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Chronic Cognitive Dysfunction After Traumatic
Brain Injury Is Improved With A
Phosphodiesterase 4B Inhibitor
Titus DJ, Wilson NM, Freund JE, Carballosa MM, Sikah KE, Furones C, Dietrich WD, Gurney
ME, Atkins CM. 2016. Journal of Neuroscience 36(27):7095-7108
Patricia Olson, MS3, PhD
SIGN Journal Club
October 11, 2016
Disclosures
There are no potential conflicts of interest to disclose.
Background
• TBI
• Learning and Memory
– Hippocampus
– LTP and synaptic plasticity
• cAMP, CREB, PDE4B
Traumatic Brain Injury
• Leading cause of death in North America for individuals ages 145yo
– motor vehicle accidents, violence, combat-related trauma
• Incidence: 538.2 per 100,000
• Prevalence of long-term disability related to TBI in the US: 3.25.3 million; approx 1-2% of population
Clinical Diagnosis
• Glasgow Coma Scale (GCS) is universally accepted tool for TBI
classification
– 15-point scale: Eye, Motor, Verbal
– Limited by confounding factors:
• sedation, paralysis, intoxication, endotracheal intubation
• Full Outline of UnResponsiveness (FOUR) Score
– 17-point scale: Eye, Motor, Brainstem, Breathing
• CT-based grading scales: Marshall scale and Rotterdam scale
Predicts the risk of increased ICP and outcome in adults accurately, but lacks
reproducibility in patients with multiple types of brain injury.
Pathophysiology: Primary Brain Injury
• Occurs at the time of trauma
– Direct impact
– rapid acceleration/deceleration
penetrating injury
blast waves
• External mechanical forces transfer to intracranial contents
– Shearing mechanisms lead to diffuse axonal injury (DAI)
– Focal cerebral contusions
– Extra-axial hematomas
Pathophysiology: Secondary Brain Injury
• Cascade of molecular injury mechanisms
• Initiated at the time of initial trauma, can continue for hours or days
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Neurotramitter-mediated excitotoxicity of glutamate causing free-radical injury
Cellular injury causing second-messenger dysfunction
Electrolyte imbalances
Mitochondrial dysfunction
Inflammatory responses
Apoptosis
Secondary ischemia from vasospasm, focal microvasuclar occulsion, vascular injury
Cellular injury causing second-messenger dysfunction:
• TBI results in CREB activation deficits during learning
• Decreased cAMP signaling and reduced cAMP-responseelement-binding protein (CREB) activation.
Current Treatment:
• Phosphodiesterase 4 Inhibitors
– Rolipram (pan-PDE4 inhibitor)
– Roflumilast
• Side effects include
– GI: Diarrhea, Nausea, Emesis, Abdominal Pain, Decreased Appetite,
Weight loss
– CNS: Headache, Insomnia, Depression
– Worsens pathology despite improving cognition
Problem:
Over 3 million individuals are living w/chronic TBI disabilities and 7085% report learning and memory impairments.
Hypothesis:
A selective PDE4B inhibitor (A33) can decrease learning and memory
impairments and thus decreasing the expression of hippocampal LTP,
thus reversing the learning deficits induced by TBI.
Memory: Implicit and Explicit
Implicit Memory
• Associative and Non-Associative
– Fear Conditioning involves the Amygdala
– Operant conditioning involves the striatum and cerebellum
– Classical Conditioning, sensitization, and habituation involve the
sensory and motor systems
– Simple reflexes involve the spinal cord
Explicit Memory:
– Four processes: Encoding, Consolidation, Storage, Retrieval
The Hippocampus and LTP: the Storage of Declarative Memory
A high-frequency train of stimuli applied to
fibers afferent to the hippocampus increase
the amplitude of EPSPs in the target neurons.
