Brain Sections Research Articles

Sex-Dependent Effects of Angiotensin Type 2 Receptor Expressing Medial Prefrontal Cortex (mPFC) Interneurons in Fear Extinction Learning

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC). Their function and mechanism in the mPFC, however, remain unexplored. We therefore used a combination of neuroanatomical, chemogenetic, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female brains were perfused and used for immunohistochemistry. Brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus. Mice then underwent Pavlovian auditory fear conditioning and anxiety-like and locomotor behavior testing using open field (OF) and elevated plus maze (EPM) tests. Results: IHC revealed that AT2R is expressed throughout the mPFC in males ( 208.6±48.8 cells/mm2) and females ( 139.0±59.8 cells/mm2) and has low co-expression on glutamatergic neurons ( TBR1 co-staining, males: 7.1%±1.8%; females: 13.2%±4.2%). Of the interneuron markers tested, AT2R is primarily expressed on somatostatin interneurons, and has higher colocalization in the mPFC of females than males ( males: 16.5%±1.2%, females: 31.8%±4.2%, p=0.02). Following fear conditioning and extinction, mPFC-AT2R deletion impaired extinction in female ( p=0.03) but not male ( p=0.76) mice. Locomotion in the OF was unaltered by mPFC-AT2R deletion in males or females ( total distance travelled; males: GFP 44.11m±5.92m, Cre 56.00m±10.29m, p=0.35; females: GFP 42.86m±4.85m, Cre 42.39m±4.97m, p=0.95), while AT2R-deleted females had increased exploratory behavior in the EPM ( open arm entries; males: GFP 12.38±1.02, Cre 13.22±1.54, p=0.66; females: GFP 12.50±0.96, Cre 20.00±2.81, p=0.02). Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel somatostatin subgroup that influences fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences, and increases the understanding of circuitry and function of the brain RAS in disordered fear learning. This may lead to improved therapeutic treatments for PTSD. This research and PJM was supported by NIH1R01HL137103-01A1 and the American Heart Association 15CSA24340001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Multimodal MRI reveals brainstem connections that sustain wakefulness in human consciousness.

Consciousness is composed of arousal (i.e., wakefulness) and awareness. Substantial progress has been made in mapping the cortical networks that underlie awareness in the human brain, but knowledge about the subcortical networks that sustain arousal in humans is incomplete. Here, we aimed to map the connectivity of a proposed subcortical arousal network that sustains wakefulness in the human brain, analogous to the cortical default mode network (DMN) that has been shown to contribute to awareness. We integrated data from ex vivo diffusion magnetic resonance imaging (MRI) of three human brains, obtained at autopsy from neurologically normal individuals, with immunohistochemical staining of subcortical brain sections. We identified nodes of the proposed default ascending arousal network (dAAN) in the brainstem, hypothalamus, thalamus, and basal forebrain. Deterministic and probabilistic tractography analyses of the ex vivo diffusion MRI data revealed projection, association, and commissural pathways linking dAAN nodes with one another and with DMN nodes. Complementary analyses of in vivo 7-tesla resting-state functional MRI data from the Human Connectome Project identified the dopaminergic ventral tegmental area in the midbrain as a widely connected hub node at the nexus of the subcortical arousal and cortical awareness networks. Our network-based autopsy methods and connectivity data provide a putative neuroanatomic architecture for the integration of arousal and awareness in human consciousness.

Radiation attenuation characterization of some biological samples by using the Klein–Nishina formula

