Clinical trial NCT02512809
Conditions: Toxicity Hydrocephalus
Sponsor: Nationwide Children's Hospital
Start date: 2015-07-01; Completion date: 2017-10-31
Enrollment: 25 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The investigators will quantify inflammatory biomarkers in cerebrospinal fluid (CSF) and serum of children with hydrocephalus who are undergoing ventriculoperitoneal shunt placement under isoflurane anesthesia.

Detailed description:
Background \& Significance. Each year, millions of children receive general anesthesia. Isoflurane, a GABA type A (GABAA) receptor agonist is an inhaled anesthetic commonly used in clinical anesthesia practice worldwide. Anesthetics traditionally have been assumed to be safe as long as severe hypotension and hypoxia are avoided. Interfering with the balance of excitatory and inhibitory neurotransmitters in the developing brain may interfere with the formation of synaptic connections and enhance the normal apoptotic processes that lead to neuronal pruning and may induce neuroinflammation. Such enhanced neuronal pruning and neuroinflammation could lead to excessive neuronal loss at critical times during brain development and consequently cause later learning disabilities. Recent reports of neurotoxicity induced with isoflurane have triggered significant concern about the safety of this agent.

Investigations into neuroinflammation and apoptosis after anesthetic exposure in human children have been limited due to ethical and methodological concerns. Studies that have been done have been largely retrospective, population-based studies. Results from these studies have been mixed, some showing a decline in neurocognitive performance, some showing no change. Further, the vast majority of prospective, hypothesis-driven research has been undertaken in animal models. Developing a clinically relevant animal model and testing resultant hypotheses in humans are critical steps in determining the underlying cause of these changes as well as identifying possible therapeutic targets. The investigators hypothesize that piglets exposed to commonly used anesthetics will exhibit increased neuroinflammation when compared with controls. It is further hypothesized children undergoing neurosurgery with isoflurane anesthesia will show evidence of increased central nervous system inflammation. Finally, the investigators hypothesize that significantly less robust inflammation will be seen in patients undergoing MRI with general anesthesia (no surgery), indicating a role of surgical stress in the modulation of isoflurane-induced neuroinflammation.

Clinical/Translational Investigation (NCH): Surgery Group. Patients aged 0-5 years undergoing general anesthesia for ventriculoperitoneal shunt (VPS) repair will have CSF and serum sampled at the beginning of surgery (after induction but before surgical incision) and at the end of surgery (after closure but before emergence). Each of these patients will be randomized to a standardized anesthetic. An additional CSF sample will be taken prior to post anesthesia care unit (PACU) discharge. These samples will be analyzed as described above. Control Group. Patients undergoing diagnostic MRI under general anesthesia for non-neurologic pathology will receive a standardized anesthetic. Serum will be collected at the beginning and end of the procedure and will be analyzed for inflammation as above.

Short term goals: Demonstrate that isoflurane has a role in modulation of neuroinflammation in humans. Determine if surgical stress also has a role in modulation of this inflammation. Develop a clinically relevant animal model for hypothesis-driven anesthetic neuroinflammation research.

Long term goals: Identify which anesthetic regimens, if any, are safe for use in the developing brain. Identify therapeutic targets for prevention or treatment of anesthetic-induced neuroinflammation. Obtain independent federal funding for future research and establish an experimental neuroscience program on the Nationwide Children's campus.

Arms: arm 1: Pediatric patients, aged 0-5 years, diagnosed with hydrocephalus undergoing a surgical (non-bedside) shunting procedure with general anesthesia. Pts will receive a standardized general anesthetic with isoflurane. arm 2: Pediatric patients, aged 0-5 years, diagnosed with hydrocephalus undergoing a surgical (non-bedside) shunting procedure with general anesthesia. Pts will receive a standardized general anesthetic with dexmedetomidine and remifentanil infusions.. arm 3: Otherwise healthy pediatric patients, aged 0-5 years, undergoing MRI with general anesthesia for evaluation of non-neurologic disease. Patients will receive a standardized general anesthetic with isoflurane.
Interventions: intervention 1: Isoflurane intervention 2: Dexmedetomidine
Intervention descriptions: intervention 1: None intervention 2: None

Protocol documents:
Prot_SAP_000.pdf: 
Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
Title: Isoflurane-induced neuroinflammation in children with hydrocephalus: A bench-to-bedside, translational 
study of molecular pathways and therapeutic approaches. 
 
Overall Hypothesis. Children with hydrocephalus (HC) exposed to isoflurane anesthesia show increased 
neuroinflammatory markers in serum and cerebrospinal fluid when compared to children not exposed to 
isoflurane. Moreover, children undergoing isoflurane anesthesia for diagnostic procedures demonstrate 
increases in serum inflammatory markers despite lack of neuropathology or surgically-induced stress. 
 
