Center for Blood-Brain Barrier Research
The members of the Center for Blood-Brain Barrier Research include 11 PIs from three
Departments (Biomedical Sciences, Immunotherapeutics & Biotechnology, and Pharmaceutical
Sciences) and their teams (currently 17 Graduate students and five postdocs).
The Center's mission is to discover and develop novel therapeutics that will advance
drug therapy in diseases affecting the central nervous system. The Center will foster
research to characterize new drug targets based on understanding of both receptor
and transport mechanisms and of metabolic pathways.
By encouraging collaboration with colleagues across School of Pharmacy departments,
the Center will raise the pharmacy school's research profile, enhance the competitiveness
of Center members for extramural funding and increase the attractiveness of the TTUHSC
School of Pharmacy to prospective faculty members.
Members of the Center and Their Research Interests
Please click on a name to link to each faculty member's individual web page.
Dr. Abbruscato’s group investigates the role of the glucose carrier GLUT1 and of the
Sodium Dependent Glucose Transporter (SGLT) at the blood-brain barrier (BBB). In particular,
they focus on the regulation of SGLT under pathophysiological conditions such as ischemia/reperfusion
injury (stroke). Selective pharmacological modulation of SGLT could have clinical
implications: (i) inhibition of SGLT at the BBB could be beneficial in stroke by limiting
excessive glucose delivery to brain and brain edema (because SGLT transfers 210 molecules
of water for each Na+ ion and glucose molecule); (ii) modulating SGLT activity in
neurons could be protective by reducing oxidative stress and cellular edema; Further
studies on the expression and regulation of SGLT in vivo with temporary focal ischemia
combined with reperfusion would lead to a better understanding as to how it can be
utilized as a therapeutic target in stroke and other CNS disorders. These studies
are funded by the National Institutes of Neurological Disorders and Stroke in collaboration
with Drs. Bickel and Smith.
Based on substantial work in his lab and by others, he believes that the Na+, K+,
2Cl- Cotransporter (NKCC) plays a critical role in cellular edema associated with
stroke. NKCC protein expression and function has been proven to be modulated by PKC
in ischemia in our lab and could provide a promising drug target. One exciting avenue
to pursue is the �cross-talk� between NKCC and opioid receptors. Classically, opioid
receptor activation stimulates K+ channels and suppresses Ca2+ current into the cytoplasm
of neurons. Opioid receptor activation during stoke has been shown to modulate other
key ion channels by the activation of protein kinase C (PKC). Publications from Drs.
Abbruscato and Karamyan labs have also shown that the selective δ agonist, DPDPE,
μ agonist, DAMGO and δ/μ receptor agonist, biphalin, significantly decrease water
gain induced in hippocampus slices subjected to oxygen-glucose deprivation. Biphalin
has also been shown to reduce infarction and edema ratios, penumbral injury, and neuronal
NKCC activation via PKC (Yang et al., JPET 2011; Yang et al., Brain Research 2011).
Current experiments in collaboration with Dr. Karamyan are characterizing the regional
expression of opioid receptor types in the ischemic brain during ischemia-reperfusion
injury. Structure activity experiments are being conducted in collaboration with Dr.
Victor Hruby (University of Arizona. Department of Chemistry) characterizing novel
opioid agonists that are designed to target the predominant opioid receptors expressed
during stroke injury. Multi-functional opioid receptor agonists are being designed
with a variety of pharmacophores positioned differently by a variety of linking strategies.
The spectrum of methods in Dr. Abbruscato’s laboratory includes the following in vitro and in vivo techniques:
Models of the BBB (bovine primary, mouse cell line); hypoxia chamber for reduced oxygen/glucose
experiments; trans-endothelial cell resistance measurement; mouse primary neuronal
and astrocytic cell culture; hippocampal brain slicing and oxygen glucose deprivation
experiments; Western blot analysis. Available in vivo techniques include permanent
and reversible mouse MCAO; infarct and edema ratio; locomotor testing for neurologic
outcome post ischemia; Blood-CNS pharmacokinetics; in situ brain perfusion, genetic
and experimental models of diabetes mellitus; mouse or rat brain endothelial cell
The main focus of Dr. Bickel’s lab is the identification of novel, BBB-specific targets
for drug delivery. Receptor systems like the transferrin receptor or insulin receptor
are highly expressed at the BBB and have been successfully used in preclinical studies
for brain targeting, but these receptors are obviously not brain-specific. A vascular
targeting strategy based on receptors only expressed at the BBB has thus far been
elusive. This is an attractive goal, because it may reduce the risk of unwanted effects
in peripheral organs, and potentially allow regional targeting to brain tissue affected
by disease (e.g., brain tumors or neuroinflammatory conditions associated with ischemia/reperfusion
injury). We are collaborating with Dr. Jon Weidanz (Immunotherapy Center, Texas Tech
School of Pharmacy, Abilene) on the characterization of peptide-MHC complexes on brain
endothelial cells as unique targets for specific T-cell receptor mimic antibodies
(TCRm). These studies received initial funding by a NIH R21 grant. A current collaborative
project (with Dr. Jon Weidanz and Dr. Paul Lockman, TTUHSC School of Pharmacy, Amarillo)
explores the potential therapeutic effect of TCRm on brain metstasis of breast cancer.