Radiation has beneficial applications in medicine and nuclear sciences, but it can also be harmful due to its ionizing properties. So, radiation usage has a significant role in different research fields. In the present study, the Klein–Nishina (K–N) approach was used to evaluate the electronic and energy-transfer cross sections of Water, bone, adipose, soft, long, breast, brain and skin tissues. Compton mass attenuation coefficients (CMAC) and Compton mass energy transfer coefficients (CMACtr) were determined in the energy range 0.284–15 MeV. Different methods, based on the effective charge, were used to determine the parameters of the materials for the energy range. The cross sections were firstly calculated for elements that made up the materials. The investigated parameters of the materials were then determined using the relevant cross sections in the energy range. The results were compared with each other and some possible results from the literature. Klein–Nishina electronic cross sections, Compton mass attenuation coefficients and Compton mass energy transfer coefficients of the studied materials were decreased with increasing γ-ray energy like the elements. A good agreement was observed for CMACs and CMACtrs based on Zeff1/A and Zeff3/A (Diff. ≤ 16.4%) for Bone Compact. In addition, a comparison with the literature was done for CaCO3 in CMAC for some photon energies. The maximum difference (%) between used methods and literature was observed as ≤ 19.0%. The reported data should be useful using the gamma rays in Compton scattering energy region.

Anatomy of the brain of capybara (Hydrochoerus hydrochaeris) using magnetic resonance imaging.

Capybara is considered the largest living rodent and is widespread distributed in the South America and in the Brazilian territory. The purpose of this study was to provide the anatomical description of the brain in the capybara (Hydrochoerus hydrochaeris) using magnetic resonance imaging (MRI). Brains of ten normal capybaras were imaged and sectioned in the anatomical studies. MRI was acquired on 0.25 Tesla equipment, promoting good-quality images capable to identify and classify the main anatomical structures of clinical interest. MRI reference images were validated by comparing them with gross anatomical sections. The capybara sulci and gyri were named for its similar location and orientation to those described in the previous descriptions in the capybara and in the domestic dog. Capybaras presented prominent cerebral sulcus and gyrus in relation to other caviomorph rodents, but in reduced number when compared to domestic animals and other wild mammals such as elephants and giraffes. The findings of this study indicate that the shape of the capybara brain is remarkably similar to that of the caviomorph rodents with a higher neocortilization. The capybara rhinencephalon was well-developed implying a good sense of smell. Due to this development of the rhinencephalon, we can suggest that capybara brain is a macrosmatic brain. The MRI and gross anatomical sections of capybara brain may help veterinary researchers and clinicians increase the accuracy of brain MRI scans interpretation in these animals.

Cross-modality mapping using image varifolds to align tissue-scale atlases to molecular-scale measures with application to 2D brain sections

This paper explicates a solution to building correspondences between molecular-scale transcriptomics and tissue-scale atlases. This problem arises in atlas construction and cross-specimen/technology alignment where specimens per emerging technology remain sparse and conventional image representations cannot efficiently model the high dimensions from subcellular detection of thousands of genes. We address these challenges by representing spatial transcriptomics data as generalized functions encoding position and high-dimensional feature (gene, cell type) identity. We map onto low-dimensional atlas ontologies by modeling regions as homogeneous random fields with unknown transcriptomic feature distribution. We solve simultaneously for the minimizing geodesic diffeomorphism of coordinates through LDDMM and for these latent feature densities. We map tissue-scale mouse brain atlases to gene-based and cell-based transcriptomics data from MERFISH and BARseq technologies and to histopathology and cross-species atlases to illustrate integration of diverse molecular and cellular datasets into a single coordinate system as a means of comparison and further atlas construction.

Ex vivo method for rapid quantification of post traumatic brain injury lesion volumes using ultrasound

BackgroundStudies of traumatic brain injury often involve the quantification of the lesion volume as a major outcome measure. The determination of lesion volume typically employs the cutting and mounting of brain tissue, and the calculation of the cross-sectional area of the lesion within each section of brain after histological staining. This is a time consuming and laborious task often requiring many weeks to determine the lesion volume for an individual brain. MethodsIn this report we present a method for determining the lesion volume within the brain following traumatic brain injury that involves the use of ultrasound imaging. With this process the lesion volume can be determined within a time period of 90 min per brain rather than weeks and months. Moreover, we have developed a pipeline that will combine the cross-sectional ultrasound images of the brain with the Allen Mouse Brain Atlas to provide the precise anatomical structures that are affected by traumatic injury to the brain. The anatomical detail was lastly paired with behavioral data showing neurological deficits correlated with specific areas of brain injury. ResultsThe accuracy and precision of this method was shown to be highly consistent with the traditional histological approach. Additionally, the mapping process and behavioral data show that neurological recovery from 1 to 3 weeks post injury is not correlated with gross anatomical recovery of the TBI lesion in our TBI model. ConclusionTogether these approaches will enhance the pipeline for processing brain tissue in experimental conditions where the lesion volume is an important outcome parameter and provide more high resolution information about the identity of the damaged regions of the brain.