Innovation. 
Evaluation of anesthetic risks in human children.  
Clinical, prospective pediatric studies evaluating effects of anesthetic exposure on the central nervous 
system (CNS) are inexistent due to ethical and methodological considerations. 78% of children with HC, once 
treated, go on to suffer neurologic deficits.1-4 Neuroinflammation and neurotoxic effects during anesthesia 
might be in cause.5  
The role of inflammation in HC remains poorly understood. Adult animal models show that active 
inflammation correlates with severity and can be measured in CSF.6 This has not been explored in humans. It 
is critical to quantify HC-induced neuroinflammation, evaluate how anesthesia modulates this inflammation, 
and measure its contributions to HC-related morbidity. Children with HC undergoing ventriculoperitoneal shunt 
(VPS) insertion have been selected as our study population due to a number of circumstances making them 
ideal for this study.  While obtaining CSF and serum for research in healthy children is ethically inappropriate 
and methodologically challenging, CSF sampling is routine care during VPS insertion. CSF cytokine and micro-
RNA (miRNA) levels are used to measure the severity of neuroinflammation. Healthy children scheduled for 
MRI with anesthesia for non-neurologic disease will serve as control.  
CSF cytokine levels are markers of CNS inflammation, and are reported elevated in adult patients 
undergoing VPS insertion after subarachnoid hemorrhage.7 Few studies have evaluated inflammation in 
children with HC, with or without anesthesia.8,9 CSF biomarkers are likely to reflect these changes. An increase 
in CSF cytokine levels in certain types of HC has been reported, but the elevation was not statistically 
significant.19 We plan to stratify the results by age to avoid bias while allowing comparison with previous 
investigations. 
miRNAs represent a novel method to quantify inflammation in biological systems. miRNAs are small 
(18-25 nucleotides) non-coding nucleic acid molecules. They regulate (usually in an inhibitory fashion) gene 
expression at the post-transcriptional level.10-12 500 human miRNA sequences are known, however, specific 
targets of individual miRNAs remain elusive. Although a mystery, miRNA targets may potentially lead to 
therapeutic intervention. miRNAs have a critical role in the development of the CNS, including neural induction 
and differentiation, neural patterning, cell specification, axonal pathfinding, and neural pruning (apoptosis).10-16  
Our model is a novel use of four miRNAs in quantifying neuroinflammatory response in children with 
HC undergoing anesthesia. Elevated CSF miRNAs, particularly the four molecules we plan to measure, have 
been reported in neuroinflammatory conditions.17-19 
The present study will demonstrate neuroinflammation in patients with HC anesthetized with isoflurane 
and determine pathways involved in the neuroinflammatory process. These observations could lead to specific 
targets for therapeutic interventions and contribute to decreasing morbidity and improving quality of life of 
these children. We intend to identify the relationship between neuroinflammation and severity of disease and 
hope to make future therapeutic recommendations.  
This novel, clinical, human model will help elucidate the role of inflammation in human HC and examine 
the effects of one anesthetic agent. Results will help expand our understanding of anesthesia-induced 
neuroinflammation. If neuroinflammation is demonstrated, the potential for treatment innovations could 
dramatically enhance the quality of life of children with this devastating disease. 
 
Translational Significance 
Each year, millions of children receive general anesthesia. Isoflurane, a GABA type A (GABAA) 
receptor agonist, is an inhaled anesthetic commonly used in clinical anesthesia worldwide. Anesthetics have 
traditionally been assumed to be safe as long as severe hypotension and hypoxia are avoided. Anesthesia 
may induce neuroinflammation and alter the formation of normal synaptic connections. These changes could 
lead to excessive neuronal loss or disruption of synaptogenesis at critical times during brain development and 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
consequently cause cognitive dysfunction. Recent reports of neurotoxicity induced with isoflurane and other 
anesthetics in animal models have triggered significant concerns about the safety of these agents.20 
Basic research data on the effects of isoflurane on brain development originate mainly from rodent 
studies21-23 with very limited work in animals with gyrencephalic brains, such as swine and non-human 
primates.24,25  These studies have significant limitations and vagaries in interpretation, having used solely an 
antibody-based immunohistochemical (IHC) technique to identify the presence of triggered “apoptotic” cells. 
Unequivocal end-stage apoptosis and cell death were not clearly demonstrated histologically in these 
studies.24,25  Many previous studies showing anesthesia-induced neurotoxicity may have overestimated the 
true degree of neuronal injury and its true clinical implications. Most importantly, no known study has examined 
the effects of anesthetics on neuroinflammation, an arguably more important process when considering lasting 
neurodegeneration that leads to later neurocognitive deficits. CNS inflammatory response in children after 
anesthesia exposure has become our primary focus in an attempt to elucidate the true consequence of these 
agents on child brain development and cognitive function.  
 
Specific Aims 
1. To establish the neuroinflammation profile in positive-control (lipopolysaccharide (LPS) treated) 
and isoflurane-treated neonatal piglets. 
Hypothesis: Piglets treated with LPS (an E. coli endotoxin) have increased peripheral blood and 
CSF proinflammatory cytokine levels; increased peripheral blood and CSF proinflammatory microRNA 
(miRNA) concentrations; and increased brain microglial activation when compared to controls. This 
“positive control” will allow comparison with piglets exposed to isoflurane.  
Further, piglets administered isoflurane anesthesia show increased peripheral blood and CSF 
cytokine levels; increased peripheral blood and CSF proinflammatory miRNA concentrations; and 
increased brain microglial activation when compared to controls. 
 
2. To establish the neuroinflammation profile in human children with hydrocephalus before and 
after VPS insertion with isoflurane anesthesia. 
Hypothesis: Children with hydrocephalus have increased peripheral blood and CSF cytokine 
and proinflammatory miRNA levels after isoflurane anesthesia when compared with blood and CSF 
sampled before anesthesia and surgery, despite surgical relief of hydrocephalus. This exacerbation of 
neuroinflammation shall persist for 24 hours and longer.  
It is further hypothesized that anesthesia-induced neuroinflammation will be reduced or 
eliminated using a total intravenous anesthesia (TIVA) technique with dexmedetomidine and 
remifentanil instead of isoflurane. This suggests a direct role of isoflurane in the neuroinflammatory 
process. Children undergoing inhaled anesthesia for magnetic resonance imaging (MRI) without 
tracheal intubation or surgery will serve as a control. 
 