It is supported by an IDEA Award of the Breast Cancer Research Program (DoD).
Another project in Dr. Bickel’s group addresses the question, whether protection of
the BBB by targeted delivery of anti-inflammatory drugs to brain microvascular endothelial
cells can be beneficial in stroke therapy. Our studies in models of transient and
permanent brain ischemia in mice showed significant therapeutic effect of anti-inflammatory
oligonucleotide drugs when delivered via BBB transferrin receptors. Currently our
goal is to characterize at the cellular level, where the pharmacological effect seen
in the whole animal models actually occur (e.g., activity on endothelial cells, neurons,
and/or glial cells?). This research is supported by a grant from the American Heart
Association. In addition to the middle cerebral artery occlusion (MCAO) stroke model,
we are using brain microdialysis to monitor pharmacological effects in vivo under
The following methods are routinely used in Dr. Bickel’s laboratory:
In vitro techniques:
Brain endothelial cell culture (cell lines from human and murine origin); production
and purification of monoclonal antibodies from hybridoma supernatant; transport studies
with endothelial cell monolayers (Transwell); cell adhesion assays ; flowcytometry
; radiotracer labeling; radioligand binding assays; ELISA; Northern blotting; Real
time PCR; gel shift assays; HPLC and FPLC for analytical and semipreparative purpose;
protein conjugations; supravital staining of brain slices (TTC); immunohistochemistry;
wide field fluorescence microscopy and confocal microscopy.
Ex vivo methods:
Autoradiography of brain cryostat sections following administration of radioactive
tracers in vivo; capillary depletion technique to characterize brain uptake; brain
capillary and endothelial cell isolation.
In vivo techniques:
Middle Cerebral Artery Occlusion (MCAO) by intraluminal suture for induction of permanent
or transient brain ischemia in mice; brain microdialysis; Laser Doppler Flowmetry;
carotid artery catheterization in mice (and rats) for arterial blood sampling in pharmacokinetic
studies; carotid artery catheterization for vascular brain perfusion; sensorimotor
testing in mice (Corner test).
Dr. Cucullo’s group research focus investigates three areas of research revolving
around the physiology and pathophysiology of the blood-brain barrier: 1) Pathophysiology of tobacco smoke toxicity at the neurovascular unit. The goal of this research project is to investigate both in vivo (rodent models)
and in vitro differential effects of tobacco/cigarette smoke exposure from “reduced-exposure”
versus regular cigarette brands, and to identify potential prodromic factors for the
pathogenesis and progression of cerebrovascular impairment and brain disorders. The
study encompasses cross-interaction between systemic inflammation and neuronal activity
and involves In vitro and In vivo assessment of peripheral blood immune cells interaction
via inflammatory mediators with the blood-brain barrier endothelium as well as pathogenesis
and progression of secondary brain injuries. The study makes use of immunohistochemistry
and electroencephalographic monitoring techniques (brain microsurgery and stereotactic
electrode placement) as well as brain histology, and characterization by FACS analysis
and cell sorting of sub-populations of circulating white blood cells.
2) In vitro modeling of the human cerebrovascular system. The goal of this research study is to optimize current hollow fiber technology with
the purpose of developing a dynamic (flow-based) artificial models of the human brain
vascular network to enable/facilitate the study of the effect of hemodynamic (e.g.,
stroke, transient ischemia) and pro-inflammatory challenges on BBB integrity and function
as well as provide an ideal artificial testing and optimization platform for rationale
CNS drug design and delivery methods. The study encompasses the characterization of
the pharmacokinetic and pharmacodynamic properties of the BBB including mechanisms
of pharmacoresistance; BBB induction and maintenance; evaluation of BBB function in
response to endogenous and exogenous stimuli (e.g., inflammation, stroke, hypoperfusion,
ischemia, brain trauma, etc).