Autoradiographic labelling of metabotropic glutamate type 2/3 receptors in the hemi-parkinsonian rat brain

L-3,4-dihydroxyphenylalanine (L-DOPA) is the treatment of choice for Parkinson’s disease (PD) motor symptoms, but its chronic use is hindered by complications such as dyskinesia. Pre-clinical studies discovered that activation of metabotropic glutamate type 2 and 3 (mGlu2/3) receptors alleviates L-DOPA-induced dyskinesia. To gain mechanistic insight into the anti-dyskinetic activity of mGlu2/3 activation, we performed autoradiographic binding with [3H]-LY-341,495 in brain sections from L-DOPA-treated 6-hydroxydopamine (6-OHDA)-lesioned rats that developed mild or severe dyskinesia, as well as L-DOPA-untreated 6-OHDA-lesioned and sham-lesioned animals. In the ipsilateral hemisphere, mildly dyskinetic 6-OHDA-lesioned rats showed a decrease in [3H]-LY-341,495 binding in the entopeduncular nucleus (EPN, 30 % vs sham-lesioned rats, P<0.05), globus pallidus (GP, 28 % vs sham-lesioned rats, P<0.05; 23 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001), and primary motor cortex (49 % vs sham-lesioned rats, P<0.05; 45 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001). Severely dyskinetic 6-OHDA-lesioned rats exhibited an increase in binding in the primary motor cortex (43 % vs mildly dyskinetic 6-OHDA-lesioned rats, P<0.05). In the contralateral hemisphere, mildly dyskinetic 6-OHDA-lesioned rats harboured a decrease in binding in the EPN (30 % vs sham-lesioned rats; 24 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05), GP (34 % vs sham-lesioned rats, P<0.05; 23 % vs L-DOPA-untreated 6-OHDA-lesioned rats, P<0.001), and primary motor cortex (50 % vs sham-lesioned rats; 44 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05). Severely dyskinetic 6-OHDA-lesioned rats presented a decrease in binding in the GP (30 % vs sham-lesioned rats; 19 % vs L-DOPA-untreated 6-OHDA-lesioned rats, both P<0.05). Abnormal involuntary movements scores of 6-OHDA-lesioned animals were positively correlated with [3H]-LY-341,495 binding in the ipsilateral striatum, ipsilateral EPN, ipsilateral primary motor cortex and contralateral primary motor cortex (all P<0.05). These results suggest that alterations in mGlu2/3 receptor levels may be part of an endogenous compensatory mechanism to alleviate dyskinesia.

Synergistic action of vitamin D3 and A on motor activity regulation in mice model of extrapyramidal syndrome: Correlational insights into astrocyte regulation, cytokine modulation, and dopaminergic activity