 
 
 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
Summary Illustration. Please see Figure 1 for a graphic representation of the experiment. 
 
 
 
 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
Preliminary Data. Two years have 
been invested in developing a valid 
piglet model to study anesthesia-
induced neurotoxicity. We can now 
safely and effectively use this model 
to easily test a number of different 
conditions and anesthetic regimens. 
We are confident it is the most 
clinically relevant animal model for 
neuro-inflammation investigations. It 
reproduces exactly the same surgical 
and anesthetic conditions children 
undergo in the operating room. The 
equipment 
includes 
a 
standard 
anesthesia machine used clinically 
for the care of children at NCH 
(Figure 2). All routine physiological 
monitoring devices are used. Piglets 
receive an inhalational induction of 
anesthesia, a peripheral intravenous 
catheter, and tracheal intubation. 
Their 
lungs 
are 
mechanically 
ventilated and they are monitored 
throughout the experimental and recovery period. An infusion of dextrose-containing isotonic fluid is given. 
Arterial blood gases and chemistries are monitored every hour 
to prevent acid-base abnormalities and electrolyte disturbances. 
Normothermia is actively maintained. Perioperative antibiotics 
are used at an appropriate per kilogram human dose to prevent 
wound infection. The piglets are demographically similar. Care is 
taken to prevent confounders that could make interpretation of 
data problematic. Please see Table 1 for a summary of average 
vital signs and selected laboratory values for this animal study.  
To date, 27 animals have been studied. Microscopic 
evaluation of the brains of these animals was performed by a 
pediatric neuropathologist at NCH, and has yielded several 
exciting 
findings. 
Surprisingly, 
the 
typical 
“footprint” 
of 
neuroinflammation was not observed either with anesthesia or 
LPS. One may speculate that brain tissue was harvested too 
early after the insult, i.e., 48 hours. It is possible that when 
harvesting brain tissue after 48 hours of convalescence, 
manifestations of acute inflammation are not yet apparent. In one rodent study, hallmarks of inflammation and 
neurodegeneration were not seen until 72 hours had elapsed, and in some cases it took as long as 14 days.26 
Cytokines, however, have been shown to be significantly elevated a few hours after the initial insult.27 
Therefore, to identify early inflammation, performing ELISA for inflammatory cytokines (TNF-α, IL-1β, MCP-1, 
PGE2, and NF-ĸB p65), and qPCR for proinflammatory miRNAs will be essential. Immunofluorescent labeling 
will be used to identify activated inflammatory cells in the brain tissue of these animals. It is most likely to 
support early inflammation, which will guide in making further improvements to our research model. For this 
reason, we have elected to allow 72 hours for convalescence after experimental exposure. 
The most concerning findings on microscopic examination of piglets’ brains were twofold: 1) a 
significant morphological and structural change in the isoflurane and LPS treated animals when compared to 
controls, and 2) an acute ischemic infarct in one of three isoflurane-treated animals that was not present in 
either of the other groups.  


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
The 
first 
concerning 
finding is related to a significant 
change in the morphology and 
structure in the entorhinal cortex 
and CA1 hippocampus. In control 
animals, prevalent clusters of 
small, round cells in the neuronal 
layers of both areas were noted 
(Figure 3, panels A, B, and C). 
Based on review of the literature 
and consultation with a pediatric 
neuropathologist, they are most 
likely immature neurons. They 
could also represent microglial 
cells, 
astrocytes, 
or 
oligodendrocytes in development. 
These 
cells 
were 
virtually 
obliterated in isoflurane and LPS-
treated animals. The loss of 
these cells was seen in every 
LPS or isoflurane-treated animal 
studied to date. We hypothesize 
that the cells may be dying as a 
result 
of 
neuroinflammation, 
ischemia, or both when animals 
are treated with isoflurane or 
LPS. Identifying their implications 
and the mechanisms involved in 
their loss will be essential to 
determine the significance of this 
observation. To achieve this goal, 
immunohistochemical 
labeling 
will be utilized for immature 
neurons 
(NeuN), 
astrocytes 
(GFAP), microglia (MHC II), and 
oligodendrocytes (Olig2). 
The second and most 
concerning 
finding 
in 
the 
isoflurane-treated animals was an 
acute basal ganglia ischemic 
infarct. Our colleague pediatric neuropathologist is of the opinion that the infarct, which was seen in the basal 
ganglia, is 12-24 hours old (Figure 4, panels D, E, and F). This is extremely concerning given the fact that 
brain infarcts in children are often “subclinical” and found incidentally when cerebral imaging is performed later 
for another reason. Kim et al. reported that 21% of children had abnormalities, some of which were described 
as “unspecified white matter abnormality of unclear etiology”.28 So-called “silent” strokes are shockingly 
common in children with sickle cell disease.29,30 It is not impossible that anesthetics, particularly when 
repeated, may be causing areas of ischemic infarction that are never detected because they do not cause 
typical stigmata of stroke, such as weakness, facial droop, dysarthria, or gait disturbance.  We are concerned 
that if subclinical infarcts are occurring, they may be partially responsible for the neurocognitive deficits that 
have been reported in children after multiple anesthetics.31 It is vital to learn whether this is a common effect of 
anesthesia in animals with complex (gyrencephalic) brains in order to make anesthesia safer for infants and 
children. 
 