3) Role of Altered Glycemia in the Pathogenesis of CNS Disorders through alterations
of BBB function and integrity. A hallmark of the chronic complications associated with diabetes mellitus (particularly
type 2) is compromised microvascular integrity. The complexity of the processes that
cause CNS disease in diabetes and the difficulty of detecting vascular impairments
in the large and heterogeneous brain microvascular bed make the study and characterization
of this pathology extremely difficult. The main goal of this study is to assess the
impact and unfold the mechanism/s by which hyper- and hypoglycemia can impair BBB
integrity and function.
General Methods in use in Dr.Cucullo’s lab include the following:
Models of the BBB (bovine primary, human primary, Human cell lines, Rodent (rat) cell
line); hypoxia chamber for reduced oxygen/glucose experiments; real time trans-endothelial
cell resistance measurement; Static (Transwell) and flow-based (based on capillary-like
hollow fiber technology) BBB multi-culture models; In vitro flow cessation-reperfusion
w/wo circulating immune cells; Western blot analysis, ELISA, Immunocytochemistry;
online real time NO, H2O2, and glucose measurements, etc.
Our group is studying nature and function of the brain renin-angiotensin system, and
its regulation at the level of receptors and proteolytic enzymes in health and disease.
In the last several years our main research efforts were focused on pharmacological
and biochemical characterization of a novel, non-AT1, non-AT2 angiotensin binding
site discovered in our laboratory. Most recently, we identified the novel angiotensin
binding site as membrane-bound variant of metalloendopeptidase neurolysin (EC 184.108.40.206).
Our current studies are focused on understanding the (patho)physiological role of
neurolysin in neurogenic hypertension and ischemic stroke.
Additionally, in collaboration with Dr. Tom Abbruscato we study the role of opioid
receptors for neuroprotection in stroke. This project involves (patho)physiological
and pharmacological studies to understand alterations of opioid receptors in pathophysiology
of stroke, and development of new opioid ligands (in collaboration with medicinal
chemists) to target these receptors.
The following techniques/methods are regularly used in our laboratory:
primary brain cultures (neuronal, astroglial and capillary endothelial), cell-viability
assays, radioligand receptor binding assays (whole cell and membrane), functional
assays for GPCRs, in vitro autoradiography, immunoblotting, immunocytochemistry, fluorescence
microscopy, protein modification and purification, proteolytic studies, enzymatic
assays, supravital staining of brain slices (TTC), middle cerebral artery occlusion
(MCAO) by intraluminal suture for induction of permanent or transient brain ischemia
in mice, Laser Doppler Flowmetry; sensorimotor testing in mice; blood pressure monitoring
in mice and rats using radiotelemetry, dosing and sampling methods in rats and mice.
Dr. Mehvar’s research is focused on the pharmacological approaches to reduce ischemia-reperfusion
(IR) injury in the liver. Hepatic ischemia-reperfusion injury is a serious, yet so
far unavoidable, complication of many clinical situations such as liver transplantation,
major liver resection surgery (Pringle maneuver), and hemorrhagic and septic shock.
Although all of the in vivo conditions resulting in the interruption of blood supply
to the liver produce warm IR injury, liver transplantation is unique in that, in addition
to a variable degree of warm IR injury during organ retrieval and/or transplantation,
it is also subjected to cold (storage) IR injury during organ preservation. Initially,
we focused on the pharmacokinetic-based delivery approaches to target anti-inflammatory
drugs to the liver for prevention of IR injury. Our current emphasis, however, is
on the role of cytochrome P450 in generation of reactive oxygen species (ROS) and
pharmacological approaches to inhibit P450 generation of ROS.
Part of this project is the live-cell imaging of hypoxia/reoxygenation injury in hepatocytes
by confocal microscopy, which recently stimulated collaboration with Dr. Bickel’s
Major techniques used in our laboratory are:
In vivo warm hepatic IR model in rats; ex vivo warm hepatic IR model in rats; ex vivo
cold hepatic IR model in rats; rat liver transplantation model; macromolecular-based
drug delivery to the liver; primary cultures of rat hepatocytes; P450 content and
activity assays; HPLC analysis of small molecules and macromolecules in biological
samples; pharmacokinetic dosing and sampling methods in rodents; and pharmacokinetic
and drug metabolism data analysis.
The members of this laboratory are using various in vivo and in vitro approaches to
study brain metabolism.