BackgroundExtrapyramidal syndromes (EPS) represent neurological side effects of antipsychotic medications, characterized by motor disturbances. While previous studies have indicated the neuroprotective effects of vitamin D and A against EPS, the underlying mechanisms of this protection remain unclear. MethodsTwenty-four adult mice were categorized into four groups: positive and negative control groups, one receiving a dopamine antagonist, and the other receiving both a dopamine antagonist and vitamins D and A. Sections of the corticobasal loop, specifically the motor cortex (M1) and basal nuclei (CPu), were prepared for Immunohistochemistry (IHC) and stained with Glial Fibrillary Acidic Protein (GFAP) to visualize reactive astrocytes. ELISA assays for TNF-α, IL-6, IL-4, IL-13, and dopamine levels were performed on homogenized brain sections. ResultsThe EPS group exhibited a significant increase in TNF-α and IL-6 levels in M1 and CPu. Treatment with dopamine agonists and vitamin D&A resulted in significant reductions in IL-6 levels. Only the Vitamin D&A group showed a significant decline in TNF-α. The EPS group recorded significant decreases in IL-4 and IL-13, with IL-13 significantly elevated in the dopamine agonist and Vitamin D&A groups. IL-4 was notably increased in the Vitamin D&A groups. Dopamine concentration significantly declined in the EPS group, with improvements observed in the groups treated with dopamine agonists, and vitamin D&A. Reactive astrocytes were significantly expressed in the M1 and CPu of the EPS group but poorly expressed in other groups. ConclusionsEPS is linked to astrocyte activation, an upsurge in pro-inflammatory cytokines, a decline in anti-inflammatory cytokines, and dopamine in the corticobasal loop. Administration of vitamin D3 and A was found to suppres pro-inflammatory cytokines and repress anti-inflammatory cytokines associated with astrocyte activation.

Astroglial Activation Is Exacerbated in a Down Syndrome Mouse Model

Down syndrome (DS), also known as trisomy 21, is one of the most common chromosomal disorders associated with intellectual disability. Mouse models are valuable for mechanistic and therapeutic intervention studies. The purpose of this study was to investigate astroglial anomalies in Dp16, a widely used DS mouse model. Brain sections were prepared from one-month-old Dp16 mice and their littermates, immunostained with an anti-GFAP or anti-S100B antibody, and imaged to reconstruct astroglial morphology in three dimensions. No significant difference in the number of astrocytes was found in either the hippocampal CA1 region or cortex between Dp16 and WT mice. However, the average astroglial volume in Dp16 was significantly (P < 0.05) greater than that in WT, suggesting the astroglial activation. Reanalysis of the single-nucleus RNA sequencing data indicated that the genes differentially expressed between WT and Dp16 astrocytes were associated with synapse organization and neuronal projection. In contrast, in vitro cultured neonatal astrocytes did not exhibit significant morphological changes. The expression of Gfap in in vitro cultured Dp16 astrocytes was not increased as it was in in vivo hippocampal tissue. However, after treatment with lipopolysaccharides, the inflammatory response gene IFNβ increased significantly more in Dp16 astrocytes than in WT astrocytes. Overall, our results showed that the increase in astrogliogenesis in DS was not apparent in the early life of Dp16 mice, while astrocyte activation, which may be partly caused by increased responses to inflammatory stimulation, was significant. The inflammatory response of astrocytes might be a potential therapeutic target for DS intellectual disability.

Pre-clinical evaluation of 99mTc-labeled chalcone derivative for amyloid-β imaging post-head trauma.

Aβ42 plaque formation is one of the preliminary pathologic events that occur post traumatic brain injury (TBI) which is also among the most noteworthy hallmarks of AD. Their pre symptomatic detection is therefore vital for better disease management. Chalcone-picolinic acid chelator derivative, 6-({[(6-carboxypyridin-2-yl)methyl](2-{4-[(2E)-3-[4-(dimethyl amino)phenyl]prop-2-enoyl]phenoxy}ethyl)amino}methyl)pyridine-2-carboxylic acid, Py-chal was synthesized to selectively identify amyloid plaques formed post head trauma using SPECT imaging by stable complexation to 99mTc with > 97% efficiency without compromising amyloid specificity. The binding potential of the Py-chal ligand to amyloid plaques remained high as confirmed by in vitro binding assay and photophysical spectra. Further, the Py-chal complex stained amyloid aggregates in the brain sections of rmTBI mice model. In vivo scintigraphy in TBI mice model displayed high uptake followed by high retention while the healthy rabbits displayed higher brain uptake followed by a rapid washout attributed to absence of amyloid plaques. Higher uptake in brain of TBI model was also confirmed by ex vivo biodistribution analysis wherein brain uptake of 3.38 ± 0.2% ID/g at 2min p.i. was observed for TBI mice model. This was followed by prolonged retention and more than twofold higher activity as compared to sham mice brain. This preliminary data suggests the specificity of the radiotracer for amyloid detection post head trauma and applicability of 99mTc labeled Py-chal complex for TBI-induced β-amyloid SPECT imaging.