Materials and Methods  
 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
Aim 1 (Tzagournis Medical Research Facility (OSU)): All neonatal piglet (10-14 days old, 2-3 kg) 
experimentations will be performed following The OSU Institutional Animal Care and Use Committee (IACUC) 
policy (protocol number 2014A00000018, already underway).  Animals will arrive in the vivarium 24 hours 
before experimentation for acclimation to the environment.  
The positive control (LPS) arm receives E. coli endotoxin intraperitoneally (100 mcg/kg). This induces 
acute inflammation with minimal morbidity.32 For the experimental (isoflurane) arm, animals are 
anesthetized with 5% isoflurane in 100% O2 via face cone. Tracheal intubation and mechanical ventilation to 
ensure normocapnia and normoxia are used. Isoflurane 2% in 50% oxygen/50% air is administered during 
vascular cannulation and throughout the study period. The femoral vein and artery catheters are placed via 
surgical cut-down, allowing medication and fluid administration, continuous arterial blood pressure monitoring, 
and blood sampling. End-tidal CO2 and rectal temperature are monitored continuously. Arterial blood gases are 
measured hourly throughout. Anesthesia exposure lasts 3 hours after which vascular catheters are removed. 
The surgical incision is closed and infiltrated with local anesthetic. The piglet is awakened, the trachea 
extubated, and the animal is returned to its cage. Subcutaneous buprenorphine is given every three hours for 
pain management, supervised by experienced animal care personnel. For both arms: based on our 
preliminary observations (see above), piglets are allowed to convalesce for 72 hours to ensure better 
pathological identification of the presence of neuroinflammation. The piglets convalesce in temperature-
controlled cages and receive commercial piglet milk replacer. After 72 hours, anesthesia with 5% isoflurane in 
100% oxygen is given for non-survival surgery. Peripheral blood is obtained via venipuncture and CSF 
collected via lumbar puncture. The CSF and serum are stored at -80oC. Animals receive transaortic 
heparinized phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) and the brains harvested and 
saved for later analysis. 
 
Serum and CSF analysis. Serum and CSF are analyzed for miRNA (miRNA) and inflammatory cytokine 
levels. miR-155, miR-146a, miR-146b, and miR-142-3p are quantified using qPCR. The following cytokines are 
measured using enzyme-linked immunosorbent assay (ELISA): TNF-α, IL-1β, MCP-1, PGE2, and NF-ĸBp65. 
 
Immunohistochemistry. The hippocampus is dissected from the brain and fixed. Hippocampal tissues are 
sectioned to 40µm thickness, mounted on slides, and stained according to established procedures for MHC II, 
GFAP, S100β, and anti-ionized calcium binding adaptor molecule 1. 
 
Aim 2 (Nationwide Children’s Hospital): Human Subjects.  
 
VPS Patients. Children aged 1 month to 16 years with hydrocephalus undergoing VPS insertion will be 
randomized to one of two standardized general anesthetics necessitating tracheal intubation. Clinician’s 
discretion will be used to determine doses and rates of infusion. 
 
Conventional Anesthetic Group: 
 
Induction: 
- Propofol 2-3 mg/kg 
- Fentanyl§ 1-2 µg/kg when indicated 
 
 
 
 
- Rocuronium dose at provider discretion 
Maintenance: - Isoflurane 1-2% in 50% O2 in air or sevoflurane (2-4%) in 50% O2 in air 
- Rocuronium if needed 
- Isotonic fluid 10-30 mL/kg (lactated Ringer’s or normal saline) 
- Neuromuscular blockade reversal: glycopyrrolate 0.01 mg/kg / neostigmine 0.07 
mg/kg or sugammadex 2-4 mg/kg 
- Fentanyl§ at provider discretion 
Total intravenous anesthesia (TIVA) Group:  
 
Induction: 
- Nitrous oxide for IV start (if needed)  
- Fentanyl§ 1-2 µg/kg  
- Dexmedetomidine§ 1 µg/kg (over 10 minutes) 
 
 
 
 
- Rocuronium dose at provider discretion 
 
 
Maintenance: - FiO2: 50% (in 50% air) 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
- Dexmedetomidine§ infusion at a dose of 1-2 µg/kg/hr 
titrated to a BIS of <60 
- Remifentanil infusion at 0.2-0.8 µg/kg/min 
 
 
 
 
- Rocuronium if needed 
- Isotonic fluid 10-30 mL/kg (lactated Ringer’s or normal saline) 
- Reversal of neuromuscular blockade as in isoflurane group 
- Fentanyl§ at provider discretion  
*Rocuronium 1.2 mg/kg for rapid sequence induction if indicated 
§Dexmedetomidine and fentanyl are not approved by the FDA for use in children <18 years, but have 
been routinely used safely in pediatric anesthesia of all ages for years.33-35 An Investigational New Drug 
(IND) application for each drug has been submitted and is pending. 
 
Rocuronium will not be administered until BIS is <60. 
 
