Subjects:• Adult male Sprague-Dawley rats, approximately 275-325g (N=70)• Rats were pair-housed and provided with 20g (+/-1) of food per day.

• Purina Test Diets compounded 2% dried whole blueberry powder into standard rodent chow. Controls were fed the same diet, without the blueberry enhancement.

• Subjects in the blueberry diet groups were provided the special diet beginning the day of injury or sham-injury and were continued for the rest of their survival time or 6 weeks, whichever came first.

Surgery and Brain Injury: • A 4mm modified syringe hub was implanted over a craniotomy hole covering the right parietal cortex.

• Two bone screws were placed 1mm rostral to bregma and 1mm caudal to lamba and dental acrylic was applied over the

entire device, leaving the hub accessible, to secure the device to the skull.

• Skin was stitched and rats were allowed 24 hours to recover from the general anesthetic.

• Mild/moderate fluid percussion injury (2.12-2.26 atm, measured by an oscilloscope) was administered under isoflurane

• Surgical stitches were opened and the female fitting of the surgically implanted hub was connected to the male fitting of

injury device.

• A pendulum-style hammer was released, impacting a fluid filled tube, creating a fluid pulse that impacted the brain.

• Injured rats had significantly suppressed reflexes, indicating loss of consciousness consistent with a mild/moderate TBI.

Startle Chamber: • Animals were placed in a Coulbourn acoustic startle chamber one day prior to euthanasia.

• Eight trial types were presented in a series of random blocks:

Duffy, K.B., Spangler, E.L., Devan, B.D., Guo, Z., Bowker, J.L., Janas, A.M., et al. (2008). A blueberry-enriched diet provides cellular protection

against oxidative stress and reduces a kainate-induced learning impairment in rats. Neurobiology of Aging, 29, 1680-1689. [Abstract]

Esposito, L., Raber, J., Kekonius, L., Yan, F., Yu, G.Q., Bien-Ly, N., et al. (2006). Reduction in mitochondrial superoxide dismutase modulates

Alzheimer’s disease-like pathology and accelerates the onset of behavioral changes in human amyloid precursor protein transgenic mice.

Journal of Neuroscience, 26, 5167-5179.

Hoge, C. (2008, July). Mild traumatic brain injury and post-concussive symptoms in military personnel. Abstract and presentation, The 16 Annual

National Neurotrauma Symposium, Orlando, Fl.

Langlois, J.A., Rutland-Brown, W., & Thomas, K.E. (2004). Traumatic brain injury in the United States: Emergency department visits,

hospitalizations, and deaths. Atlanta: Centers for Disease Control and Prevention, National Center for Injury Prevention and Control.

Stromberg, I, Gemma, C., Vila, J., & Bickford, P.C. (2005). Blueberry- and spirulina- enriched diets enhance striatal dopamine recovery and induce

a rapid, transient microglia activation after injury of the rat nigrostriatal dopamine system. Experimental Neurology, 196, 298-307.

Tadros, M.G., Khalifa, A.E., Abdel-Naim, A.B., & Arafa, H.M. (2005). Neuroprotective effect of taurine in 3-nitropropionic acid-induced

experiemental animal model of Huntington’s disease phenotype. Pharmacology, Biochemistry, and Behavior, 82, 574-582.

Torri, E., Lemos, M., Caliari, V., Kassuva, C.A., Bastos, J.K., & Andrade, S.F. (2007). Anti-inflammatory and antinociceptive properties of blueberry

extract (Vaccinium corymbosum), The Journal of Pharmacy and Pharmacology, 59, 591-596.

Wiley, J.L., Compton, A.D., Pike, B.R., Temple, M.D., McElderberry, J.W., & Hamm, R.J. (1996). Reduced sensorimotor reactivity following

traumatic brain injury in rats. Brain Research, 716, 47-52.

- No significant injury impairments were found for ACS or PPI in rats with mild/moderate TBI- Wiley et al. found significant moderate/severe TBI induced deficits in ACS and PPI,

concluding deficits were likely due to higher structure influence (such as the hippocampus)

on the startle reflex (1996).- The current study revealed no significant hippocampus-mediated deficiencies in a water

maze task (data not shown), indicating the rat hippocampi were not significantly impaired

by the milder TBI.- Groups fed the blueberry diet had significantly better PPI than those on the control diet.

- Only true at 90db – likely the 80db was not perceived by the older rats- The sham-blueberry group had better PPI, but not the injured-blueberry group.- Separate analysis of the 3 week and 3 month groups revealed the improvement in PPI was

only present at the 3 week time point. There are 2 possible explanations:

1. PPI decline due to age (fits with an acoustic perception deficit with 80db pre-pulse)

2. The cessation of the blueberry diet at 6 weeks may indicate the improved PPI is only

present while the diet is being consumed- The 1 year survival group will be divided, with half eating the blueberry diet at the time of

ACS/PPI testing and the other half remaining on the control diet. They will be tested with

multimodal startle to avoid interference due to reduced acoustic perception.