Improved Detection of Tryptic Peptides from Tissue Sections Using Desorption Electrospray Ionization Mass Spectrometry Imaging.

DESI-MSI is an ambient ionization technique used frequently for the detection of lipids, small molecules, and drug targets. Until recently, DESI had only limited use for the detection of proteins and peptides due to the setup and needs around deconvolution of data resulting in a small number of species being detected at lower spatial resolution. There are known differences in the ion species detected using DESI and MALDI for nonpeptide molecules, and here, we identify that this extends to proteomic species. DESI MS images were obtained for tissue sections of mouse and rat brain using a precommercial heated inlet (approximately 450 °C) to the mass spectrometer. Ion mobility separation resolved spectral overlap of peptide ions and significantly improved the detection of multiply charged species. The images acquired were of pixel size 100 μm (rat brain) and 50 μm (mouse brain), respectively. Observed tryptic peptides were filtered against proteomic target lists, generated by LC-MS, enabling tentative protein assignment for each peptide ion image. Precise localizations of peptide ions identified by DESI and MALDI were found to be comparable. Some spatially localized peptides ions were observed in DESI that were not found in the MALDI replicates, typically, multiply charged species with a low mass to charge ratio. This method demonstrates the potential of DESI-MSI to detect large numbers of tryptic peptides from tissue sections with enhanced spatial resolution when compared to previous DESI-MSI studies.

Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories.

Brain banks provide small tissue samples to researchers, while gross anatomy laboratories could provide larger samples, including complete brains to neuroscientists. However, they are preserved with solutions appropriate for gross-dissection, different from the classic neutral-buffered formalin (NBF) used in brain banks. Our previous work in mice showed that two gross-anatomy laboratory solutions, a saturated-salt-solution (SSS) and an alcohol-formaldehyde-solution (AFS), preserve antigenicity of the main cellular markers (neurons, astrocytes, microglia, and myelin). Our goal is now to compare the quality of histology and antigenicity preservation of human brains fixed with NBF by immersion (practice of brain banks) vs. those fixed with a SSS and an AFS by whole body perfusion, practice of gross-anatomy laboratories. We used a convenience sample of 42 brains (31 males, 11 females; 25-90 years old) fixed with NBF (N = 12), SSS (N = 13), and AFS (N = 17). One cm3 tissue blocks were cut, cryoprotected, frozen and sliced into 40 μm sections. The four cell populations were labeled using immunohistochemistry (Neurons = neuronal-nuclei = NeuN, astrocytes = glial-fibrillary-acidic-protein = GFAP, microglia = ionized-calcium-binding-adaptor-molecule1 = Iba1 and oligodendrocytes = myelin-proteolipid-protein = PLP). We qualitatively assessed antigenicity and cell distribution, and compared the ease of manipulation of the sections, the microscopic tissue quality, and the quality of common histochemical stains (e.g., Cresyl violet, Luxol fast blue, etc.) across solutions. Sections of SSS-fixed brains were more difficult to manipulate and showed poorer tissue quality than those from brains fixed with the other solutions. The four antigens were preserved, and cell labeling was more often homogeneous in AFS-fixed specimens. NeuN and GFAP were not always present in NBF and SSS samples. Some antigens were heterogeneously distributed in some specimens, independently of the fixative, but an antigen retrieval protocol successfully recovered them. Finally, the histochemical stains were of sufficient quality regardless of the fixative, although neurons were more often paler in SSS-fixed specimens. Antigenicity was preserved in human brains fixed with solutions used in human gross-anatomy (albeit the poorer quality of SSS-fixed specimens). For some specific variables, histology quality was superior in AFS-fixed brains. Furthermore, we show the feasibility of frequently used histochemical stains. These results are promising for neuroscientists interested in using brain specimens from anatomy laboratories.

