergence of global Atorney Docket No. 047162-7357W01(02701) electrical network activity. To ensure the absence of electrical global network activity during the perfusion, we formulated the perfusate to specifically inhibit neuronal excitability. To evaluate the formulation's efficacy, we conducted evoked response tests on freshly harvested porcine hippocampal tissue using multi-electrode arrays (MEA). The results showed that exposure to the perfusate led to an 80% reduction in stimulated synaptic activity compared to a standard recording solution (FIGS. 1D-1E). Additionally, global electrical activity was continuously monitored via clinical -grade surface grid electrodes and electrocorticography (ECoG) from the dorsal cerebral cortex. Throughout the perfusion, no spontaneous global activity' was detected (FIG. IF). Collectively, these data provided evidence that the perfusion platform is capable of exerting stringent control over, and effectively silencing, global neuronal network activity.

[0119] Example 2: Cellular uptake and intracellular transport of chemical tracers

[0120] To validate that the perfusion platform is amenable to the mapping of long-range circuits, a critical first step is to confirm the viability of the cells, especially their ability to uptake neuroactive vectors, commonly by endocytosis, and active transport between axons and soma. First analyzed were the classic chemical tracers that are commonly used for anterograde (biotinylated dextran amine [BDA] 10k) and retrograde (Cholera toxin subunit b [CTb], Fluorogold [FG]) tracing (FIGS. 2A-2B). Following long-term brain perfusion, an efficient cell labeling was observed demonstrating efficient tracer uptake by the neurons at the injection sites (FIG. 2B). Notably, through signal amplification with immunostaining, detailed cell morphology' was observed, including axon terminal branches to the level of terminal boutons (FIG. 2C).

[0121] Rather than lipophilic tracers, such as carbocyanine dyes, that passively diffuse along the myelin sheaths, resulting in bidirectional labeling, the anterograde and retrograde tracer transport is maintained and guided by the intra-axonal microtubule system, namely kinesin and dynein protein family respectively. Therefore, the tracer transport relies on an energy-dependent process in the viable cells. To examine whether neurons in the perfused brain support active transport of those tracers, anterograde (BDA10K) and retrograde (BDA3K) tracers were co-injected in one exemplary cerebral cortical region, the primary' motor cortex (Ml) in the cruciate gy rus, where pyramidal neurons develop long monosynaptic corticospinal connections. It was observed that, with a 24h-48h perfusion protocol, anterograde tracer could transport with the travel velocity of 1.33mm / h along the axons from the injection site, while retrograde tracers were detected in the neuronal soma in Atorney Docket No. 047162-7357W01(02701) the associated and contralateral cortices which demonstrated an estimated distance of 2.5mm / h of transport at a globe scale in the intact brain (FIG. 2D). It has been reported that low molecular weight BDA (3K) moves faster than high molecular weight BDA (I OK), resulting in neuronal filling in a shorter time. This is consistent with the observation here of longer axonal transport distance using retrograde tracers.

[0122] In addition to cortical neuronal tracing, further were explored the potential applications of this approach in other CNS regions, especially those that are challenging to study in experimental animals and humans. Given that the brain was surgically isolated and vasculature at the intersection of the first cervical spinal cord and medulla oblongata was exposed, this provided the access to study the connectivity of cranial nen es in the brain stem. Without further optimization of the current surgical procedures, three cranial nerve pathways, namely cranial nerves XII (Hypoglossal), VII (Facial) and VI (Abducens) were investigated using retrograde tracers (BDA3K). Following long-term perfusion, robust filling of all tested cranial nen es was observed, and retrograde labeling of the lower motor neurons located in the hypoglossal and abducens nuclei of the ventral medulla was observed (FIGS. 3A-3C). Overall, these data demonstrated that with proper restoration of circulation and long-term maintenance of cell viability, neurons in the postmortem brain possess capability' for tracer uptake and active axonal transport both anterogradely and retrogradely across CNS.

[0123] Example 3: Viral labeling of neurons

[0124] Given evidence for active neuronal tracing using conventional chemical tracers, next explored was the potential of using a restored brain for viral tracing. In recent decades, the exploitation of viruses in cell type-specific labeling and neuroanatomical tract-tracing has revolutionized our understanding of brain wiring. As efficient delivery systems for genetic payloads, viral tracers allow cell-type-specific targeting, and high throughput connectivity sequencing. Notably, the recombinant Sindbis virus has been used to express unique RNA barcodes, which allows neuronal proj ection mapping with single-cell resolution by exploiting RNA sequencing (MAPseq).