Mild/Moderate TBI should impair both ACS and PPI measures

A blueberry-enhanced diet should attenuate TBI-related

deficits in ACS and PPI

INTRODUCTION

HYPOTHESES

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

Fluid Percussion Injury Device Subjects in Acoustic Startle Chamber

Blueberry Diet and Brain Injury Effects on Pre-Pulse Inhibition in Rats

Cynthia Gibson, Krystin Jansen, and Mandy MooreWashington College, Psychology Department

Acoustic Startle Response (ASR):

- Tests of the initial vs. final startle response revealed equal habituation across groups and ages

- Tests of peak startle (ACS) revealed no significant differences across groups or ages

Pre-Pulse Inhibition (PPI):

- A 4 (group) x 2 (survival time) ANOVA on peak PPI with an 80db pre-pulse tone revealed a significant main effect for time

- F(1,57) = 5.20, p < .05, ηp2 = .084 = .084

- Animals tested at 3 weeks (M = 68.44, SD = 183.39) had significantly better PPI than at 3 months (M = .0606, SD = 39.4)

- A 4 (group) x 2 (diet) ANOVA on peak PPI with a 90db pre-pulse tone revealed a significant main effect for group

- F(3,62) = 2.729, p < .05, ηp2 = .117

- The Sham-BB diet group had significantly better PPI (M = 81.45, SD = 22.55) than either the Sham-Control diet (M = 42.38,

SD = 54.94) or the Injured-Control diet (M = 28.38, SD = 83.03). The Injured-BB diet group (M = 53.29, SD = 31.51) was

not significantly different from any other group.

- The effects of diet were tested separately for the 3 week survival groups and the 3 month survival groups

- The 3 week blueberry groups had significantly better 90db peak PPI than the control diet groups, t(34) = 2.021, p = .05.

- The 3 month blueberry and control diet groups were not significantly different, t(32) = 1.391, p > .05

Traumatic Brain Injury (TBI):

• Nearly 1.5 million annual TBIs with 90,000 new cases of long term disability yearly in America (Langois, Rutland-Brown, & Thomas, 2004). TBI is also the signature injury of military personal in Iraq and Afghanistan (Hoge, 2008).

• TBI results in excessive neurotransmitter release, diffuse axonal injury, and alters intracellular cascades, mitochondria, and glial cells.

• Post-TBI pathologies include oxidative stress, inflammation, and cell loss in the hippocampus and other vulnerable brain area

Blueberries and TBI::

• Blueberries reduce pain and oxidative stress (Stromberg, Gemma, Vila, & Bickford, 2005; Torri et al., 2007), as well as attenuating hippocampal cell loss due to inflammatory brain injury (Duffy et al., 2008).

• Extensive research into Alzheimer’s disease, aging, and various brain injury models have demonstrated cognitive and physiological benefits to a blueberry-enriched diet, but no studies have examined blueberry dietary supplementation in TBI.

• Antioxidants may have beneficial effects on pre-pulse inhibition of startle, as demonstrated in an injection model of Huntington’s disease (Tadros, Khalifa, Abdel-Naim, & Arafa, 2005) and a transgenic mouse model of Alzheimer’s disease (Esposito et al., 2006).

Acoustic Startle, Prepulse Inhibition, and TBI:

• Extensive TBI research has focused on hippocampus-mediated deficits in learning and memory, with less focus on simpler measures such as the startle reflex.

• Brainstem and spinal cord structures are involved in the acoustic startle reflex (ACS), while limbic structures are implicated in regulation of pre-pulse inhibition (PPI), a model of cognitive gating of ACS.

• The cortex, hippocampus, and various limbic structures are selectively vulnerable to TBI damage.

• Moderate/Severe TBI in rats produced deficits in ACS and PPI at 8 and 30 days post-injury (Wiley et al., 1996)

• Wiley et al. ruled out motor impairment and auditory structural damage as explanations, but PPI deficits could have been due to severely reduced ACS

STUDY DESIGN

3 Week Survival

3 Month Survival

1 Year Survival

Sham - Control Diet 11 8 8

Injured – Control Diet 8 9 10

Sham – BB Diet 10 7 8

Injured – BB Diet 7 10 10

Numbers in cells represent final number of subjects per group. BB = Blueberry.Grayed groups are still in progress.

Figure 3.Figure 3. Mean 90db PPI was not significantly different Mean 90db PPI was not significantly different for blueberry and control diets 3 months post-injury or for blueberry and control diets 3 months post-injury or sham injury. Error bars = +/-SDsham injury. Error bars = +/-SD

1. Initial Startle (1st 6 trials @ 120db) 3. Startle Stimulus (120db) 5. 90db pre-pulse tone only 7. 90db pre-pulse + Startle Stimulus

2. No Stimulus 4. 80db pre-pulse tone only 6. 80db pre-pulse + Startle Stimulus 8. Final Startle (last 6 trials @ 120db)

Figure 1.Figure 1. Time means for peak 80db PPI. *p<.05. Time means for peak 80db PPI. *p<.05. Error bars = +/-SEMError bars = +/-SEM

Figure 2.Figure 2. Mean 90db PPI for blueberry and control Mean 90db PPI for blueberry and control diets 3 weeks post-injury or sham injury. *p<.05. diets 3 weeks post-injury or sham injury. *p<.05. Error bars = +/-SEMError bars = +/-SEM

Figure 2.Figure 2. Group means for peak 90db PPI. *p<.05. Group means for peak 90db PPI. *p<.05. Error bars = +/-SEMError bars = +/-SEM

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