# The roles of the necroptotic and excitotoxic pathways in diisopropyl fluorophosphate-induced neuronal necrosis

> **NIH VA I01** · VA GREATER LOS ANGELES HEALTHCARE SYSTEM · 2021 · —

## Abstract

Our objective in this project is to determine the destructive seizure-inducing effects of the
systemically administered organophosphate diisopropyl fluorophosphate (DFP) on rat brain, and to
determine the roles of the necroptotic and exitotoxic pathways of programmed necrosis in DFP-
induced neuronal necrosis. For Specific Aim 1 we will determine the time course and degree of
neuronal necrosis (using the hematoxylin and eosin [H & E] stain) and nuclear translocation of
receptor-interacting protein (kinase)-1 (RIP1), receptor-interacting protein (kinase)-3 (RIP3) and
mixed lineage kinase domain-like protein (MLKL), using immunofluorescence microscopy, 6, 24 and
72 h after DFP administration in dorsal hippocampal CA1-CA3 and hilus. For Specific Aim 2 we will
determine if co-administration of diazepam (the current standard of care) with either the RIP1 inhibitor
7-Cl-O-necrostatin-1 (7-Cl-O-nec-1) or the NMDA-receptor antagonist MK-801 (dizocilpine), given 60
min after DFP injection, are neuroprotective and if 7-Cl-O-nec-1 reduces nuclear translocation of
RIP1, RIP3 and MLKL in the hippocampus 24 h (or 6 or 72 h, depending on the results of Specific
Aim 1) after DFP injection. For Specific Aim 3 we will determine if co-administration of diazepam and
both 7-Cl-O-nec-1 and MK-801 60 min after DFP injection provides greater hippocampal
neuroprotection than either given by itself. For Specific Aim 4 we will determine if 7-Cl-O-Nec-1
and/or MK-801 (depending on the results of Specific Aims 2 and 3) provides greater neuroprotection
and better spatial learning and memory in DFP-treated rats compared to those given vehicle two one
months after DFP-induced status epilepticus (SE). The methods that we will use are (1) in situ
transcardiac perfusion-fixation of the brain with 4% phosphate-buffered paraformaldehyde for light
and immunofluorescence microscopy; (2) the stereological optical fractionator for unbiased estimates
of hippocampal CA1-3 and hilar neurons after 60-µm-thick coronal sections are stained with H & E for
light microscopy (Specific Aims 1-4) or RIP1, RIP3 and MLKL antibodies for immunofluorescence
microscopy (Specific Aim 2); (3) subcellular fractionation and western blots as a separate approach to
confirm the degree of nuclear translocation of RIP1, RIP3 and MLKL from DFP-induced SE (Specific
Aim 2); and (4) the Barnes maze, to test rats’ spatial learning and memory two one months after DFP
injection, and to correlate findings with the numbers of remaining normal neurons in dorsal
hippocampus (Specific Aim 4). We will analyze the data with multi-factor ANOVA and post-hoc t-tests,
using pooled standard deviations and α = 0.05. We will determine if inhibition of RIP1 is
neuroprotective and if optimal neuroprotection is provided by both 7-Cl-O-nec-1 and MK-801 given
together, which, if true, should influence the standard of care following organophosphate exposure.

## Key facts

- **NIH application ID:** 10124989
- **Project number:** 5I01BX004632-02
- **Recipient organization:** VA GREATER LOS ANGELES HEALTHCARE SYSTEM
- **Principal Investigator:** DENSON G FUJIKAWA
- **Activity code:** I01 (R01, R21, SBIR, etc.)
- **Funding institute:** VA
- **Fiscal year:** 2021
- **Award amount:** —
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10124989

## Citation

> US National Institutes of Health, RePORTER application 10124989, The roles of the necroptotic and excitotoxic pathways in diisopropyl fluorophosphate-induced neuronal necrosis (5I01BX004632-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10124989. Licensed CC0.

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