[0125] Importantly, the rapid and high expression of RNA barcodes enables efficient labeling of neurons as short as 24h in rodents, which is well suited for the time frame that one can repeatedly achieve with this perfusion platform. Additionally, many in vitro studies have shown efficient infection of virus carrying transgenes in organotypic brain slice cultures while viral transduction becomes a lab routine for tissue culture for cells derived from Atorney Docket No. 047162-7357W01(02701) different species. These studies collectively suggested that with virus that can rapidly generate genetic materials, and with sufficient incubation time and condition to maintain cell viability ex vivo, it is possible to virally label cells in the isolated postmortem brain for brain mapping. To determine whether viral tracing is achievable in the ex vivo brain with global structural and cell functional features, we co-injected the chemical tracer (BDA10K) with the Sindbis virus carrying GFP into the postmortem porcine prefrontal cortex. Following 24h of perfusion, the GFP RNA signals were visualized using RNAscope and robust GFP labeling in the injection site was observed, demonstrating successful gene delivery (FIG. 4A). Additionally, observed was a well preserved endogenous RNA expression in brain cells using layer specific marker Cux2 (Cut Like Homeobox 2) and Fezf2 (zinc- finger transcription factor), suggesting that the extended perfusion metabolically supports the energy requirements for both normal brain molecular functions and rapid viral RNA expression (FIG. 8A).

[0126] To assess the feasibility' of employing the perfusion system for neuronal tracing with MAPseq. a sindbis viral library encoding a diverse collection of barcode sequences was injected into the dmPFC. Following a 24-hour perfusion in the brains injected with MAPSeq virus, the barcode mRNA from the injection site and various target regions of interest were extracted and sequenced (FIG. 4B). The projection patterns of 338 neurons from the dmPFC to 6 brain regions: the ipsilateral anterior cingulate cortex (ACC), the ipsilateral dorsal lateral PFC (dlPFC), the ipsilateral ventral medial PFC (vmPFC), contralateral ACC (c-ACC), and contralateral dmPFC (c-dmPFC), contralateral dlPFC (c-dlPFC) were successfully traced. Given the significant anatomical distances between each gyrus in the porcine brain, the possibility of virus spreading from one region to another, as seen in rodents, can be discounted. It was observed that 42.32% of the neurons project to ACC, 41.74% to c-dmPFC, 8.99% to dlPFC, 4.35% to c-ACC, 1.45% to vmPFC and 1.16% to c- dlPFC (FIG.4C-4F). Most cells project to only one region (97.93%), 2.07% project to two regions, and none project to three regions. Among all tested projection targets, the ACC received the highest level of innervation from neurons originating in the dmPFC. This supports existing know ledge of the ACC's interconnected relationship with the dmPFC in governing emotional and cognitive control.

[0127] Further explored was the versatility of the perfusion platform in facilitating the expression of genetic material carried by a range of widely used viral vectors for neuronal tracing, namely lentivirus, AAV2, and AAV9 (FIGS. 8B-8D). A robust labeling of the reporter genes in all tested viral vectors following the perfusion was observ ed. This result Atorney Docket No. 047162-7357W01(02701) suggests the potential for cell type-specific neuronal tracing in the postmortem brain.

[0128] Example 4:

[0129] In this study, it has been shown that the perfusion platform can sustain cellular viability and tissue integrity7in isolated postmortem large mammalian brains for extended time, up to 24 and even 48h. The prolongation of brain viability, achieved without eliciting global electrical activity, substantiates and refines the prior findings that cellular deterioration in postmortem brains follows an extended, rather than an acute, temporal course and that certain molecular process can be altered with appropriate interventions. Importantly, the findings revealed the preservation of not just global cerebral circulation but also the integrity of the neurovascular architecture- Blood-Brain barrier - throughout the perfusion process.

[0130] Using the perfusion platform, provided herein is the proof of concept of applying active neuronal tracing in postmortem, intact, large mammalian brains. Employing well- established chemical tracers, efficient cellular uptake and energy-dependent axonal transport was observed, highlighting the capability for extensive neuronal connectivity studies that surpass the limitations of rodent models. Additionally, robust viral-mediated gene expression was observed and MAPseq was successfully applied for high-throughput connectome mapping, extending the platform's utility for potential cell-type-specific targeting and comprehensive connectome mapping in larger species.