 
This dose of dexmedetomidine has been used for many types of procedures in children. At our 
institution, we use a dose range of 0.3 mcg/kg/hr – 3 mcg/kg/hr depending on surgical need. Per our 
usual clinical practice, all doses are titrated based on clinical response. A dose of 1 mcg/kg/hour was 
used in one study for sedation for MRI scans.49 Another study reported doses in the 1-2 mcg/kg/hour 
range for oral surgery.50 Finally, high-dose dexmedetomidine infusions have been reported to be safe in 
children. In fact, one report describes an infant who received sixty times the intended dose with no 
serious sequelae.51,52  
 
MRI Patients. Children 6 months to 10 years undergoing MRI under general anesthesia for non-CNS 
pathology. Standard general anesthesia will include: 
 
Induction:  
- Sevoflurane induction  
 
 
 
 
- Peripheral intravenous catheter  
 
 
 
 
- Placement of an LMA 
 
 
Maintenance:  - FiO2: 50% in air 
- Isoflurane 1-2% in 50% O2 in air 
- Isotonic fluid (lactated Ringer’s or normal saline) 10-30 mL/kg 
- Infants <10kg: D5LR titrated to deliver glucose at 5 mg/kg/min 
 
Note: To ensure anesthesia consistency and reduce a Pearson Coefficient effect, anesthesiologists will be 
recruited to provide anesthesia for the study. 
 
Research Plan  
Rationale for the piglet model. The piglet is a well-accepted study alternative to larger animal models 
used for neuroscience. We pioneered its utilization for pediatric anesthesia neuroinflammation evaluation. 
Rodent and non-human primate models, used in most prior studies, have not proved to be the ideal animal 
models to investigate anesthetic neurotoxicity because they are not closely applicable to human neonates. 
Very few pediatric anesthesia neurotoxicity studies have been done in non-human primates. However, the cost 
is prohibitive and the animals are extremely sensitive to early rearing conditions, particularly stress and 
maternal separation.36-38 The relevance of an animal model must take into consideration the context of human 
pathology by appreciating the brain maturity and pathobiology involved when compared to the human infant. 
Very few non-human primate genes for neurotransmitter receptors have been cloned, meaning that crucial 
receptor-ligand affinities, allosteric modulators, post-translational modifications, alternative splicing variants, 
and receptor subunit compositions, all relevant to anesthetic-mediated neural injury, are unknown for this 
species. On the other hand, porcine GABAA and NMDA receptors have been cloned.39  


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
In contrast to newborn rodents, the percentage of 
adult brain weight at birth in piglets is much closer to that 
of humans (Figure 5).40 The brain of the pig more closely 
resembles the human brain and is gyrencephalic, 
whereas the rodent brain is lissencephalic. Neonatal 
swine cranial geometries including cortical and basal 
ganglia topology are similar to human infants. Important 
similarities between swine and humans have also been 
demonstrated in hippocampus, entorhinal cortex, basal 
ganglia, and brainstem, which are the areas of interest in 
this study.15 The pig and human brain are similar in growth 
patterns36-38 and piglet brain level of development is more 
analogous to that of full-term gestation human newborns 
than to ruminants with prenatal brain growth spurts, or 
rodents with postnatal growth spurts.36 The multilayer 
cortical sub-plate of the developing pig brain and the 
expression pattern of reelin, a glycoprotein that influences 
neuronal migration, are strongly analogous to the 
developing human brain.41,42 Grey and white matter 
patterns and distributions are similar in human neonates 
and piglets, and the maturation of the postnatal pig brain 
is comparable to humans with respect to myelination and 
electrical activity.40 Based on previous work, 10-14 day old piglets correspond to 2 month old human 
infants with respect to the period of rapid brain growth and maturation.40  
Using this clinically relevant pediatric swine model, we propose to evaluate an anesthetic commonly 
used in clinical pediatric anesthesia worldwide. Development of a preclinical animal model to study anesthesia-
related neuroinflammation is critical to identifying safe pharmacologic regimens for infants and children. 
 
Rationale for anesthetic selection (HC patients). For the conventional anesthesia group, isoflurane is 
selected for several reasons. Firstly, isoflurane is the most commonly used inhaled anesthetic for 
neuroanesthesia, likely due to its minimal effects on intracranial pressure and cerebral perfusion pressure.40 
Drugs planned for induction (lidocaine, rocuronium, fentanyl) have not been reported to be neurotoxic or to 
cause neuroinflammation in regimens used clinically. Isoflurane, however, has been shown to be toxic in 
multiple animal studies, whether used alone41 or in combination with other agents.45 We expect isoflurane to 
exacerbate neuroinflammation as evidenced by an increase in CSF inflammatory biomarkers after exposure.  
 
For the TIVA group, we selected an anesthetic regimen designed to reduce or eliminate the anesthetic-
induced neuroinflammatory response. These patients will receive a total intravenous anesthesia (TIVA) with an 
infusion of dexmedetomidine, an α2-agonist and hypnotic agent, and remifentanil, an ultra-short-acting µ opioid 
agonist medication. Neither dexmedetomidine nor remifentanil have been shown to cause neuroinflammation 
or neurotoxicity. In fact, dexmedetomidine may attenuate the inflammatory effects caused by other 
anesthetics.47,48 This anesthesia regimen will help isolate isoflurane as a strong contributor to 
neuroinflammation as opposed to surgical insult. The combination of remifentanil and dexmedetomidine is 
successfully used as a general anesthetic for infants and children undergoing invasive neurological 
procedures.49,50 
 
Rationale for the anesthesia control group (MRI patients). Because direct laryngoscopy, tracheal 
intubation, and surgery are significant hemodynamic events, these patients will not be subjected to these 
physiological stresses and consequently represent an excellent, ethical control group. Anesthesia with 
isoflurane alone is routinely used in children in order to ensure optimal MRI conditions. No other drugs will be 
given. Anesthesia exposure will be similar in terms of drug, dose and duration in these patients exempted of 
intracranial pathology or neuroinflammatory disease. 
 