The increase lasts for days or weeks and
requires activation of several afferent axons
together. This property has been termed
cooperativity, and it results from the
requirement of NMDA receptors that
glutamate bind them and that the cell be
hypopolarized, the binding opens the channel
and the hypopolarization displaces Mg++ that
blocks the channel lumen. Also required is
that the pre-and post-synaptic cells both be
active at the same time. This property is
termed associativity.
LTP, Learning and Memory, and CREB
• Long-term memory is represented at the cellular level by
activity-dependent modulation of both the function and the
structure of specific synaptic connections
• these synapses depend on the activation of specific patterns
of gene expression
• Inhibition of transcription or translation can block the
formation of long-term memory
Early Phase vs. Late Phase
NMDAR = coincidence detectors
Problem:
Over 3 million individuals are living w/chronic TBI disabilities and 7085% report learning and memory impairments.
Hypothesis:
A selective PDE4B inhibitor (A33) can decrease learning and memory
impairments and thus decreasing the expression of hippocampal LTP,
thus reversing the learning deficits induced by TBI.
Methods
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Fluid-Percussion Injury Surgery
Drug administration
Electrophysiological recordings
Eliza, Western
Fear Conditioning, Water Maze
Fluid-Percussion Injury
• Reproducible, Scalable (mild, moderate, severe injury)
• Brain injury is induced by a rapid (~20ms) fluid pulse through a
craniotomy onto the intact dura that follows the inner curvature
of the skull and creates an elastic decompression of the brain
• The mechanical forces disrupt cell membranes, blood vessels, and
neuronal processes.
Electrophysiology
• 12 weeks post-surgery
• ipsilateral hippocampus dissected into 440μM transverse slices
• Field EPSPs were recorded from CA1 striatum radiatum
– Hippocampal slices were treated w/vehicle or 300nM A33
– Stepwise current increases from 20 to 240μA
• Paired-pulse facilitation was measured
• LTP was induced by high-frequency stimulation using a single
train of 100 pulses delivered at 100Hz
Schaffer Collateral Pathway
The experimental setup for demonstrating
LTP is shown here:
Recordings are made intracellularly from CA1
neurons of the hippocampus while
stimulation is applied to Schaffer Collaterals
of CA3 neurons. The amplitudes of the EPSPs
in the CA1 neurons are shown in B. For a
single stimulus, the amplitude of the EPSPs is
plotted at 100%. When a train of stimuli is
applied instead, the amplitude of the EPSPs
augment to about 150%, whereas with 4
such trains, the amplitude increases to 250%.
Many people think that long-term
potentiation is an example of Hebb’s rule at
work and that it is the physiological basis of
memory.
Hypothesis
A selective PDE4B inhibitor (A33) can decrease learning and memory
impairments and thus decreasing the expression of hippocampal LTP,
thereby reversing the learning deficits induced by TBI.
Fig 1A: A33 bath
application to TBI
slices reversed the
depression in basal
synaptic transmission.
Fig 1B: A33 did not
rescue the
decrease in PPF
caused by TBI
Fig 1C: A33
improved LTP
expression
impaired by
TBI
Fig 1D and 1E: A33 significantly rescued the maintenance phase
of LTP and these changes were not due to differences in
depolarization during the tetanus
Early Phase vs. Late Phase
NMDAR = coincidence detectors
A33 and TNF-α
• PDE4B regulates expression of proinflammatory cytokine TNF-α
in circulating leukocytes and resident MΦ
• To determine whether TNF-α was elevated in the injured brain
at 6 hours post-injury, the ipsilateral parietal cortex and
hippocampus were assayed by ELISA for TNF-α levels
• 0.33mg/kg A33 administered 30 min post-injury and 1 h before
animal was sacked.