[0131] The brain connectivity research has historically benefited from methodologies predominantly developed for rodent models, given their suitability7for experimental manipulation and troubleshooting. On the other hand, postmortem brain tissue, with its inherent advantages of bypassing bioethical concerns, holds immeasurable value for studying brain functions and structures. The perfusion platform, in this context, serves as a groundbreaking approach that bridges these two resources, combining them in an unprecedented manner.

[0132] Enumerated embodiments

[0133] The following exemplary7embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0134] Embodiment 1 provides a method comprising: a. perfusing a mammalian brain with a perfusate using a perfusion device; Atorney Docket No. 047162-7357W01(02701) b. introducing a tracer into the mammalian brain while the mammalian brain is being perfused with the perfusate; and c. monitoring movement of the tracer in the mammalian brain.

[0135] Embodiment 2 provides a method according to embodiment 1 , comprising, based on the monitoring, mapping the mammalian brain.

[0136] Embodiment 3 provides the method according to any one of embodiments 1-2, wherein the mapping of the mammalian brain is a mesoscale connectivity7mapping of the mammalian brain.

[0137] Embodiment 4 provides the method according to any one of embodiments 1-3, comprising, based on the monitoring, examining long-range neuronal connections of the mammalian brain.

[0138] Embodiment 5 provides the method according to any one of embodiments 1-4. wherein the tracer comprises at least one anterograde tracer and at least one retrograde tracer.

[0139] Embodiment 6 provides the method according to any one of embodiments 1-5, wherein the at least one anterograde tracer comprises BDA10K. and the at least one retrograde tracer comprises BDA3K.

[0140] Embodiment 7 provides the method according to any one of embodiments 1-6, wherein the tracer comprises at least one chemical tracer.

[0141] Embodiment 8 provides the method according to any one of embodiments 1-7, wherein the at least one chemical tracer comprises one or more chemical tracers selected from the group consisting of: Fluorescein-conjugated dextran 10k, FluoroGold. CTb.

[0142] Tetramethylrhodamine-conjugated dextran 10k. Fluorescein-conjugated dextran 3k, and combinations thereof.

[0143] Embodiment 9 provides the method according to any one of embodiments 1-8, wherein the tracer comprises at least one viral tracer. Atorney Docket No. 047162-7357W01(02701)

[0144] Embodiment 10 provides the method according to any one of embodiments 1-9, wherein the at least one viral tracer comprises one or more viruses selected from the group consisting of: Sindbis virus, Lentivirus, Adeno-associated virus 2 (AAV 2), Adeno-associated virus 9 (AAV 9), and combinations thereof.

[0145] Embodiment 11 provides the method according to any one of embodiments 1-10, wherein the at least one viral tracer comprises recombinant Sindbis virus.

[0146] Embodiment 12 provides the method according to any one of embodiments 1-11, wherein the at least one viral tracer carries green fluorescent protein (GFP).

[0147] Embodiment 13 provides the method according to any one of embodiments 1-12, comprising visualizing GFP RNA signals using RNAscope.

[0148] Embodiment 14 provides the method according to any one of embodiments 1-13, wherein the tracer is injected into at least one of a primary motor cortex of the mammalian brain or a dorsal medial prefrontal cortex of the mammalian brain.

[0149] Embodiment 15 provides the method according to any one of embodiments 1-14, wherein the mammalian brain is perfused for at least 6 hours.

[0150] Embodiment 16 provides the method according to any one of embodiments 1-15, wherein the mammalian brain is perfused for 6-24 hours.

[0151] Embodiment 17 provides the method according to any one of embodiments 1-16, wherein the mammalian brain is perfused for 12-24 hours.

[0152] Embodiment 18 provides the method according to any one of embodiments 1-17, wherein the mammalian brain is perfused for 18-24 hours.

[0153] Embodiment 19 provides the method according to any one of embodiments 1-18, comprising removing the mammalian brain from a body of a mammal and connecting the mammalian brain to the perfusion device. Atorney Docket No. 047162-7357W01(02701)

[0154] Embodiment 20 provides the method according to any one of embodiments 1-19, wherein connecting the mammalian brain to the perfusing device comprises connecting an artery of the mammalian brain to a fluid line of the perfusion device.

[0155] Embodiment 21 provides the method according to any one of embodiments 1-20, wherein connecting the mammalian brain to the perfusing device comprises connecting ascending pharyngeal arteries of the mammalian brain to a fluid line of the perfusion device.