1. Epilepsy. In collaboration with Dr Karin Borges, Univ Queensland, Australia, we showed an anaplerotic
diet rich in triheptanoin could alter seizure parameters in two mouse models of epilepsy.
Current research is focused on identifying the molecular mechanisms of this protective
response. Pharmacological manipulations of metabolic pathways will allow us to identify
pathways that are affected by triheptanoin and determine if they are involved in the
antiepileptic response. Further, we showed that voluntary exercise in mice alters
seizure development in the pilocarpine/status epilepticus model. Ongoing experiments
are testing if exercise and triheptanoin share a common mechanism of action.
2. Lipocalin-2 (LCN2). This iron-binding protein is induced as part of the innate immune response and is
induced early in epileptogenesis. In an in vitro model, a necrotic cell extract can
induce LCN2 in the C6 astroglioma cell line. Experiments are underway to identify
the mechanism of induction. As inflammation contributes to the development of epilepsy,
a long-term goal of this project is to identify mechanisms by which LCN2 contributes
to the development of or protection from epilepsy.
3. Choline transporter. Choline is an essential nutrient important for growth and maintenance of cells. The
protein responsible for uptake into cells has not been convincingly demonstrated.
We are using chemical and molecular biological approaches to identify this protein.
Present research involves development of a photoaffinity probe to identify the transporter.
Once identified, our long-term goals are to identify the role of this protein in tissue
injury and repair.
A wide range of experiment approaches and techniques are in use in the laboratory.
We are currently using in vivo models for epilepsy. There is extensive experience
in primary neural cell culture along with use of culture cell lines. Molecular expertise
includes all routine biochemical and molecular techniques such as chromatography,
cloning and expression of proteins, quantitative PCR, immunoblotting and histochemistry,
microscopy, and RNA silencing.
The main focus of Dr. Trippier’s research is drug discovery and medicinal chemistry.
The group uses the principles of rational and in silico drug design to generate compounds
with desirable activity. The techniques of synthetic medicinal chemistry are used
to probe the structure-activity relationship of the molecules and enhance the observed
biological activity. Of specific interest is the design of highly specific and potent
kinase inhibitors, a class of enzyme that represents excellent targets for therapeutic
intervention of a range of neurodegenerative diseases such as Alzheimer’s and the
neuromuscular disease Spinal Muscular Atrophy. The group is also interested in designing
small molecule probes to elucidate the complex signaling pathways involved in axon
Major techniques used in our laboratory include: Organic synthesis; compound purification; chemical biology; HPLC; nuclear magnetic
resonance structure elucidation; mass spectrometry; molecular modeling; enzyme-based
assays; cell-based assays.
With the collaboration of Dr. Ulrich Bickel (TTUHSC) and Dr. George Gokel (University
of Missouri, St. Louis), I recently submitted an RO1 proposal to synthesize and test
a series of compounds that are predicted to increase eNOS activity. Our preliminary
studies showed that triacsin C, the lead compound in the series, inhibits long chain
fatty acyl CoA synthetase, inhibits eNOS palmitoylation, increases NO production in
both cultured endothelial cells and intact aorta, and enhances methacholine induced
relaxation of vascular smooth muscle. It is anticipated that these compounds may have
clinical utility in treatment of hypertension. There is preliminary evidence that
triacsin C and like compounds may have neuroprotective effects in an experimental
model of stroke. In both a temporary (1 hour of occlusion followed by 24 hours reperfusion)
and permanent (24 hours occlusion) stroke model, triacsin C significantly reduced
the cerebral infarct volume. Other data indicates that triacsin C may not only increase
eNOS activity, it may also inhibit iNOS expression. Hypertension is the single most
important risk factor for stroke. It is estimated that about 80% of stroke victims
are hypertensive. Our observations with triacsin C suggest that it is possible to
develop an antihypertensive drug that would also have the benefit of reducing neurologic
damage in the event of a stroke. The Ischemia/Reperfusion Injury Center would provide
an important mechanism for feedback and discussion of this research.
Major Biochemical Techniques used in my laboratory include: Assay of catalytic activities of long chain fatty acyl CoA synthetase and nitric
oxide synthase, radioimmunoassay of prostaglandins, Griess reaction, and extraction
and analysis of total tissue/cellular lipid. We also do cell culture (human and rat
coronary endothelial cells), endothelial shear stress measurement, western blotting,
immunoprecipitation, and siRNA. In addition, I have extensive experience with and
am fully equipped to do the Langendorf perfused heart preparation.
Information on this page is current as of February 2013 >/em>