Human Subjects. Recruitment and Informed Consent. Participants will not be responsible for the cost of the 
study. Neither the patients nor the parents will be compensated for their participation. Seventy-five (75) 
children will be recruited for the study. Parents/guardians will be approached by study personnel during their 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
preoperative visit in the surgery unit (for hydrocephalus patients) or in the MRI suite (for MRI patients). 
Parents/guardians will be informed about this study in which their child could be enrolled and which may help 
improve the anesthesia care of hydrocephalus patients. If they indicate that they are not interested, they will be 
thanked for their time and no further discussion will occur. If they are interested, study information will be 
provided. Written informed consent will be signed if they agree to have their child participate. The option of 
withdrawing their child from the study at any time will be indicated if they become uncomfortable with the 
participation. 
 
Inclusion/Exclusion Criteria for Human Subjects. 
Inclusion Criteria (HC study patients): Pediatric patients, aged 1 month to 16 years, diagnosed with 
hydrocephalus undergoing a primary or revision VPS procedure under general anesthesia. Participants must 
have a parent/guardian who is entitled to provide written informed consent in accordance with human 
investigation committee guidelines. 
 
Exclusion Criteria (HC study patients): Infection of any kind within the last 14 days, treatment in the 
last 48 hours with corticosteroid medications, anticoagulant administration in the last 48 hours, clinically 
unstable patients, patients with an American Society of Anesthesiologists physical status ≥4 (severe, life-
threatening disease), infants born more than 4 weeks premature if less than 2 years of age, infant born earlier 
than 28 weeks gestation if greater than 2 years of age but less than 3 years of age. 
Inclusion Criteria (MRI with general anesthesia: peripheral blood patients; anesthesia but no 
surgery): Otherwise healthy pediatric patients, aged 6 months to 10 years, undergoing MRI with general 
anesthesia for evaluation of non-neurologic, non-inflammatory disease. Participants must have a 
parent/guardian who is entitled to provide written informed consent in accordance with HIC guidelines. 
 
Exclusion Criteria (MRI with general anesthesia: peripheral blood patients; anesthesia but no 
surgery): Infection of any kind within the last 14 days, known central nervous system disease, treatment in the 
last 48 hours with corticosteroid medications, treatment with any drug known to induce or suppress 
inflammation, patients that have an American Society of Anesthesiologists physical status ≥4 (severe, life-
threatening disease), infants born more than 4 weeks premature.  
 
Early Withdrawal Criteria (All Patients): Hypoxemia (arterial oxygen saturation <90% for more than 2 
minutes or <80% for greater than 30 seconds); hypotension (systolic or diastolic blood pressure reduced ≥20% 
from preoperative values for greater than 3 minutes).  
 
Sample Procurement and Analysis.  
VPS Patients: 500µL, the minimum volume of CSF required for testing, will be drawn by the 
neurosurgery team after induction of anesthesia. 5mL of peripheral blood will be obtained before surgery by 
the anesthesia team via the patient’s existing peripheral intravenous (PIV) line. If removal of blood from the PIV 
line is not possible, a peripheral venipuncture will be performed. At the conclusion of surgery, similar samples 
will be obtained.  An additional 5mL sample of peripheral blood will be drawn in the post-anesthesia care unit 
(PACU) as described above. All samples will be transported on ice and stored frozen at -80oC within 30 
minutes of collection pending further analysis. Cytokines (blood and CSF) will be analyzed using ELISA for 
TNF-α, IL-1β, MCP-1, PGE2, and NF-ĸB p65. miR-155, miR-146a, miR-146b, and miR-142-3p will be 
quantified using qPCR.  
 
MRI Patients. Patients without neurologic or systemic inflammatory disease scheduled for diagnostic 
MRI under general anesthesia will be recruited for the study. A standardized anesthetic will be given. 
Peripheral blood will be drawn as above (5mL), before anesthesia, once the peripheral intravenous line is 
placed as part of routine clinical care. At the conclusion of the scan, another sample of peripheral blood (5mL) 
will be drawn via the peripheral intravenous line before the child is awakened. Serum handling and analysis will 
be identical to the above mentioned. 
 
The MRI group will have the same adverse event monitoring as the surgical group: 
 
 
Continuously during MRI 
 
12 hours, 24 hours, and 1 week after MRI 
 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
 
 
 
Additional Clinical Data to be Collected and Reviewed. The following data will also be collected and 
reviewed to minimize confounding variables: patient weight, sex, preoperative hematology and chemistry 
values, intraoperative vital signs, and postoperative vital signs per routine care in anesthesia. 
 
Statistical Analysis. We have consulted with three biostatisticians in the development of this study. The 
sample size (n=25/group) was determined to avoid a Type I (>5%) and Type II (>80%) errors using power 
analysis in order to show a twofold effect size increase in neuroinflammation in the VPS group receiving 
isoflurane when compared to the TIVA group. As a pilot study, the analysis is not adjusted for the use of 
multiple markers or comparisons. Parametric data will be analyzed using a two-tailed Student t-test and 
expressed as P<0.05. Comparison with the control (MRI) group will be performed under the same conditions. 
This applies to both the human and animal portions of the study, as the same markers will be measured in both 
groups. Additionally, we will perform a post-hoc analysis using either a Tukey-Kramer test or the Student-
Newman-Keuls method for multiple comparisons.  
Feasibility: We estimate that at NCH, 2-3 primary VPS surgeries and >30 MRI scans are performed 
under general anesthesia per week, making the recruitment of the necessary sample size within 1 year very 
feasible.    
 