[0156] Embodiment 22 provides the method according to any one of embodiments 1-21, comprising placing the mammalian brain in a brain chamber of the perfusion device.

[0157] Embodiment 23 provides the method according to any one of embodiments 1-22, further comprising warming the mammalian brain to about 36 degrees Celsius.

[0158] Embodiment 24 provides the method according to any one of embodiments 1-23, wherein the perfusate is an acellular and cytoprotective perfusate.

[0159] Embodiment 25 provides the method according to any one of embodiments 1-24, wherein the perfusate is formulated to inhibit neuronal excitability in the mammalian brain.

[0160] Embodiment 26 provides the method according to any one of embodiments 1-25, comprising monitoring arteriovenous gradients of glucose consumption and maintaining a stable glucose level.

[0161] Embodiment 27 provides the method according to any one of embodiments 1-26, wherein the arteriovenous gradients of glucose consumption are monitored hourly.

[0162] Embodiment 28 provides the method according to any one of embodiments 1-27, wherein the stable glucose level is maintained by introducing glucose to the mammalian brain via the perfusate.

[0163] Embodiment 29 provides the method according to any one of embodiments 1-28, further comprising injecting vessel labeling dye through arteries of the mammalian brain at an end of the perfusion. Atorney Docket No. 047162-7357W01(02701)

[0164] Embodiment 30 provides the method according to any one of embodiments 1-29, further comprising injecting Evans blue-al bumin complex into the mammalian brain.

[0165] Embodiment 31 provides the method according to any one of embodiments 1-30, wherein the Evans blue-albumin complex is injected intra-arterially into the mammalian brain.

[0166] Embodiment 32 provides the method according to any one of embodiments 1-31, wherein the perfusion of the mammalian brain with the perfusate preserves a blood brain barrier provided by the mammalian brain.

[0167] Embodiment 33 provides the method according to any one of embodiments 1-32, further comprising monitoring electrical activity via electrodes and electrocorticography (ECoG) from a dorsal cerebral cortex of the mammalian brain.

[0168] Embodiment 34 provides the method according to any one of embodiments 1-33, further comprising monitoring hemodynamics, dissolved gasses, and electrolyte levels.

[0169] Embodiment 35 provides the method according to any one of embodiments 1-34, further comprising administering one or more metabolic supplements into the perfusate solution.

[0170] Embodiment 36 provides the method according to any one of embodiments 1-35, wherein the one or more metabolic supplements comprise one or more members selected from the group consisting of glucose, adenosine, amino acids, vitamins, and combinations thereof.

[0171] Embodiment 37 provides the method according to any one of embodiments 1-36, further comprising injecting a dye into the mammalian brain via a fluid line of the perfusion device.

[0172] Embodiment 38 provides the method according to any one of embodiments 1-37, wherein the dye is Evans blue dye.

[0173] Embodiment 39 provides the method according to any one of embodiments 1-38, further comprising perfusing the mammalian brain with albumin after injecting the dye into the mammalian brain to remove the dye from vessels in the mammalian brain. Atorney Docket No. 047162-7357W01(02701)

[0174] Embodiment 40 provides the method according to any one of embodiments 1-39, further comprising sectioning the mammalian brain.

[0175] Embodiment 41 provides the method according to any one of embodiments 1-40, further comprising imaging sections of the mammalian brain.

[0176] Embodiment 42 provides the method according to any one of embodiments 41, further comprising processing samples taken from the mammalian brain.

[0177] Other Embodiments

[0178] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0179] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attomey Docket No. 047162-7357W01(02701)CLAIMSWhat is claimed is:

1. A method comprising: a. perfusing a mammalian brain with a perfusate using a perfusion device; b. introducing a tracer into the mammalian brain while the mammalian brain is being perfused with the perfusate; and c. monitoring movement of the tracer in the mammalian brain.

2. The method of claim 1, comprising, based on the monitoring, mapping the mammalian brain.

3. The method of claim 2, wherein the mapping of the mammalian brain is a mesoscale connectivity mapping of the mammalian brain.

4. The method of claim 1, comprising, based on the monitoring, examining long-range neuronal connections of the mammalian brain.

5. The method of claim 1, wherein the tracer comprises at least one anterograde tracer and at least one retrograde tracer.

6. The method of claim 5, wherein the at least one anterograde tracer comprises BDA10K, and the at least one retrograde tracer comprises BDA3K.