In vitro characterization of [125I]HY-3-24, a selective ligand for the dopamine D3 receptor.

Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [125I]HY-3-24. In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. HY-3-24 showed high potency at D3R (Ki = 0.67 ± 0.11 nM, IC50 = 1.5 ± 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R Ki = 86.7 ± 11.9 nM and D4R Ki > 1,000). The Kd (0.34 ± 0.22 nM) and Bmax (38.91 ± 2.39 fmol/mg) values of [125I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [125I]HY-3-24. Autoradiography results demonstrated that [125I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. These results suggest that [125I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [125I]HY-3-24 can be used as the specific D3R radioligand.

Long-term Elevation of Complement Factors in Cerebrospinal Fluid of Patients With Borna Disease Virus 1 Encephalitis.

Borna disease virus 1 (BoDV-1) causes rare but severe zoonotic infections in humans, presenting as encephalitis. The case-fatality risk is very high and no effective countermeasures have been established so far. An immunopathology is presumed, while data on immune responses in humans are limited. Evidence of a role of the complement system in various neurological disorders and in viral infections of the central nervous system is increasing and specific inhibitors are available as therapeutic options. In this study, we investigated factors of the complement system in the cerebrospinal fluid (CSF) of patients with BoDV-1 infections (n = 17) in comparison to noninflammatory control CSF samples (n = 11), using a bead-based multiplex assay. In addition, immunohistochemistry was performed using postmortem brain tissue samples. We found an intrathecal elevation of complement factors of all complement pathways and an active cascade during human BoDV-1 infections. The increase of certain complement factors such as C1q was persistent, and C3 complement deposits were detected in postmortem brain sections. Intrathecal complement levels were negatively correlated with survival. Further investigations are warranted to clarify whether targeting the complement cascade by specific inhibitors might be beneficial for patients suffering from severe BoDV-1 encephalitis.

Reliable detection of RNA in hippocampus sections of mice by FISH up to a post-mortem delay of 24 h

Proteins can be successfully localized in post-mortem (PM) brain tissue sections if the time until PM tissue sampling is not too long. In this study, we show that this also applies to the localization of RNA and in particular to the RNA of microglia-specific receptor proteins using the probes and the RNAscope™ Multiplex Fluorescent Detection Kit v2 from Advanced Cell Diagnostics. Brains were removed from killed mice after different PM delays and processed into paraffin sections. In sections of brains from animals whose cadavers had been kept at room temperature (21 °C) before tissue removal, ubiquitously expressed RNAs of genes with low to high expression levels (Polr2a, PPIB, and UBC) were reliably detected in the brain sections even if tissue removal was delayed by up to 48 h. In addition, microglia-specific G protein-coupled receptor RNA (Gpr34, P2ry12) could be reliably assigned to microglia by simultaneous labeling of the microglia with microglia-specific antibodies (Iba1 or P2ry12). Only after a delay of 48 h until tissue removal were the receptor RNA signals significantly lower. The reduction in receptor RNA signals could be delayed if the animal cadavers were stored at 4 °C until the brains were removed. Tissue sections of PM brain samples allow the spatial and cellular localization of specific RNA, at least if the sampling takes place within the first 24 h of PM.

