Attachment 2 Neurohistology SOW.pdf

Statement of Work (SOW) Project Title: Neurohistology of Mouse Brain Hemispheres

Organization: Uniformed Services University of the Health Sciences (USUHS)

Date: March 16, 2026

1. Background

Survivors of hematopoietic ARS (H-ARS) are at risk of developing delayed effects of acute radiation exposure (DEARE), a spectrum of chronic illnesses involving multiple organ systems and occurring months to years after radiation exposure. DEARE remains an understudied area of radiation injury. Importantly, data regarding central nervous system (CNS) DEARE following H-ARS is scarce, which is necessary to determine if survivors would be cognitively capable of returning to duty. In an on-going AFRRI Intramural project, we assessed a variety of cognitive and mood tests in male and female mice that survived H-ARS following mixed field radiation exposure (65%/35% n/γ and 35%/65% n/γ). We tested these animals from 30 days to 6 months following exposure. Several important results were found in exposed mice, including: 1) significant increases in anxiety-like behavior for at least 3 months following exposure, 2) altered patterns of locomotor activity, and 3) decreased problem-solving abilities. Interestingly, these deficits were similar in irradiated mice regardless of the type of mixed field they were exposed to. Finally, these results suggest that H-ARS survivors experience CNS DEARE, which could significantly limit their ability to return to their pre-exposure military duties.

Importantly, there are currently no medical countermeasures (MCMs) specifically for CNS DEARE, nor do we know if our current MCMs can attenuate or prevent these cognitive and mood changes in survivors. One method to assess the potential mechanisms of CNS DEARE is to evaluate molecular and cellular brain changes. The current project will assess changes in neuroinflammation (Iba1 staining), senescence and autophagy (LAMP-1 staining), and the integrity of the blood brain barrier, all potential mechanisms underlying these radiation-induced behavioral changes that could be utilized for future MCM development to protect the brain and preserve cognitive function in exposed individuals.

2. Scope of Work

The vendor shall to embed, slice, and stain multiple mouse brains together on the same plane of slicing, where 40 mouse brains are embedded together in a single block, sectioned on the coronal plane, slices collected, and stained for cellular markers of neuroinflammation, autophagy, and blood brain barrier compromise, cover slipped, and returned to USUHS for digital imaging and quantification. Slices shall be approximately 30- 40 microns thick. Staining shall be done at a standard interval throughout the entire cortex on every 10-15th section to quantify signals across multiple brain regions. Finally, unstained sections shall be appropriately preserved and sent back to our lab for potential staining in the future.

3. Tasks/Requirements

Task 1: Embedding and sectioning into a single block, freeze-sectioned at 35μ thickness in the Coronal plane and collected into cups containing antigen preserve solution. The first series of 12 cups shall collect the tissue through the cortex (cerebrum) of the specimen, ~9mm, sectioned at 35μ. The second series of 12 cups shall collect tissue through the cerebellum/brainstem, ~5mm, sectioned at 35μ.

Task 2: The following staining shall be performed through the cortex (cerebrum) on every 12th section spaced at 420μm intervals, yielding ~21 2 x 3 slides per stain series per block:

-- Blood Brain Barrier Compromise (BBB) Stain to reveal compromise of the blood brain barrier, counterstained with Thionine to reveal cell bodies.

- Iba1 Immunofluorescence to reveal microglia, double-labeled with Lamp-1 Immunofluorescence to reveal autophagy, counterstained with Blue fluorescent stain to reveal nucleic acid.

Task 3: Placement of coverslips, labeling, serial ordering and placement of slides in slide boxes.

Tasks 4: The slides shall be shipped to USUHS. 22 cups of cut, unstained, free-floating sheets of sections shall be sent in antigen preserve solution, approximately 30 days after the shipment of the stained slides. This delay will allow time to review the stained slides and request additional stains on this reserve of sections. 10 cups from the first series shall contain ~21 sheets of sections each and 12 cups from the cerebellum/brainstem shall contain ~12 sheets of sections each. The total number of sheets of sections to be sent unstained and free-floating in Antigen Preserve is ~354 per block.

4. Deliverables

Free Floating Sections Finished Slides

5. Period of Performance

The vendor shall include delivery schedule after contract award and receipt of the 40 Mouse Brain Hemispheres from USUHS.

6. Place of Performance

The vendor’s facility.

7. Government Furnished Equipment (GFE) and Facilities

USUHS shall provide 40 appropriately fixed and stored mouse brain hemispheres.

8. Performance Standards:

This study shall be conducted in accordance with Good Laboratory Practice (GLP) principles, However, the complete set of GLP requirements such as QA audited reports are not required.