7. The method of claim 1, wherein the tracer comprises at least one chemical tracer.

8. The method of claim 7, wherein the at least one chemical tracer comprises one or more chemical tracers selected from the group consisting of: Fluorescein-conjugated dextran 10k, FluoroGold, CTb, Tetramethylrhodamine-conjugated dextran 10k, Fluorescein-conjugated dextran 3k, and combinations thereof.

9. The method of claim 1, wherein the tracer comprises at least one viral tracer.Attomey Docket No. 047162-7357W01(02701)10. The method of claim 9, wherein the at least one viral tracer comprises one or more viruses selected from the group consisting of: Sindbis virus. Lentivirus. Adeno- associated virus 2 (AAV2), Adeno-associated virus 9 (AAV9), and combinations thereof.

11. The method of claim 9, wherein the at least one viral tracer comprises recombinant Sindbis virus.

12. The method of claim 9, wherein the at least one viral tracer carries green fluorescent protein (GFP).

13. The method of claim 12, comprising visualizing GFP RNA signals using RNAscope.

14. The method of claim 1, wherein the tracer is injected into at least one of a primary7motor cortex of the mammalian brain or a dorsal medial prefrontal cortex of the mammalian brain.

15. The method of claim 1, wherein the mammalian brain is perfused for at least 6 hours.

16. The method of claim 15. wherein the mammalian brain is perfused for 6-24 hours.

17. The method of claim 16, wherein the mammalian brain is perfused for 12-24 hours.

18. The method of claim 17, wherein the mammalian brain is perfused for 18-24 hours.

19. The method of claim 1, comprising removing the mammalian brain from a body of a mammal and connecting the mammalian brain to the perfusion device.

20. The method of claim 19. wherein connecting the mammalian brain to the perfusing device comprises connecting an artery of the mammalian brain to a fluid line of the perfusion device.Attomey Docket No. 047162-7357W01(02701)21. The method of claim 19, wherein connecting the mammalian brain to the perfusing device comprises connecting ascending pharyngeal arteries of the mammalian brain to a fluid line of the perfusion device.

22. The method of claim 19, comprising placing the mammalian brain in a brain chamber of the perfusion device.

23. The method of claim 19, further comprising warming the mammalian brain to about 36 degrees Celsius.

24. The method of claim 1, wherein the perfusate is an acellular and cytoprotective perfusate.

25. The method of claim 1, wherein the perfusate is formulated to inhibit neuronal excitability in the mammalian brain.

26. The method of claim 1, comprising monitoring arteriovenous gradients of glucose consumption and maintaining a stable glucose level.

27. The method of claim 26. wherein the arteriovenous gradients of glucose consumption are monitored hourly.

28. The method of claim 26, wherein the stable glucose level is maintained by introducing glucose to the mammalian brain via the perfusate.

29. The method of claim 1, further comprising injecting vessel labeling dye through arteries of the mammalian brain at an end of the perfusion.

30. The method of claim 1, further comprising injecting Evans blue-albumin complex into the mammalian brain.

31. The method of claim 30, wherein the Evans blue-albumin complex is injected intraarterially into the mammalian brain.Attomey Docket No. 047162-7357W01(02701)32. The method of claim 1, wherein the perfusion of the mammalian brain with the perfusate preserves a blood brain barrier provided by the mammalian brain.

33. The method of claim 1, further comprising monitoring electrical activity via electrodes and electrocorticography (ECoG) from a dorsal cerebral cortex of the mammalian brain.

34. The method of claim 1, further comprising monitoring hemodynamics, dissolved gasses, and electrolyte levels.

35. The method of claim 1, further comprising administering one or more metabolic supplements into the perfusate solution.

36. The method of claim 35, wherein the one or more metabolic supplements comprise one or more members selected from the group consisting of glucose, adenosine, amino acids, vitamins, and combinations thereof.

37. The method of claim 1, further comprising injecting a dye into the mammalian brain via a fluid line of the perfusion device.

38. The method of claim 37, wherein the dye is Evans blue dye.

39. The method of claim 37, further comprising perfusing the mammalian brain with albumin after injecting the dye into the mammalian brain to remove the dye from vessels in the mammalian brain.

40. The method of claim 39, further comprising sectioning the mammalian brain.

41. The method of claim 40. further comprising imaging sections of the mammalian brain.

42. The method of claim 1, further comprising processing samples taken from the mammalian brain.Attomey Docket No. 047162-7357W01(02701)43. The method of claim 1, wherein the mammalian brain is a pig brain.

44. The method of claim 1, wherein the mammalian brain is a human brain.

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