415 Intraoperative Molecular Imaging of Gliomas using Indocyanine-Conjugated Choline Kinase Alpha Inhibitor

OBJECTIVES/GOALS: Distinguishing tumor tissue from normal brain parenchyma remains a major challenge during the resection of gliomas, leading to the persistence of tumor cells. This study aims to assess the choline kinase alpha-targeting fluorophore JAS239 as a novel fluorescent agent to intraoperatively visualize gliomas in an orthotopic murine model. METHODS/STUDY POPULATION: The human glioblastoma-derived U87 MG-Luc2 cell line will be intracranially implanted in nude mice and tumor growth will be assessed using bioluminescence imaging. After 14 days, the mice will be treated with either antiangiogenic therapy (10 mg/kg bevacizumab, twice/week) or saline (control). Tumor growth will be monitored until 21-28 days after initial implantation, at which point JAS239 (4.0 mg/kg, 90 min before sacrifice) and Evans Blue (4 ml/kg, 60 min before sacrifice) will be administered. The mice will be sacrificed, and their brains will be harvested and sectioned for near-infrared imaging. The brain sections will be processed for histopathologic analysis, allowing for the correlation of observed fluorescence with the distribution of tumor and comparison of signal-to-background ratios. RESULTS/ANTICIPATED RESULTS: JAS239 is an indocyanine-based choline mimetic (excitation 745 nm, emission 775 nm) that has been shown to cross the blood-tumor barrier (BTB) in rodent glioblastoma studies. PET imaging with choline-based radiotracers like 18F-choline has also been shown to delineate both contrast-enhancing tumor (CET) and non-contrast-enhancing tumor (NCET) regions, supporting the hypothesis that JAS239 will be able to visualize heterogeneous glioma tissue in our mouse model. Evans Blue is a passive dye in the visible light spectrum (excitation 620 nm, emission 680 nm) expected to only fluoresce in CET regions due to the disruption of the BTB. JAS239 is expected to fluoresce in both CET and NCET regions, which will be assessed by the fluorescence in mice treated with bevacizumab (expected to renormalize the BTB and model NCETs). DISCUSSION/SIGNIFICANCE: JAS239 may allow for real-time visualization of heterogeneous glioma tissue, which is important because there are no current intraoperative imaging agents for NCETs. Future research and clinical translation of this class of agents may allow surgeons to maximize the safe resection of gliomas, improving progression-free and overall survival rates.

4R-cembranoid suppresses glial cells inflammatory phenotypes and prevents hippocampal neuronal loss in LPS-treated mice.

Chronic neuroinflammation has been implicated in neurodegenerative disease pathogenesis. A key feature of neuroinflammation is neuronal loss and glial activation, including microglia and astrocytes. 4R-cembranoid (4R) is a natural compound that inhibits hippocampal pro-inflammatory cytokines and increases memory function in mice. We used the lipopolysaccharide (LPS) injection model to study the effect of 4R on neuronal density and microglia and astrocyte activation. C57BL/6J wild-type mice were injected with LPS (5 mg/kg) and 2 h later received either 4R (6 mg/kg) or vehicle. Mice were sacrificed after 72 h for analysis of brain pathology. Confocal images of brain sections immunostained for microglial, astrocyte, and neuronal markers were used to quantify cellular hippocampal phenotypes and neurons. Hippocampal lysates were used to measure the expression levels of neuronal nuclear protein (NeuN), inducible nitrous oxide synthase (iNOS), arginase-1, thrombospondin-1 (THBS1), glial cell-derived neurotrophic factor (GDNF), and orosomucoid-2 (ORM2) by western blot. iNOS and arginase-1 are widely used protein markers of pro- and anti-inflammatory microglia, respectively. GDNF promotes neuronal survival, and ORM2 and THBS1 are astrocytic proteins that regulate synaptic plasticity and inhibit microglial activation. 4R administration significantly reduced neuronal loss and the number of pro-inflammatory microglia 72 h after LPS injection. It also decreased the expression of the pro-inflammatory protein iNOS while increasing arginase-1 expression, supporting its anti-inflammatory role. The protein expression of THBS1, GDNF, and ORM2 was increased by 4R. Our data show that 4R preserves the integrity of hippocampal neurons against LPS-induced neuroinflammation in mice.

Alteration of mechanical stresses in the murine brain by age and hemorrhagic stroke.