PDE4B regulates expression of proinflammatory cytokine
TNF-α in circulating leukocytes and resident MΦ
Fig 2B: A33 treatment
significantly reduced
TNF-α levels in TBI
animals
Fig 2C and 2D: Although PDE4 isoforms were present in the
hippocampus 3 months post-surgery, there was not a difference in
protein levels between sham and TBI animals
Does PDE4B inhibition by A33 improve chronic
learning and memory deficits after TBI
• Post-surgery recovery: 3 months w/o treatment
• Animals received A33 (0.3mg/kg i.p.) or vehicle 30 minutes
before training
• Then tested serially on:
– fear conditioning 12 and 16 weeks post-surgery
– water maze 13 weeks post-surgery
– working memory 14 weeks post-surgery
A33 or vehicle were administered 30 minutes before behavioral training on the
indicated days (arrows).
Fig 3B—Contextual Test
At both 24h and 1 month after
training, TBI animals treated
w/vehicle demonstrated
significantly less contextual fear
conditioning compared w/sham
animals treated w/vehicle or TBI
animals treated w/ A33
Fig 3C—Cued Test
There was no significant
interaction of surgery x drug
treatment for either time
point in baseline freezing;
however, when freezing in
response to the cue was
assessed, there was
significant interaction of
surgery x drug treatment at
both time points.
Fig 3D: These fear
conditioning differences were
not due to changes in shock
sensitivity since the minimal
shock threshold to elicit a
flinch, jump, or vocalization
was not significantly different
between animal groups.
To determine whether A33 would improve learning in another
hippocampal-dependent learning task, the same cohort of animals were
tested in a water maze at 13 weeks post-surgery using a hidden platform.
Escape latency and
path length to find the
hidden platform were
significantly impaired
in TBI animals
compared to sham
animals.
There was a main
effect of A33 drug
treatment for path
length during
acquisition.
A33 treatment significantly improves long-term spatial
memory retention.
Fig 3F and 3G: TBI animals treated with
vehicle spent significantly less time in
the target quadrant compared with
sham animals treated with vehicle or
A33, or TBI animals treated with A33.
The number of platform zone crossings
were also significantly less in TBI
animals treated with vehicle versus
A33, or sham animals treated with
vehicle or A33.
Fig 5: Working memory
A modified water maze to assess spatial
working memory at 14 weeks post-surgery:
The animals were trained to locate a hidden
platform that remains invariant only between
pairs of trials by 5sec.
TBI animals had significantly longer escape
latencies compared with sham animals and
A33 treatment improved working memory in
both sham and TBI animals.
Fig 6: Cortical and
hippocampal volume was
measured at the
completion of behavioral
testing: Significant cortical
atrophy was observed in
TBI animals.
There was no effect of A33
treatment.
These results indicate that
intermittent A33
treatment did not improve
pathology in the chronic
TBI recovery period.
Conclusions
• A33 (a selective PDE4B inhibitor) has procognitive benefits
when administered 3 months postinjury
• A33 was also shown to
– Improve hippocampal LTP
– Cross the BBB at relevant concentrations
– Improve performance in learning tasks
• Tx w/a PDE4B inhibitor may address hypofunction in the
signaling pathways that use CREB phosphorylation, namely
those involved in memory formation in the hippocampus
Take-Away
Short-term treatment w/a PDE4B inhibitor improves synaptic
plasticity, learning and memory when delivered at the time of
learning at 3 months after TBI, and thus can alter chronic
reduction of neurogenesis
Strengths
• Comprehensive study
– Molecular to behavioral
• Translatable
Weaknesses
Neither the study nor the discussion addressed
• The role of the amygdala in fear conditioning
• The role of synaptic function in the context of an ensemble of
neurons in a neural circuit in the formation of complex
memories
• The role of modulatory transmitters (DA, Ach) in the retrieval
process.
References
• Hemphill JC, Phan N. 2016. Traumatic brain injury: Epidemiology,
classification, and pathophysiology. Up to Date.
• Kandel ER. 2012. The molecular biology of memory: cAMP, PKA, CRE,
CREB-1, CREB-2, and CPEB. Molecular Brain 5:14.
• Lifshitz, J. 2009. Animal Models of Acute Neurological Injuries pp369384
• Mann, M. 2011. Learning and Memory.