Adverse Events.    
• 
Common adverse reactions seen with dexmedetomidine include hypotension, bradycardia, transient 
hypertension during loading dose, nausea, and dry mouth. These events are usually self-limited and 
resolve without treatment. 
• 
Adverse reactions associated with infusions >24h include acute respiratory distress syndrome (ARDS), 
respiratory failure, and agitation. 
• 
Rare serious adverse events (SAEs) of dexmedetomidine include cardiac arrest. 
• 
Common adverse reactions seen with fentanyl include respiratory depression, apnea, rigidity and 
bradycardia; if these remain untreated, respiratory arrest, circulatory depression or cardiac arrest could 
occur. 
• 
Other less common adverse reactions include hypertension, hypotension, dizziness, blurred vision, 
nausea, emesis, laryngospasm and diaphoresis. 
 
The study will be terminated early if >20% of patients experience hypotension or bradycardia (defined 
as blood pressure or heart rate diminished 20% from baseline for more than one minute). The study will also 
be terminated early if >20% of patients have an adverse reaction at a dexmedetomidine dose of  ≥ 1 
mcg/kg/hr.  Study termination will occur if adverse reactions occur that have a “reasonable possibility” of being 
attributable to the investigational drug(s). “Reasonable possibility” is defined according to 21 CFR 312.32(a) 
under Safety Reporting Requirements for INDs and BA/BE Studies. The investigational drug in this case will be 
unblinded and standard treatment initiated.  
 
 
Early withdrawal criteria related to heart rate will be age-dependent due to variability in the normal heart 
rate for children of different ages. 
6-12 mos 
HR < 80 
12-24 mos 
HR < 60 
24 mos - <11 years: 
HR < 50 
 
 
Adverse event capture will begin as soon as the patient enters the operating room. Adverse event 
capture will end once the patient leaves the recovery room. Depending on the type of adverse event, the follow 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
up period will be 48 hours-4 weeks.  All adverse events and the intensity of these events, whether related to 
study drug or not, will be recorded for the duration of this study.   Adverse events that occur will be assessed 
for causality.    
The cause of adverse events in relation to the study medications and procedures will be termed as: 
Certain - event or laboratory test abnormality, with plausible time relationship to drug intake; cannot be 
explained by disease or other drugs; response to withdrawal plausible. 
Likely - event or laboratory test abnormality, with reasonable time relationship to drug intake; unlikely to be 
attributed to disease or other drugs; response to withdrawal clinically reasonable. 
Possible - event or laboratory test abnormality, with reasonable time relationship to drug intake; could also be 
explained by disease or other drugs; information on drug withdrawal may be lacking or unclear. 
Unlikely - event or laboratory test abnormality, with a time to drug intake that makes a relationship improbable 
(but not impossible); disease or other drugs provide plausible explanations. 
Unassessible - event or laboratory test abnormality; cannot be judged because information is insufficient or 
Contradictory; data cannot be supplemented or verified. 
The severity of adverse events will be graded ordinally with the following designations:  mild (1), 
moderate (2), severe (3), life-threatening (4). 
Dexmedetomidine 
Adverse Event 
Mild (1) 
Moderate (2) 
Severe (3) 
Life-threatening (4) 
Hemodynamic 
Disturbance 
Bradycardia or 
hypotension (HR 
or BP decreased 
>20% from 
patient's baseline) 
for 0-30 sec 
Bradycardia or 
hypotension (HR 
or BP decreased 
>30% from 
patient's 
baseline) for 0-30 
sec 
Bradycardia or 
hypotension (HR or 
BP decreased >30% 
from patient's 
baseline) for >30 sec 
or requiring treatment 
Cardiac Arrest 
Allergic 
Reaction 
Rash, not 
including hives 
Hives 
Bronchoconstriction, 
non-airway tissue 
swelling 
Leads to anaphylaxis 
 
Fentanyl 
Adverse Event 
Mild (1) 
Moderate (2) 
Severe (3) 
Life-threatening (4) 
Respiratory 
Depression 
Respiratory rate 
<10 
Respiratory rate 
<8 
Respiratory rate <6 or 
requiring intervention 
Respiratory arrest 
Allergic 
Reaction 
Rash, not 
including hives 
Hives 
Bronchoconstriction, 
non-airway tissue 
swelling 
Leads to anaphylaxis 
Hemodynamic 
Disturbance 
Bradycardia or 
hypotension (HR 
or BP decreased 
>20% from 
patient's baseline) 
for 0-30 sec 
Bradycardia or 
hypotension (HR 
or BP decreased 
>30% from 
patient's 
baseline) for 0-30 
sec 
Bradycardia or 
hypotension (HR or 
BP decreased >30% 
from patient's 
baseline) for >30 sec 
or requiring treatment 
Cardiac Arrest 
Nausea 
Temporary, no 
vomiting, requires 
Vomiting, 
requires one 
Vomiting, requires two 
therapeutic 
Vomiting, requires 
more than 2 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
no treatment 
therapeutic 
medication 
medications 
therapeutic 
medications 
 