Residual mechanical stresses, also known as solid stresses, emerge during rapid differential growth or remodeling of tissues, as observed in morphogenesis and tumor growth. While residual stresses typically dissipate in most healthy adult organs, as the growth rate decreases, high residual stresses have been reported in mature, healthy brains. However, the origins and consequences of residual mechanical stresses in the brain across health, aging, and disease remain poorly understood. Here, we utilized and validated a previously developed method to map residual mechanical stresses in the brains of mice across three age groups: 5-7 days, 8-12 weeks, and 22 months. We found that residual solid stress rapidly increases from 5-7 days to 8-12 weeks and remains high in mature 22 months mice brains. Three-dimensional mapping revealed unevenly distributed residual stresses from the anterior to posterior coronal brain sections. Since the brain is rich in negatively charged hyaluronic acid, we evaluated the contribution of charged extracellular matrix (ECM) constituents in maintaining solid stress levels. We found that lower ionic strength leads to elevated solid stresses, consistent with its unshielding effect and the subsequent expansion of charged ECM components. Lastly, we demonstrated that hemorrhagic stroke, accompanied by loss of cellular density, resulted in decreased residual stress in the murine brain. Our findings contribute to a better understanding of spatiotemporal alterations of residual solid stresses in healthy and diseased brains, a crucial step toward uncovering the biological and immunological consequences of this understudied mechanical phenotype in the brain.

Parvalbumin interneuron deficiency in the prefrontal and motor cortices of spontaneously hypertensive rats: an attention-deficit hyperactivity disorder animal model insight.

Attention deficit hyperactivity disorder (ADHD) is characterized by impairments in developmental-behavioral inhibition, resulting in impulsivity and hyperactivity. Recent research has underscored cortical inhibition deficiencies in ADHD via the gamma-aminobutyric acid (GABA)ergic system, which is crucial for maintaining excitatory-inhibitory balance in the brain. This study explored postnatal changes in parvalbumin (PV) immunoreactivity, indicating GABAergic interneuron types, in the prefrontal (PFC) and motor (MC) cortices of spontaneously hypertensive rats (SHRs), an ADHD animal model. Examining PV- positive (PV+) cells associated with dopamine D2 receptors (D2) and the impact of dopamine on GABA synthesis, we also investigated changes in the immunoreactivity of D2 and tyrosine hydroxylase (TH). Brain sections from 4- to 10-week-old SHRs and Wistar Kyoto rats (WKYs) were immunohistochemically analyzed, comparing PV+, D2+ cells, and TH+ fiber densities across age-matched SHRs and WKYs in specific PFC/MC regions. The results revealed significantly reduced PV+ cell density in SHRs: prelimbic (~20% less), anterior cingulate (~15% less), primary (~15% less), and secondary motor (~17% less) cortices. PV+ deficits coincided with the upregulation of D2 in prepubertal SHRs and the downregulation of TH predominantly in pubertal/postpubertal SHRs. Reduced PV+ cells in various PFC regions could contribute to inattention/behavioral alterations in ADHD, while MC deficits could manifest as motor hyperactivity. D2 upregulation and TH deficits may impact GABA synthesis, exacerbating behavioral deficits in ADHD. These findings not only shed new light on ADHD pathophysiology but also pave the way for future research endeavors.

Advancing Biological Research: New Automated Analysis of Immunofluorescence Signals

In recent years, optical imaging and efficient computational approaches have improved the ability to analyse and understand biological phenomena. Immunofluorescence (IF) is a widely used immunochemical technique that provides information about protein localisation and expression levels. However, the manual analysis of IF images can present important limitations, such as operator workload and interpretative bias. Thus, the development of automated tools for IF signal computation is crucial. Several software programs have been proposed to address this challenge, but there is still a need for more accurate and reliable systems. In this work, we present Q-IF, a software for automatically measuring cellular IF signals with an intuitive and easy-to-use interface. We describe the software and validate its results in different biological scenarios using SH-SY5Y neuroblastoma cells, human fibroblasts, and rat brain sections. The Q-IF system automatically carries out the entire process, from IF signal quantification to statistical analysis, thus evading operator biases and speeding up the analysis workflow. Our results demonstrate the accuracy and reliability of the Q-IF system, highlighting its potential as a valuable tool for IF analysis in biological research.