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Preliminary Studies

Strategies for Success

Pitfalls to Avoid

New Research Plan Components

Specific Aims

Background and Significance

Preliminary Studies/Progress Report
Research Design and Methods
Inclusion Enrollment Report
Progress Report Publication List
Human Subjects Sections.
protections, women/minorities, enrollment,

Other Research Plan Sections.

animals, select agents, MPI, consortium,
support, resource sharing



New Research Strategy Section

Current Application

New Application

Background and

Research Strategy
a. Significance
Research Design and b. Innovation
c. Approach

Studies for New
Progress Report

Preliminary Studies
PIs preliminary studies, data and/or
experiences pertinent to the
Helps to establish likelihood of
success of proposed application


Changes to Application Forms and Instructions

Preliminary Studies:
Strategies for Success
Describe research team expertise
this now goes in Biosketch

Organize PS by specific aim

Summarize significance of PS for proposed
Present PS findings concisely
Describe relevance of PS findings for
proposed study
Use consistent format for PS

Expertise of Research Team

The P.I. and her team of co-investigators have conducted several
studies that directly support this intervention. Dr. Insel, PhD, RN has
spent the last 12 years investigating the role of cognitive function in
older persons and its relationship to adherence.

Gillis Einstein, PhD, co-investigator is an expert in cognitive aging

and prospective memory research as demonstrated in the background
in the description of foundational behavioral laboratory findings that
are ready for translation.
Daniel G. Morrow, PhD, co-investigator is an expert in cognitive aging
and medication adherence. Dr. Morrow has expertise in patient
centered instructions including text comprehension to promote
medication adherence and in health literacy to promote health literacy.
He specifically addressed issues of text comprehension and literacy
throughout the intervention manual.

Dr. Insel and her co-investigators have been collaborating for the past
8 years in different capacities and participate in ongoing
communication. All have played an active role in developing the grant

Expertise of Research Team

Drs. Moore & Hockenberry have over 20 years of clinical and research
experience in pediatric oncology. They have served as PI on extramurally
funded research projects. Dr. Moore has conducted clinical studies on the
long-term effects of CNS treatment and laboratory-based studies on
mechanisms of tissue injury. Dr. Hockenberry is well known for her
research on fatigue and sleep disturbances among children with cancer.

Dr. Montgomery has 35 years of basic science laboratory research and

extensive expertise in the use of a wide variety of assays and methods.
Examples of his research expertise include SDS-PAGE, 2-D
electrophoresis, PCR/RT-PCR, DNA/RNA agrose gels, Northern/Southern
blotting, and DNA fragmentation assays.

Preliminary Studies:
Strategies for Success
Organize PS by specific aim
Use consistent format for PS
Concise presentation of PS findings
Use figures and tables to summarize PS
results (but make them reader friendly!)

Organize Studies by Specific Aim

Preliminary Studies Related to Aim 1:
Study 1: Title:
Funded by: (Source and dates)
Purpose/Aims/Hypotheses (summarized)
Sample (good idea to include gender and ethnicity)
Methods: (summarized)
Results: (only include those that are relevant)
Relevance to proposed study:

Preliminary Studies Supporting

Significance of the Proposed Project
Methotrexate-Induced Oxidative Stress in Children with ALL.
Purpose was to investigate oxidative stress as a mechanism of
methotrexate-induced CNS injury.
Oxidative stress was measured by:
Significant increases in oxidized PC were observed during the
most intensive phase of ALL therapy (F =2.98; p = 0.04) and were
greatest in children with high-risk ALL who received the most
intensive CNS treatment (F = 12.67; p < 0.001). F2-isoprostanes
increased from diagnosis to the consolidation (H = 16.9; DF = 3, p
Findings support significance of proposal by providing evidence
for methotrexate-induced markers of oxidative stress in the CNS.
We will extend these findings by studying expression of genes
involved in oxidative stress and oxidant defense in tissue
samples obtained from the hippocampus.

Preliminary Studies Supporting Feasibility

Rat Model to Study Methotrexate-Induced CNS Injury.
Purpose was to a) develop a rat model that employed
intraventricular administration of methotrexate, and b) assess
effects of methotrexate on cells in the CNS.
Summary of Methods: Fisher 344 male rats were anesthetized
and methotrexate (4 mg/kg) was administered through the left
lateral ventricle with an Alzet osmotic pump and brain infusion
cannula. After rats regain consciousness, they were returned to
their cages, and examined daily for signs of CNS toxicity,
infection, neurological damage, and/or failure to thrive.
Animals received either methotrexate (n = 4) or artificial CSF (n
= 3), and were euthanized 2 days later. Histological studies of
sections from brain cortex were assessed for astrocyte
reactivity using glial fibrillary acidic protein (GFAP), a
commonly used marker to identify astrocytosis in a wide range
of neurological injury studies. Figure 1 shows GFAP positive
cells in brain cortex taken after 2 days of treatment with
methotrexate or artificial CSF.

Preliminary Studies Supporting Feasibility

Figure 1: GFAP Staining of Brain Cortex from Rats Treated
with Methotrexate (a) or Artificial CSF (b)

a. Methotrexate

b. Control

Summarize PS Results with

Figures & Tables

GFAP positive cells per microscopic field were quantified. Results from the
Mann-Whitney Rank Sum Test showed that the median number of GFAP
positive cells per field was significantly greater in methotrexate treated
animals compared to controls (T = 1694, p <0.0001). Figure 2 below is a box
plot of GFAP positive cells per field in methotrexate treated (MTX) and control

Findings support feasibility by demonstrating development of the rat

model and positive staining for astrocytosis with GFAP and show that
the drug increased astrocytosis

Strategy: Use PS to Support Methods

Aim 2: Investigate gene expression changes in
selected regions of the hippocampus following
intraventricular administration of methotrexate.
Animals in the experimental group will be treated
with methotrexate as described above, and animals
in the control group will be treated with artificial
CSF. Brains will be removed as described in Aim 1
above and delivered to the Tissue Acquisition and
Cellular/Molecular Share Service for paraffin
embedded sectioning. Sections of the hippocampus
will be stained with Cresyl Violet. Regions that are
either densely populated or relatively deplete of
neurons will be removed using LCM (See Figure 3).

Preliminary Studies Supporting Methods

H & E Staining Of Hippocampus Regions Enriched and
of Nuclei
H & E Staining
Of Deplete
Enriched and Relatively Deplete of Nuclei

Nuclei Enriched

Nuclei Depleted

Summary of Preliminary Studies

In summary, preliminary studies demonstrate that cognitive
processes, specifically a composite of executive function and
working memory thought to underlie prospective memory, predict
medication adherence among self-managing community dwelling
older adults.
Studies on patient centered design instructions indicate that
comprehension and memory for medication taking instructions
improve with this support. Studies on the influence of external
aids, specifically the medication chart used in this intervention,
reveal that they improve older adult understanding of provider
A preliminary test of a multifaceted prospective memory
intervention based on memory strategies indicates that the inhome nurse delivered intervention can be implemented in a
community setting with older adults and adherence is improved.
These findings form the basis for the expanded, refined
intervention in this application.

Preliminary Studies:
Pitfalls to Avoid
Findings should be foundational for
proposed study or not included
Findings should not be redundant with
proposed study
Findings should be directly relevant to
proposed work and demonstrate how
findings guide further work