If 3 or more adverse reactions occur, the study will be temporarily suspended and methodology will be 
reviewed by the study team and safety officer. If 6 or more adverse reactions occur, the study will be stopped. 
Routine preoperative evaluation will be performed on all patients by an attending pediatric 
anesthesiologist prior to surgery. This includes review of the medical record, review of relevant laboratory 
studies, history and physician examination including airway examination, and discussion of the anesthetic plan 
with the parents and/or patient. Unless clinically indicated, patients will not receive preoperative monitoring. 
Intraoperative monitoring will be in accordance with the American Society of Anesthesiologist standard and will 
include 5- lead electrocardiogram, pulse oximetry, non-invasive blood pressure, core temperature, end-tidal 
carbon dioxide, and end-tidal anesthetic concentration (when appropriate.) Monitoring in the operating room is 
continuous, except for non-invasive blood pressure, which is measured at least every 3 minutes. 
In the post-anesthesia care unit, the patient will receive the same monitoring as in the operating room 
except end tidal carbon dioxide. Again, monitoring will be continuous except for non-invasive blood pressure, 
which will be monitored at least every 15 minutes. 
 
Pitfalls and Alternatives. We expect to confirm neuroinflammation in the piglet model and in human children 
based on our preliminary work and existing literature despite not having seen the typical “footprint” of 
neuroinflammation in the small number of animals studied thus far. Although we expect to detect elevated 
cytokines in brain homogenates from animals after only 48 hours, we have revised our brain tissue harvest 
protocol to 72 hours to match reported time after which inflammation is most likely to be evident. Nonetheless, 
we have considered alternatives in the eventuality that inflammatory injury is not seen. Alternative methods to 
detect neuroinflammation can be used. Positron emission tomography (PET) and magnetic resonance imaging 
(MRI) scanning have been used to quantify neuroinflammation in children and in adults.51,52 These modalities 
could easily be adapted for use in our protocol. 
 
Future Directions. This translational, pilot study is designed to spawn multiple future studies and to provide 
the framework for Dr. Whitaker’s career in translational investigation. An option for follow-up on our study 
consent will be included, as children with HC frequently return for surgical revisions. For instance, if we 
demonstrate less inflammation in our TIVA cohort, a future randomized controlled trial evaluating the 
neurocognitive outcomes in HC patients based on anesthetic technique will be warranted.  In addition, we plan 
to collaborate with the Department of Behavioral Health here at Nationwide, as it will be important not only to 
demonstrate neurobiological but also neurocognitive changes. 
 
IRB/IACUC: The animal portion of this study will be performed at The Ohio State University, and IACUC 
approval has already been obtained. We have been working with this model for nearly 2 years and have 
received IACUC approval. For the human arm of our study, an IRB application has been submitted and has 
been approved. 
 
Timeline/Milestones:  
1. November - December 2014 (complete):  
a. Complete and submit the IRB application including informed consent form 
b. Establish protocols with investigational pharmacy; ensure study drugs will be available 
c. Educate practitioners (investigators, anesthesia, PACU and neurosurgery) about the study 
d. Order necessary supplies and materials (animal protocol and study are approved and 
underway) 
2. May 1, 2015: Begin study, pending IRB and IND approval 
3. May – July 2015 
a. Begin to recruit patients during the preoperative visit (Surgery Unit prior to neurosurgery or MRI) 
b. Collect samples (CSF and peripheral blood for VPS patients; peripheral blood for MRI patients) 
c. Assess study procedures and adjust where necessary to ensure safety and efficiency 
d. February 2, 2015: Submit the Ohio State University Davis-Bremer Pre-K Award application 
($50,000 for one year; renewable) 


Emmett Whitaker, M.D. 
 
Research Plan 
Davis-Bremer Pre-K Award 
 
Aug. 31, 2017 
 
4. August 2015: 
a. Evaluate recruitment performance and adjust accordingly  
b. Ad-hoc analysis of preliminary data 
c. Begin ELISA and qPCR analysis of collected samples 
5. July 2015– February 2016: 
a. Maintain recruitement and perform data collection 
b. Continue ELISA and qPCR analysis 
c. Completion of patient recruitment and sample collection at the end of year 2015 
6. February – May 2016: 
a. Analyze all collected data 
b. Prepare initial manuscripts 
c. Possible manuscripts to which we will submit our manuscripts:  
i. Goal manuscript submission deadline: May 30, 2016 
ii. Potential journals for manuscript submission: Anesthesiology, Anesthesia and 
Analgesia, Neuroscience 
d. Determine the national or international meetings at which we will present our data 
e. Potential annual meetings for data presentation: 
i. American Society of Anesthesiologists (ASA); Society for Pediatric Anesthesia (SPA); 
International Anesthesia Research Society (IARS); Society for Neuroscience and Critical 
Care (SNACC) 
f. Dates for annual meetings: 
i. ASA: October 2016 
ii. SPA: October 2016; March 2017 
iii. IARS: March 2017 
iv. SNACC: October 2016 
g. Principal investigator to present data at departmental Grand Rounds 
7. April 2016 – May 2016 
a. Complete data analysis 
b. Prepare abstracts and oral presentations for annual meetings 
c. Consider post-hoc analyses 
d. Determine future directions: 
i. Sex differences in anesthetic-induced neuroinflammation 
ii. Positron-emission tomography quantification of anesthetic-induced neuroinflammation 
iii. Central nervous system - enteric nervous system interactions and their influence on 
anesthetic-induced neuroinflammation 
8. May 30, 2016: Complete study 
 
 


Emmett Whitaker, M.D. 
Appendix 
Davis-Bremer Pre-K Award Application 
February 2, 2015
 
 
14 
 
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February 2, 2015
 
 
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February 2, 2015
 
 
16 
 
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